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• Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy. Ghosh, Arindam; Meub, Mara; Helmerich, Dominic A.; Weingart, Julia; Eiring, Patrick; Nerreter, Thomas; Kortüm, K. Martin; Doose, Sören; Sauer, Markus. In Science, 387(6730), S. eadq4510-. American Association for the Advancement of Science, 2025.
  • [ Abstract ]
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  Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable ~15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration?dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley

  @article{ghoshdecoding, abstract = {Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable  15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration?dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley}, author = {Ghosh, Arindam and Meub, Mara and Helmerich, Dominic A. and Weingart, Julia and Eiring, Patrick and Nerreter, Thomas and Kortüm, K. Martin and Doose, Sören and Sauer, Markus}, booktitle = {Science}, journal = {Science}, keywords = {home}, number = 6730, pages = {eadq4510–}, publisher = {American Association for the Advancement of Science}, title = {Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy}, volume = 387, year = 2025 }

  %0 Journal Article %1 ghoshdecoding %A Ghosh, Arindam %A Meub, Mara %A Helmerich, Dominic A. %A Weingart, Julia %A Eiring, Patrick %A Nerreter, Thomas %A Kortüm, K. Martin %A Doose, Sören %A Sauer, Markus %B Science %D 2025 %I American Association for the Advancement of Science %J Science %N 6730 %P eadq4510-- %R 10.1126/science.adq4510 %T Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy %U https://doi.org/10.1126/science.adq4510 %V 387 %X Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable ~15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration?dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley

• Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy. Raj, Gowtham; Vasudev, D. S.; Christopher, Sarah; Babulal, Anupama; Harsha, P.; Ram, Soumakanya; Tiwari, Mehul; Sauer, Markus; Varghese, Reji. In Nanoscale, 16(7), S. 3755–3763. The Royal Society of Chemistry, 2024.
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  The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host–guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic β-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.

  @article{raj2024multifunctional, abstract = {The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host–guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic β-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.}, author = {Raj, Gowtham and Vasudev, D. S. and Christopher, Sarah and Babulal, Anupama and Harsha, P. and Ram, Soumakanya and Tiwari, Mehul and Sauer, Markus and Varghese, Reji}, journal = {Nanoscale}, keywords = {sauer}, number = 7, pages = {3755–3763}, publisher = {The Royal Society of Chemistry}, title = {Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy}, volume = 16, year = 2024 }

  %0 Journal Article %1 raj2024multifunctional %A Raj, Gowtham %A Vasudev, D. S. %A Christopher, Sarah %A Babulal, Anupama %A Harsha, P. %A Ram, Soumakanya %A Tiwari, Mehul %A Sauer, Markus %A Varghese, Reji %D 2024 %I The Royal Society of Chemistry %J Nanoscale %N 7 %P 3755--3763 %R 10.1039/D3NR06071C %T Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy %U http://dx.doi.org/10.1039/D3NR06071C %V 16 %X The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host–guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic β-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.
• Development of Peptide-Based Probes for Molecular Imaging of the Postsynaptic Density in the Brain. Fernandes, Eduardo F. A.; Palner, Mikael; Raval, Nakul Ravi; Jeppesen, Troels E.; Danková, Daniela; Bærentzen, Simone L.; Werner, Christian; Eilts, Janna; Maric, Hans M.; Doose, Sören; Aripaka, Sanjay Sagar; Kaalund, Sanne Simone; Aznar, Susana; Kjaer, Andreas; Schlosser, Andreas; Haugaard-Kedström, Linda M.; Knudsen, Gitte M.; Herth, Matthias M.; Stro̷mgaard Kristian. In J. Med. Chem., 67(14), S. 11975–11988. American Chemical Society, 2024.
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  The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson’s disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.

  @article{fernandes2024development, abstract = {The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson’s disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.}, author = {Fernandes, Eduardo F. A. and Palner, Mikael and Raval, Nakul Ravi and Jeppesen, Troels E. and Danková, Daniela and Bærentzen, Simone L. and Werner, Christian and Eilts, Janna and Maric, Hans M. and Doose, Sören and Aripaka, Sanjay Sagar and Kaalund, Sanne Simone and Aznar, Susana and Kjaer, Andreas and Schlosser, Andreas and Haugaard-Kedström, Linda M. and Knudsen, Gitte M. and Herth, Matthias M. and Stro̷mgaard, Kristian}, booktitle = {Journal of Medicinal Chemistry}, journal = {J. Med. Chem.}, keywords = {maric}, month = {07}, number = 14, pages = {11975–11988}, publisher = {American Chemical Society}, title = {Development of Peptide-Based Probes for Molecular Imaging of the Postsynaptic Density in the Brain}, volume = 67, year = 2024 }

  %0 Journal Article %1 fernandes2024development %A Fernandes, Eduardo F. A. %A Palner, Mikael %A Raval, Nakul Ravi %A Jeppesen, Troels E. %A Danková, Daniela %A Bærentzen, Simone L. %A Werner, Christian %A Eilts, Janna %A Maric, Hans M. %A Doose, Sören %A Aripaka, Sanjay Sagar %A Kaalund, Sanne Simone %A Aznar, Susana %A Kjaer, Andreas %A Schlosser, Andreas %A Haugaard-Kedström, Linda M. %A Knudsen, Gitte M. %A Herth, Matthias M. %A Stro̷mgaard, Kristian %B Journal of Medicinal Chemistry %D 2024 %I American Chemical Society %J J. Med. Chem. %N 14 %P 11975--11988 %R 10.1021/acs.jmedchem.4c00615 %T Development of Peptide-Based Probes for Molecular Imaging of the Postsynaptic Density in the Brain %U https://doi.org/10.1021/acs.jmedchem.4c00615 %V 67 %X The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson’s disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.
• A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson’s disease. Sade, Ofir; Fischel, Daphna; Barak-Broner, Noa; Halevi, Shir; Gottfried, Irit; Bar-On, Dana; Sachs, Stefan; Mirelman, Anat; Thaler, Avner; Gour, Aviv; Kestenbaum, Meir; Gana Weisz, Mali; Anis, Saar; Soto, Claudio; Roitman, Melanie Shanie; Shahar, Shimon; Doppler, Kathrin; Sauer, Markus; Giladi, Nir; Lev, Nirit; Alcalay, Roy N.; Hassin-Baer, Sharon; Ashery, Uri. In Frontiers in Molecular Neuroscience, 17. 2024.
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  Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson’s disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001–0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.

  @article{sade2024novel, abstract = {Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson’s disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001–0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.}, author = {Sade, Ofir and Fischel, Daphna and Barak-Broner, Noa and Halevi, Shir and Gottfried, Irit and Bar-On, Dana and Sachs, Stefan and Mirelman, Anat and Thaler, Avner and Gour, Aviv and Kestenbaum, Meir and Gana Weisz, Mali and Anis, Saar and Soto, Claudio and Roitman, Melanie Shanie and Shahar, Shimon and Doppler, Kathrin and Sauer, Markus and Giladi, Nir and Lev, Nirit and Alcalay, Roy N. and Hassin-Baer, Sharon and Ashery, Uri}, journal = {Frontiers in Molecular Neuroscience}, keywords = {sauer}, title = {A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson’s disease}, volume = 17, year = 2024 }

  %0 Journal Article %1 sade2024novel %A Sade, Ofir %A Fischel, Daphna %A Barak-Broner, Noa %A Halevi, Shir %A Gottfried, Irit %A Bar-On, Dana %A Sachs, Stefan %A Mirelman, Anat %A Thaler, Avner %A Gour, Aviv %A Kestenbaum, Meir %A Gana Weisz, Mali %A Anis, Saar %A Soto, Claudio %A Roitman, Melanie Shanie %A Shahar, Shimon %A Doppler, Kathrin %A Sauer, Markus %A Giladi, Nir %A Lev, Nirit %A Alcalay, Roy N. %A Hassin-Baer, Sharon %A Ashery, Uri %D 2024 %J Frontiers in Molecular Neuroscience %T A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson’s disease %U https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2024.1431549 %V 17 %X Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson’s disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001–0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.
• One-step nanoscale expansion microscopy reveals individual protein shapes. Shaib, Ali H.; Chouaib, Abed Alrahman; Chowdhury, Rajdeep; Altendorf, Jonas; Mihaylov, Daniel; Zhang, Chi; Krah, Donatus; Imani, Vanessa; Spencer, Russell K. W.; Georgiev, Svilen Veselinov; Mougios, Nikolaos; Monga, Mehar; Reshetniak, Sofiia; Mimoso, Tiago; Chen, Han; Fatehbasharzad, Parisa; Crzan, Dagmar; Saal, Kim-Ann; Alawieh, Mohamad Mahdi; Alawar, Nadia; Eilts, Janna; Kang, Jinyoung; Soleimani, Alireza; Müller, Marcus; Pape, Constantin; Alvarez, Luis; Trenkwalder, Claudia; Mollenhauer, Brit; Outeiro, Tiago F.; Köster, Sarah; Preobraschenski, Julia; Becherer, Ute; Moser, Tobias; Boyden, Edward S.; Aricescu, A. Radu; Sauer, Markus; Opazo, Felipe; Rizzoli, Silvio O. In Nature Biotechnology. 2024.
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  The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

  @article{shaib2024onestep, abstract = {The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the  17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.}, author = {Shaib, Ali H. and Chouaib, Abed Alrahman and Chowdhury, Rajdeep and Altendorf, Jonas and Mihaylov, Daniel and Zhang, Chi and Krah, Donatus and Imani, Vanessa and Spencer, Russell K. W. and Georgiev, Svilen Veselinov and Mougios, Nikolaos and Monga, Mehar and Reshetniak, Sofiia and Mimoso, Tiago and Chen, Han and Fatehbasharzad, Parisa and Crzan, Dagmar and Saal, Kim-Ann and Alawieh, Mohamad Mahdi and Alawar, Nadia and Eilts, Janna and Kang, Jinyoung and Soleimani, Alireza and Müller, Marcus and Pape, Constantin and Alvarez, Luis and Trenkwalder, Claudia and Mollenhauer, Brit and Outeiro, Tiago F. and Köster, Sarah and Preobraschenski, Julia and Becherer, Ute and Moser, Tobias and Boyden, Edward S. and Aricescu, A. Radu and Sauer, Markus and Opazo, Felipe and Rizzoli, Silvio O.}, journal = {Nature Biotechnology}, keywords = {sauer}, title = {One-step nanoscale expansion microscopy reveals individual protein shapes}, year = 2024 }

  %0 Journal Article %1 shaib2024onestep %A Shaib, Ali H. %A Chouaib, Abed Alrahman %A Chowdhury, Rajdeep %A Altendorf, Jonas %A Mihaylov, Daniel %A Zhang, Chi %A Krah, Donatus %A Imani, Vanessa %A Spencer, Russell K. W. %A Georgiev, Svilen Veselinov %A Mougios, Nikolaos %A Monga, Mehar %A Reshetniak, Sofiia %A Mimoso, Tiago %A Chen, Han %A Fatehbasharzad, Parisa %A Crzan, Dagmar %A Saal, Kim-Ann %A Alawieh, Mohamad Mahdi %A Alawar, Nadia %A Eilts, Janna %A Kang, Jinyoung %A Soleimani, Alireza %A Müller, Marcus %A Pape, Constantin %A Alvarez, Luis %A Trenkwalder, Claudia %A Mollenhauer, Brit %A Outeiro, Tiago F. %A Köster, Sarah %A Preobraschenski, Julia %A Becherer, Ute %A Moser, Tobias %A Boyden, Edward S. %A Aricescu, A. Radu %A Sauer, Markus %A Opazo, Felipe %A Rizzoli, Silvio O. %D 2024 %J Nature Biotechnology %R 10.1038/s41587-024-02431-9 %T One-step nanoscale expansion microscopy reveals individual protein shapes %U https://doi.org/10.1038/s41587-024-02431-9 %X The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.
• In vitro characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease. Breyer, Maximilian; Grüner, Julia; Klein, Alexandra; Finke, Laura; Klug, Katharina; Sauer, Markus; Üçeyler, Nurcan. In Molecular Genetics and Metabolism Reports, 38, S. 101029-. 2024.
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  Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.

  @article{breyer2024vitro, abstract = {Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.}, author = {Breyer, Maximilian and Grüner, Julia and Klein, Alexandra and Finke, Laura and Klug, Katharina and Sauer, Markus and Üçeyler, Nurcan}, journal = {Molecular Genetics and Metabolism Reports}, keywords = {sauer}, pages = {101029–}, title = {In vitro characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease}, volume = 38, year = 2024 }

  %0 Journal Article %1 breyer2024vitro %A Breyer, Maximilian %A Grüner, Julia %A Klein, Alexandra %A Finke, Laura %A Klug, Katharina %A Sauer, Markus %A Üçeyler, Nurcan %D 2024 %J Molecular Genetics and Metabolism Reports %P 101029-- %R https://doi.org/10.1016/j.ymgmr.2023.101029 %T In vitro characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease %U https://www.sciencedirect.com/science/article/pii/S2214426923000757 %V 38 %X Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.
• Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy. Sachs, Stefan; Reinhard, Sebastian; Eilts, Janna; Sauer, Markus; Werner, Christian. In Frontiers in Cellular Neuroscience, 18. 2024.
  • [ Abstract ]
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  High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20–40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).

  @article{sachs2024visualizing, abstract = {High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using  8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20–40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).}, author = {Sachs, Stefan and Reinhard, Sebastian and Eilts, Janna and Sauer, Markus and Werner, Christian}, journal = {Frontiers in Cellular Neuroscience}, keywords = {werner}, title = {Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy}, volume = 18, year = 2024 }

  %0 Journal Article %1 sachs2024visualizing %A Sachs, Stefan %A Reinhard, Sebastian %A Eilts, Janna %A Sauer, Markus %A Werner, Christian %D 2024 %J Frontiers in Cellular Neuroscience %T Visualizing the trans-synaptic arrangement of synaptic proteins by expansion microscopy %U https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2024.1328726 %V 18 %X High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20–40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1).
• LGI1 Autoantibodies Enhance Synaptic Transmission by Presynaptic Kv1 Loss and Increased Action Potential Broadening. Ritzau-Jost, Andreas; Gsell, Felix; Sell, Josefine; Sachs, Stefan; Montanaro, Jacqueline; Kirmann, Toni; Maaß, Sebastian; Irani, Sarosh R.; Werner, Christian; Geis, Christian; Sauer, Markus; Shigemoto, Ryuichi; Hallermann, Stefan. In Neurology Neuroimmunology & Neuroinflammation, 11(5), S. e200284-. Wolters Kluwer, 2024.
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  Background and Objectives: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. Methods: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. Results: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. Discussion Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.

  @article{ritzaujostautoantibodies, abstract = {Background and Objectives: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. Methods: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. Results: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. Discussion Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.}, author = {Ritzau-Jost, Andreas and Gsell, Felix and Sell, Josefine and Sachs, Stefan and Montanaro, Jacqueline and Kirmann, Toni and Maaß, Sebastian and Irani, Sarosh R. and Werner, Christian and Geis, Christian and Sauer, Markus and Shigemoto, Ryuichi and Hallermann, Stefan}, booktitle = {Neurology Neuroimmunology & Neuroinflammation}, journal = {Neurology Neuroimmunology & Neuroinflammation}, keywords = {home}, number = 5, pages = {e200284–}, publisher = {Wolters Kluwer}, title = {LGI1 Autoantibodies Enhance Synaptic Transmission by Presynaptic Kv1 Loss and Increased Action Potential Broadening}, volume = 11, year = 2024 }

  %0 Journal Article %1 ritzaujostautoantibodies %A Ritzau-Jost, Andreas %A Gsell, Felix %A Sell, Josefine %A Sachs, Stefan %A Montanaro, Jacqueline %A Kirmann, Toni %A Maaß, Sebastian %A Irani, Sarosh R. %A Werner, Christian %A Geis, Christian %A Sauer, Markus %A Shigemoto, Ryuichi %A Hallermann, Stefan %B Neurology Neuroimmunology & Neuroinflammation %D 2024 %I Wolters Kluwer %J Neurology Neuroimmunology & Neuroinflammation %N 5 %P e200284-- %R 10.1212/NXI.0000000000200284 %T LGI1 Autoantibodies Enhance Synaptic Transmission by Presynaptic Kv1 Loss and Increased Action Potential Broadening %U https://doi.org/10.1212/NXI.0000000000200284 %V 11 %X Background and Objectives: Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening. Methods: We studied the effects of patient-derived purified polyclonal LGI1 autoantibodies on synaptic structure and function by combining direct patch-clamp recordings from presynaptic boutons and somata of hippocampal neurons with super-resolution light and electron microscopy of hippocampal cultures and brain slices. We also identified the protein domain mediating the presynaptic effect using domain-specific patient-derived monoclonal antibodies. Results: LGI1 autoantibodies dose-dependently increased short-term depression during high-frequency transmission, consistent with increased release probability. The increased neurotransmission was not related to presynaptic calcium channels because presynaptic Cav2.1 channel density, calcium current amplitude, and calcium channel gating were unaffected by LGI1 autoantibodies. By contrast, application of LGI1 autoantibodies homogeneously reduced Kv1.1 and Kv1.2 channel density on the surface of presynaptic boutons. Direct presynaptic patch-clamp recordings revealed that LGI1 autoantibodies cause a pronounced broadening of the presynaptic action potential. Domain-specific effects of LGI1 autoantibodies were analyzed at the neuronal soma. Somatic action potential broadening was induced by polyclonal LGI1 autoantibodies and patient-derived monoclonal autoantibodies targeting the epitempin domain, but not the leucin-rich repeat domain. Discussion Our results indicate that LGI1 autoantibodies reduce the density of both Kv1.1 and Kv1.2 on presynaptic boutons, without actions on calcium channel density or function, thereby broadening the presynaptic action potential and increasing neurotransmitter release. This study provides a molecular explanation for the neuronal hyperactivity observed in patients with LGI1 autoantibodies.
• Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes. Link, Fabian; Borges, Alyssa; Karo, Oliver; Jungblut, Marvin; Müller, Thomas; Meyer-Natus, Elisabeth; Krüger, Timothy; Sachs, Stefan; Jones, Nicola G; Morphew, Mary; Sauer, Markus; Stigloher, Christian; McIntosh, J Richard; Engstler, Markus. In eLife, 12, D. Soldati-Favre (Hrsg.), S. RP91194-. eLife Sciences Publications, Ltd, 2024.
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  Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

  @article{link2024continuous, abstract = {Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.}, author = {Link, Fabian and Borges, Alyssa and Karo, Oliver and Jungblut, Marvin and Müller, Thomas and Meyer-Natus, Elisabeth and Krüger, Timothy and Sachs, Stefan and Jones, Nicola G and Morphew, Mary and Sauer, Markus and Stigloher, Christian and McIntosh, J Richard and Engstler, Markus}, editor = {Soldati-Favre, Dominique}, journal = {eLife}, keywords = {sauer}, pages = {RP91194–}, publisher = {eLife Sciences Publications, Ltd}, title = {Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes}, volume = 12, year = 2024 }

  %0 Journal Article %1 link2024continuous %A Link, Fabian %A Borges, Alyssa %A Karo, Oliver %A Jungblut, Marvin %A Müller, Thomas %A Meyer-Natus, Elisabeth %A Krüger, Timothy %A Sachs, Stefan %A Jones, Nicola G %A Morphew, Mary %A Sauer, Markus %A Stigloher, Christian %A McIntosh, J Richard %A Engstler, Markus %D 2024 %E Soldati-Favre, Dominique %I eLife Sciences Publications, Ltd %J eLife %P RP91194-- %R 10.7554/eLife.91194 %T Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes %U https://doi.org/10.7554/eLife.91194 %V 12 %X Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.
• The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells. Schempp, Rebekka; Eilts, Janna; Schöl, Marie; Grijalva Yépez, Maria F.; Fekete, Agnes; Wigger, Dominik; Schumacher, Fabian; Kleuser, Burkhard; van Ham, Marco; Jänsch, Lothar; Sauer, Markus; Avota, Elita. 2024.
  • [ Abstract ]
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  The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

  @electronic{schempp2024neutral, abstract = {The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.}, author = {Schempp, Rebekka and Eilts, Janna and Schöl, Marie and Grijalva Yépez, Maria F. and Fekete, Agnes and Wigger, Dominik and Schumacher, Fabian and Kleuser, Burkhard and van Ham, Marco and Jänsch, Lothar and Sauer, Markus and Avota, Elita}, booktitle = {International Journal of Molecular Sciences}, keywords = {sauer}, number = 6, title = {The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells}, volume = 25, year = 2024 }

  %0 Generic %1 schempp2024neutral %A Schempp, Rebekka %A Eilts, Janna %A Schöl, Marie %A Grijalva Yépez, Maria F. %A Fekete, Agnes %A Wigger, Dominik %A Schumacher, Fabian %A Kleuser, Burkhard %A van Ham, Marco %A Jänsch, Lothar %A Sauer, Markus %A Avota, Elita %B International Journal of Molecular Sciences %D 2024 %N 6 %R 10.3390/ijms25063247 %T The Role of Neutral Sphingomyelinase-2 (NSM2) in the Control of Neutral Lipid Storage in T Cells %V 25 %X The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.
• Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy. Rühling, Marcel; Kersting, Louise; Wagner, Fabienne; Schumacher, Fabian; Wigger, Dominik; Helmerich, Dominic A.; Pfeuffer, Tom; Elflein, Robin; Kappe, Christian; Sauer, Markus; Arenz, Christoph; Kleuser, Burkhard; Rudel, Thomas; Fraunholz, Martin; Seibel, Jürgen. In Nature Communications, 15(1), S. 7456-. 2024.
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  Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.

  @article{ruhling2024trifunctional, abstract = {Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.}, author = {Rühling, Marcel and Kersting, Louise and Wagner, Fabienne and Schumacher, Fabian and Wigger, Dominik and Helmerich, Dominic A. and Pfeuffer, Tom and Elflein, Robin and Kappe, Christian and Sauer, Markus and Arenz, Christoph and Kleuser, Burkhard and Rudel, Thomas and Fraunholz, Martin and Seibel, Jürgen}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {7456–}, title = {Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy}, volume = 15, year = 2024 }

  %0 Journal Article %1 ruhling2024trifunctional %A Rühling, Marcel %A Kersting, Louise %A Wagner, Fabienne %A Schumacher, Fabian %A Wigger, Dominik %A Helmerich, Dominic A. %A Pfeuffer, Tom %A Elflein, Robin %A Kappe, Christian %A Sauer, Markus %A Arenz, Christoph %A Kleuser, Burkhard %A Rudel, Thomas %A Fraunholz, Martin %A Seibel, Jürgen %D 2024 %J Nature Communications %N 1 %P 7456-- %R 10.1038/s41467-024-51874-w %T Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy %U https://doi.org/10.1038/s41467-024-51874-w %V 15 %X Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.
• Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit. Erbacher, Christoph; Britz, Sebastian; Dinkel, Philine; Klein, Thomas; Sauer, Markus; Stigloher, Christian; Üçeyler, Nurcan. In eLife, 13, A. T. Chesler; K. J. Swartz; K. M. Albers; T. J. Price (Hrsg.), S. e77761-. eLife Sciences Publications, Ltd, 2024.
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  Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.

  @article{erbacher2024interaction, abstract = {Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.}, author = {Erbacher, Christoph and Britz, Sebastian and Dinkel, Philine and Klein, Thomas and Sauer, Markus and Stigloher, Christian and Üçeyler, Nurcan}, editor = {Chesler, Alexander Theodore and Swartz, Kenton J and Albers, Kathryn M and Price, Theodore J}, journal = {eLife}, keywords = {sauer}, pages = {e77761–}, publisher = {eLife Sciences Publications, Ltd}, title = {Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit}, volume = 13, year = 2024 }

  %0 Journal Article %1 erbacher2024interaction %A Erbacher, Christoph %A Britz, Sebastian %A Dinkel, Philine %A Klein, Thomas %A Sauer, Markus %A Stigloher, Christian %A Üçeyler, Nurcan %D 2024 %E Chesler, Alexander Theodore %E Swartz, Kenton J %E Albers, Kathryn M %E Price, Theodore J %I eLife Sciences Publications, Ltd %J eLife %P e77761-- %R 10.7554/eLife.77761 %T Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit %U https://doi.org/10.7554/eLife.77761 %V 13 %X Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.
• PCNA as Protein-Based Nanoruler for Sub-10 nm Fluorescence Imaging. Helmerich, Dominic A.; Budiarta, Made; Taban, Danush; Doose, Sören; Beliu, Gerti; Sauer, Markus. In Advanced Materials, 36(7), S. 2310104-. John Wiley & Sons, Ltd, 2024.
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  Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.

  @article{helmerich2024proteinbased, abstract = {Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.}, author = {Helmerich, Dominic A. and Budiarta, Made and Taban, Danush and Doose, Sören and Beliu, Gerti and Sauer, Markus}, booktitle = {Advanced Materials}, journal = {Advanced Materials}, keywords = {sauer}, month = {02}, number = 7, pages = {2310104–}, publisher = {John Wiley & Sons, Ltd}, title = {PCNA as Protein-Based Nanoruler for Sub-10 nm Fluorescence Imaging}, volume = 36, year = 2024 }

  %0 Journal Article %1 helmerich2024proteinbased %A Helmerich, Dominic A. %A Budiarta, Made %A Taban, Danush %A Doose, Sören %A Beliu, Gerti %A Sauer, Markus %B Advanced Materials %D 2024 %I John Wiley & Sons, Ltd %J Advanced Materials %N 7 %P 2310104-- %R https://doi.org/10.1002/adma.202310104 %T PCNA as Protein-Based Nanoruler for Sub-10 nm Fluorescence Imaging %U https://doi.org/10.1002/adma.202310104 %V 36 %X Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.
• Cyclophilin A is a ligand for RAGE in thrombo-inflammation. Seizer, Peter; von Ungern-Sternberg, Saskia N I; Haug, Verena; Dicenta, Valerie; Rosa, Annabelle; Butt, Elke; Nöthel, Moritz; Rohlfing, Anne-Katrin; Sigle, Manuel; Nawroth, Peter P; Nussbaum, Claudia; Sperandio, Markus; Kusch, Charly; Meub, Mara; Sauer, Markus; Münzer, Patrick; Bieber, Kristin; Stanger, Anna; Mack, Andreas F; Huber, René; Brand, Korbinian; Lehners, Moritz; Feil, Robert; Poso, Antti; Krutzke, Konstantin; Schäffer, Tilman E; Nieswandt, Bernhard; Borst, Oliver; May, Andreas E; Zernecke, Alma; Gawaz, Meinrad; Heinzmann, David. In Cardiovascular Research, 120(4), S. 385–402. 2024.
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  Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA.Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager−/− mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager−/− mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager−/− mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation.We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA–RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

  @article{seizer2024cyclophilin, abstract = {Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA.Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager−/− mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager−/− mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager−/− mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation.We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA–RAGE interaction thus represents a novel mechanism in thrombo-inflammation.}, author = {Seizer, Peter and von Ungern-Sternberg, Saskia N I and Haug, Verena and Dicenta, Valerie and Rosa, Annabelle and Butt, Elke and Nöthel, Moritz and Rohlfing, Anne-Katrin and Sigle, Manuel and Nawroth, Peter P and Nussbaum, Claudia and Sperandio, Markus and Kusch, Charly and Meub, Mara and Sauer, Markus and Münzer, Patrick and Bieber, Kristin and Stanger, Anna and Mack, Andreas F and Huber, René and Brand, Korbinian and Lehners, Moritz and Feil, Robert and Poso, Antti and Krutzke, Konstantin and Schäffer, Tilman E and Nieswandt, Bernhard and Borst, Oliver and May, Andreas E and Zernecke, Alma and Gawaz, Meinrad and Heinzmann, David}, journal = {Cardiovascular Research}, keywords = {sauer}, month = {03}, number = 4, pages = {385–402}, title = {Cyclophilin A is a ligand for RAGE in thrombo-inflammation}, volume = 120, year = 2024 }

  %0 Journal Article %1 seizer2024cyclophilin %A Seizer, Peter %A von Ungern-Sternberg, Saskia N I %A Haug, Verena %A Dicenta, Valerie %A Rosa, Annabelle %A Butt, Elke %A Nöthel, Moritz %A Rohlfing, Anne-Katrin %A Sigle, Manuel %A Nawroth, Peter P %A Nussbaum, Claudia %A Sperandio, Markus %A Kusch, Charly %A Meub, Mara %A Sauer, Markus %A Münzer, Patrick %A Bieber, Kristin %A Stanger, Anna %A Mack, Andreas F %A Huber, René %A Brand, Korbinian %A Lehners, Moritz %A Feil, Robert %A Poso, Antti %A Krutzke, Konstantin %A Schäffer, Tilman E %A Nieswandt, Bernhard %A Borst, Oliver %A May, Andreas E %A Zernecke, Alma %A Gawaz, Meinrad %A Heinzmann, David %D 2024 %J Cardiovascular Research %N 4 %P 385--402 %R 10.1093/cvr/cvad189 %T Cyclophilin A is a ligand for RAGE in thrombo-inflammation %U https://doi.org/10.1093/cvr/cvad189 %V 120 %X Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA.Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager−/− mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager−/− mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager−/− mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation.We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA–RAGE interaction thus represents a novel mechanism in thrombo-inflammation.
• Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data. Klein, Thomas; Grüner, Julia; Breyer, Maximilian; Schlegel, Jan; Schottmann, Nicole Michelle; Hofmann, Lukas; Gauss, Kevin; Mease, Rebecca; Erbacher, Christoph; Finke, Laura; Klein, Alexandra; Klug, Katharina; Karl-Schöller, Franziska; Vignolo, Bettina; Reinhard, Sebastian; Schneider, Tamara; Günther, Katharina; Fink, Julian; Dudek, Jan; Maack, Christoph; Klopocki, Eva; Seibel, Jürgen; Edenhofer, Frank; Wischmeyer, Erhard; Sauer, Markus; Üçeyler, Nurcan. In Brain Communications, 6(2), S. fcae095-. 2024.
  • [ Abstract ]
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  Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear–nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

  @article{klein2024small, abstract = {Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear–nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.}, author = {Klein, Thomas and Grüner, Julia and Breyer, Maximilian and Schlegel, Jan and Schottmann, Nicole Michelle and Hofmann, Lukas and Gauss, Kevin and Mease, Rebecca and Erbacher, Christoph and Finke, Laura and Klein, Alexandra and Klug, Katharina and Karl-Schöller, Franziska and Vignolo, Bettina and Reinhard, Sebastian and Schneider, Tamara and Günther, Katharina and Fink, Julian and Dudek, Jan and Maack, Christoph and Klopocki, Eva and Seibel, Jürgen and Edenhofer, Frank and Wischmeyer, Erhard and Sauer, Markus and Üçeyler, Nurcan}, journal = {Brain Communications}, keywords = {home}, month = {04}, number = 2, pages = {fcae095–}, title = {Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data}, volume = 6, year = 2024 }

  %0 Journal Article %1 klein2024small %A Klein, Thomas %A Grüner, Julia %A Breyer, Maximilian %A Schlegel, Jan %A Schottmann, Nicole Michelle %A Hofmann, Lukas %A Gauss, Kevin %A Mease, Rebecca %A Erbacher, Christoph %A Finke, Laura %A Klein, Alexandra %A Klug, Katharina %A Karl-Schöller, Franziska %A Vignolo, Bettina %A Reinhard, Sebastian %A Schneider, Tamara %A Günther, Katharina %A Fink, Julian %A Dudek, Jan %A Maack, Christoph %A Klopocki, Eva %A Seibel, Jürgen %A Edenhofer, Frank %A Wischmeyer, Erhard %A Sauer, Markus %A Üçeyler, Nurcan %D 2024 %J Brain Communications %N 2 %P fcae095-- %R 10.1093/braincomms/fcae095 %T Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model and pilot data %U https://doi.org/10.1093/braincomms/fcae095 %V 6 %X Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons (P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever (P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells (P < 0.001). Linear–nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.
• The neuropeptide pigment-dispersing factor signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons. Hofbauer, Benedikt; Zandawala, Meet; Reinhard, Nils; Rieger, Dirk; Werner, Christian; Evers, Jan Felix; Wegener, Christian. In European Journal of Neuroscience, 59(10), S. 2665–2685. John Wiley & Sons, Ltd, 2024.
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  The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner.

  @article{hofbauer2024neuropeptide, abstract = {The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner.}, author = {Hofbauer, Benedikt and Zandawala, Meet and Reinhard, Nils and Rieger, Dirk and Werner, Christian and Evers, Jan Felix and Wegener, Christian}, booktitle = {European Journal of Neuroscience}, journal = {European Journal of Neuroscience}, keywords = {werner}, month = {05}, number = 10, pages = {2665–2685}, publisher = {John Wiley & Sons, Ltd}, title = {The neuropeptide pigment-dispersing factor signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons}, volume = 59, year = 2024 }

  %0 Journal Article %1 hofbauer2024neuropeptide %A Hofbauer, Benedikt %A Zandawala, Meet %A Reinhard, Nils %A Rieger, Dirk %A Werner, Christian %A Evers, Jan Felix %A Wegener, Christian %B European Journal of Neuroscience %D 2024 %I John Wiley & Sons, Ltd %J European Journal of Neuroscience %N 10 %P 2665--2685 %R https://doi.org/10.1111/ejn.16294 %T The neuropeptide pigment-dispersing factor signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons %U https://doi.org/10.1111/ejn.16294 %V 59 %X The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner.

• Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane. Jobin, Marie-Lise; Siddig, Sana; Koszegi, Zsombor; Lanoiselée, Yann; Khayenko, Vladimir; Sungkaworn, Titiwat; Werner, Christian; Seier, Kerstin; Misigaiski, Christin; Mantovani, Giovanna; Sauer, Markus; Maric, Hans M.; Calebiro, Davide. In Nature Communications, 14(1), S. 34-. 2023.
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  The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.

  @article{jobin2023filamin, abstract = {The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.}, author = {Jobin, Marie-Lise and Siddig, Sana and Koszegi, Zsombor and Lanoiselée, Yann and Khayenko, Vladimir and Sungkaworn, Titiwat and Werner, Christian and Seier, Kerstin and Misigaiski, Christin and Mantovani, Giovanna and Sauer, Markus and Maric, Hans M. and Calebiro, Davide}, journal = {Nature Communications}, keywords = {werner}, number = 1, pages = {34–}, title = {Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane}, volume = 14, year = 2023 }

  %0 Journal Article %1 jobin2023filamin %A Jobin, Marie-Lise %A Siddig, Sana %A Koszegi, Zsombor %A Lanoiselée, Yann %A Khayenko, Vladimir %A Sungkaworn, Titiwat %A Werner, Christian %A Seier, Kerstin %A Misigaiski, Christin %A Mantovani, Giovanna %A Sauer, Markus %A Maric, Hans M. %A Calebiro, Davide %D 2023 %J Nature Communications %N 1 %P 34-- %R 10.1038/s41467-022-35708-1 %T Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane %U https://doi.org/10.1038/s41467-022-35708-1 %V 14 %X The γ-aminobutyric acid type B (GABAB) receptor is a prototypical family C G protein-coupled receptor (GPCR) that plays a key role in the regulation of synaptic transmission. Although growing evidence suggests that GPCR signaling in neurons might be highly organized in time and space, limited information is available about the mechanisms controlling the nanoscale organization of GABAB receptors and other GPCRs on the neuronal plasma membrane. Using a combination of biochemical assays in vitro, single-particle tracking, and super-resolution microscopy, we provide evidence that the spatial organization and diffusion of GABAB receptors on the plasma membrane are governed by dynamic interactions with filamin A, which tethers the receptors to sub-cortical actin filaments. We further show that GABAB receptors are located together with filamin A in small nanodomains in hippocampal neurons. These interactions are mediated by the first intracellular loop of the GABAB1 subunit and modulate the kinetics of Gαi protein activation in response to GABA stimulation.
• Bioorthogonal azido-S1P works as substrate for S1PR1. Sternstein, Christine; Schlegel, Jan; Sauer, Markus; Seibel, Jürgen. In Journal of Lipid Research, 64(1), S. 100311-. 2023.
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  @article{sternstein2023bioorthogonal, author = {Sternstein, Christine and Schlegel, Jan and Sauer, Markus and Seibel, Jürgen}, journal = {Journal of Lipid Research}, keywords = {sauer}, number = 1, pages = {100311–}, title = {Bioorthogonal azido-S1P works as substrate for S1PR1}, volume = 64, year = 2023 }

  %0 Journal Article %1 sternstein2023bioorthogonal %A Sternstein, Christine %A Schlegel, Jan %A Sauer, Markus %A Seibel, Jürgen %D 2023 %J Journal of Lipid Research %N 1 %P 100311-- %R https://doi.org/10.1016/j.jlr.2022.100311 %T Bioorthogonal azido-S1P works as substrate for S1PR1 %U https://www.sciencedirect.com/science/article/pii/S0022227522001444 %V 64
• Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects. Kraft, Nadine; Muenz, Thomas S.; Reinhard, Sebastian; Werner, Christian; Sauer, Markus; Groh, Claudia; Rössler, Wolfgang. In Cell and Tissue Research, 393(3), S. 489–506. 2023.
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  The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain.

  @article{kraft2023expansion, abstract = {The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain.}, author = {Kraft, Nadine and Muenz, Thomas S. and Reinhard, Sebastian and Werner, Christian and Sauer, Markus and Groh, Claudia and Rössler, Wolfgang}, journal = {Cell and Tissue Research}, keywords = {werner}, number = 3, pages = {489–506}, title = {Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects}, volume = 393, year = 2023 }

  %0 Journal Article %1 kraft2023expansion %A Kraft, Nadine %A Muenz, Thomas S. %A Reinhard, Sebastian %A Werner, Christian %A Sauer, Markus %A Groh, Claudia %A Rössler, Wolfgang %D 2023 %J Cell and Tissue Research %N 3 %P 489--506 %R 10.1007/s00441-023-03803-4 %T Expansion microscopy in honeybee brains for high-resolution neuroanatomical analyses in social insects %U https://doi.org/10.1007/s00441-023-03803-4 %V 393 %X The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain.
• DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons. Andreska, Thomas; Lüningschrör, Patrick; Wolf, Daniel; McFleder, Rhonda L.; Ayon-Olivas, Maurilyn; Rattka, Marta; Drechsler, Christine; Perschin, Veronika; Blum, Robert; Aufmkolk, Sarah; Granado, Noelia; Moratalla, Rosario; Sauer, Markus; Monoranu, Camelia; Volkmann, Jens; Ip, Chi Wang; Stigloher, Christian; Sendtner, Michael. In Cell Reports, 42(6), S. 112575-. 2023.
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  Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.

  @article{andreska2023signaling, abstract = {Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.}, author = {Andreska, Thomas and Lüningschrör, Patrick and Wolf, Daniel and McFleder, Rhonda L. and Ayon-Olivas, Maurilyn and Rattka, Marta and Drechsler, Christine and Perschin, Veronika and Blum, Robert and Aufmkolk, Sarah and Granado, Noelia and Moratalla, Rosario and Sauer, Markus and Monoranu, Camelia and Volkmann, Jens and Ip, Chi Wang and Stigloher, Christian and Sendtner, Michael}, journal = {Cell Reports}, keywords = {sauer}, number = 6, pages = {112575–}, title = {DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons}, volume = 42, year = 2023 }

  %0 Journal Article %1 andreska2023signaling %A Andreska, Thomas %A Lüningschrör, Patrick %A Wolf, Daniel %A McFleder, Rhonda L. %A Ayon-Olivas, Maurilyn %A Rattka, Marta %A Drechsler, Christine %A Perschin, Veronika %A Blum, Robert %A Aufmkolk, Sarah %A Granado, Noelia %A Moratalla, Rosario %A Sauer, Markus %A Monoranu, Camelia %A Volkmann, Jens %A Ip, Chi Wang %A Stigloher, Christian %A Sendtner, Michael %D 2023 %J Cell Reports %N 6 %P 112575-- %R https://doi.org/10.1016/j.celrep.2023.112575 %T DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons %U https://www.sciencedirect.com/science/article/pii/S2211124723005867 %V 42 %X Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.
• Domain swap facilitates structural transitions of spider silk protein C-terminal domains. Rat, Charlotte; Heindl, Cedric; Neuweiler, Hannes. In Protein Science, 32(11), S. e4783. 2023.
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  Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

  @article{https://doi.org/10.1002/pro.4783, abstract = {Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.}, author = {Rat, Charlotte and Heindl, Cedric and Neuweiler, Hannes}, journal = {Protein Science}, keywords = {hannes}, number = 11, pages = {e4783}, title = {Domain swap facilitates structural transitions of spider silk protein C-terminal domains}, volume = 32, year = 2023 }

  %0 Journal Article %1 https://doi.org/10.1002/pro.4783 %A Rat, Charlotte %A Heindl, Cedric %A Neuweiler, Hannes %D 2023 %J Protein Science %N 11 %P e4783 %R https://doi.org/10.1002/pro.4783 %T Domain swap facilitates structural transitions of spider silk protein C-terminal domains %U https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.4783 %V 32 %X Abstract Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.
• The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference. Alder-Rangel, Alene; Bailão, Alexandre Melo; Herrera-Estrella, Alfredo; Rangel, Amanda E.A; Gácser, Attila; Gasch, Audrey P.; Campos, Claudia B.L; Peters, Christina; Camelim, Francine; Verde, Fulvia; Gadd, Geoffrey Michael; Braus, Gerhard; Eisermann, Iris; Quinn, Janet; Latgé, Jean-Paul; Aguirre, Jesus; Bennett, Joan W.; Heitman, Joseph; Nosanchuk, Joshua D.; Partida-Martínez, Laila P.; Bassilana, Martine; Acheampong, Mavis A.; Riquelme, Meritxell; Feldbrügge, Michael; Keller, Nancy P.; Keyhani, Nemat O.; Gunde-Cimerman, Nina; Nascimento, Raquel; Arkowitz, Robert A.; Mouriño-Pérez, Rosa Reyna; Naz, Sehar Afshan; Avery, Simon V.; Basso, Thiago Olitta; Terpitz, Ulrich; Lin, Xiaorong; Rangel, Drauzio E.N. In Fungal Biology. 2023.
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  For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

  @article{alderrangel2023international, abstract = {For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.}, author = {Alder-Rangel, Alene and Bailão, Alexandre Melo and Herrera-Estrella, Alfredo and Rangel, Amanda E.A and Gácser, Attila and Gasch, Audrey P. and Campos, Claudia B.L and Peters, Christina and Camelim, Francine and Verde, Fulvia and Gadd, Geoffrey Michael and Braus, Gerhard and Eisermann, Iris and Quinn, Janet and Latgé, Jean-Paul and Aguirre, Jesus and Bennett, Joan W. and Heitman, Joseph and Nosanchuk, Joshua D. and Partida-Martínez, Laila P. and Bassilana, Martine and Acheampong, Mavis A. and Riquelme, Meritxell and Feldbrügge, Michael and Keller, Nancy P. and Keyhani, Nemat O. and Gunde-Cimerman, Nina and Nascimento, Raquel and Arkowitz, Robert A. and Mouriño-Pérez, Rosa Reyna and Naz, Sehar Afshan and Avery, Simon V. and Basso, Thiago Olitta and Terpitz, Ulrich and Lin, Xiaorong and Rangel, Drauzio E.N}, journal = {Fungal Biology}, keywords = {terpitz}, title = {The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference}, year = 2023 }

  %0 Journal Article %1 alderrangel2023international %A Alder-Rangel, Alene %A Bailão, Alexandre Melo %A Herrera-Estrella, Alfredo %A Rangel, Amanda E.A %A Gácser, Attila %A Gasch, Audrey P. %A Campos, Claudia B.L %A Peters, Christina %A Camelim, Francine %A Verde, Fulvia %A Gadd, Geoffrey Michael %A Braus, Gerhard %A Eisermann, Iris %A Quinn, Janet %A Latgé, Jean-Paul %A Aguirre, Jesus %A Bennett, Joan W. %A Heitman, Joseph %A Nosanchuk, Joshua D. %A Partida-Martínez, Laila P. %A Bassilana, Martine %A Acheampong, Mavis A. %A Riquelme, Meritxell %A Feldbrügge, Michael %A Keller, Nancy P. %A Keyhani, Nemat O. %A Gunde-Cimerman, Nina %A Nascimento, Raquel %A Arkowitz, Robert A. %A Mouriño-Pérez, Rosa Reyna %A Naz, Sehar Afshan %A Avery, Simon V. %A Basso, Thiago Olitta %A Terpitz, Ulrich %A Lin, Xiaorong %A Rangel, Drauzio E.N %D 2023 %J Fungal Biology %R https://doi.org/10.1016/j.funbio.2023.04.006 %T The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference %U https://www.sciencedirect.com/science/article/pii/S1878614623000521 %X For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.
• Azido-Ceramides, a Tool to Analyse SARS-CoV-2 Replication and Inhibition—SARS-CoV-2 Is Inhibited by Ceramides. Brenner, Daniela; Geiger, Nina; Schlegel, Jan; Diesendorf, Viktoria; Kersting, Louise; Fink, Julian; Stelz, Linda; Schneider-Schaulies, Sibylle; Sauer, Markus; Bodem, Jochen; Seibel, Jürgen. 2023.
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  Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.

  @electronic{brenner2023azidoceramides, abstract = {Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.}, author = {Brenner, Daniela and Geiger, Nina and Schlegel, Jan and Diesendorf, Viktoria and Kersting, Louise and Fink, Julian and Stelz, Linda and Schneider-Schaulies, Sibylle and Sauer, Markus and Bodem, Jochen and Seibel, Jürgen}, booktitle = {International Journal of Molecular Sciences}, keywords = {sauer}, number = 8, title = {Azido-Ceramides, a Tool to Analyse SARS-CoV-2 Replication and Inhibition—SARS-CoV-2 Is Inhibited by Ceramides}, volume = 24, year = 2023 }

  %0 Generic %1 brenner2023azidoceramides %A Brenner, Daniela %A Geiger, Nina %A Schlegel, Jan %A Diesendorf, Viktoria %A Kersting, Louise %A Fink, Julian %A Stelz, Linda %A Schneider-Schaulies, Sibylle %A Sauer, Markus %A Bodem, Jochen %A Seibel, Jürgen %B International Journal of Molecular Sciences %D 2023 %N 8 %R 10.3390/ijms24087281 %T Azido-Ceramides, a Tool to Analyse SARS-CoV-2 Replication and Inhibition—SARS-CoV-2 Is Inhibited by Ceramides %V 24 %X Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
• Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma. Munawar, Umair; Zhou, Xiang; Prommersberger, Sabrina; Nerreter, Silvia; Vogt, Cornelia; Steinhardt, Maximilian J.; Truger, Marietta; Mersi, Julia; Teufel, Eva; Han, Seungbin; Haertle, Larissa; Banholzer, Nicole; Eiring, Patrick; Danhof, Sophia; Navarro-Aguadero, Miguel Angel; Fernandez-Martin, Adrian; Ortiz-Ruiz, Alejandra; Barrio, Santiago; Gallardo, Miguel; Valeri, Antonio; Castellano, Eva; Raab, Peter; Rudert, Maximilian; Haferlach, Claudia; Sauer, Markus; Hudecek, Michael; Martinez-Lopez, J.; Waldschmidt, Johannes; Einsele, Hermann; Rasche, Leo; Kortüm, K. Martin. In Communications Biology, 6(1), S. 1299-. 2023.
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  The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.

  @article{munawar2023impaired, abstract = {The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.}, author = {Munawar, Umair and Zhou, Xiang and Prommersberger, Sabrina and Nerreter, Silvia and Vogt, Cornelia and Steinhardt, Maximilian J. and Truger, Marietta and Mersi, Julia and Teufel, Eva and Han, Seungbin and Haertle, Larissa and Banholzer, Nicole and Eiring, Patrick and Danhof, Sophia and Navarro-Aguadero, Miguel Angel and Fernandez-Martin, Adrian and Ortiz-Ruiz, Alejandra and Barrio, Santiago and Gallardo, Miguel and Valeri, Antonio and Castellano, Eva and Raab, Peter and Rudert, Maximilian and Haferlach, Claudia and Sauer, Markus and Hudecek, Michael and Martinez-Lopez, J. and Waldschmidt, Johannes and Einsele, Hermann and Rasche, Leo and Kortüm, K. Martin}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {1299–}, title = {Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma}, volume = 6, year = 2023 }

  %0 Journal Article %1 munawar2023impaired %A Munawar, Umair %A Zhou, Xiang %A Prommersberger, Sabrina %A Nerreter, Silvia %A Vogt, Cornelia %A Steinhardt, Maximilian J. %A Truger, Marietta %A Mersi, Julia %A Teufel, Eva %A Han, Seungbin %A Haertle, Larissa %A Banholzer, Nicole %A Eiring, Patrick %A Danhof, Sophia %A Navarro-Aguadero, Miguel Angel %A Fernandez-Martin, Adrian %A Ortiz-Ruiz, Alejandra %A Barrio, Santiago %A Gallardo, Miguel %A Valeri, Antonio %A Castellano, Eva %A Raab, Peter %A Rudert, Maximilian %A Haferlach, Claudia %A Sauer, Markus %A Hudecek, Michael %A Martinez-Lopez, J. %A Waldschmidt, Johannes %A Einsele, Hermann %A Rasche, Leo %A Kortüm, K. Martin %D 2023 %J Communications Biology %N 1 %P 1299-- %R 10.1038/s42003-023-05683-4 %T Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma %U https://doi.org/10.1038/s42003-023-05683-4 %V 6 %X The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM.
• Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1. Seeling, Michaela; Pöhnl, Matthias; Kara, Sibel; Horstmann, Nathalie; Riemer, Carolina; Wöhner, Miriam; Liang, Chunguang; Brückner, Christin; Eiring, Patrick; Werner, Anja; Biburger, Markus; Altmann, Leon; Schneider, Martin; Amon, Lukas; Lehmann, Christian H.K; Lee, Sooyeon; Kunz, Meik; Dudziak, Diana; Schett, Georg; Bäuerle, Tobias; Lux, Anja; Tuckermann, Jan; Vögtle, Timo; Nieswandt, Bernhardt; Sauer, Markus; Böckmann, Rainer A.; Nimmerjahn, Falk. In Immunity, 56(5), S. 1046–1063.e7. 2023.
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  Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

  @article{seeling2023immunoglobulin, abstract = {Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.}, author = {Seeling, Michaela and Pöhnl, Matthias and Kara, Sibel and Horstmann, Nathalie and Riemer, Carolina and Wöhner, Miriam and Liang, Chunguang and Brückner, Christin and Eiring, Patrick and Werner, Anja and Biburger, Markus and Altmann, Leon and Schneider, Martin and Amon, Lukas and Lehmann, Christian H.K and Lee, Sooyeon and Kunz, Meik and Dudziak, Diana and Schett, Georg and Bäuerle, Tobias and Lux, Anja and Tuckermann, Jan and Vögtle, Timo and Nieswandt, Bernhardt and Sauer, Markus and Böckmann, Rainer A. and Nimmerjahn, Falk}, journal = {Immunity}, keywords = {sauer}, number = 5, pages = {1046–1063.e7}, title = {Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1}, volume = 56, year = 2023 }

  %0 Journal Article %1 seeling2023immunoglobulin %A Seeling, Michaela %A Pöhnl, Matthias %A Kara, Sibel %A Horstmann, Nathalie %A Riemer, Carolina %A Wöhner, Miriam %A Liang, Chunguang %A Brückner, Christin %A Eiring, Patrick %A Werner, Anja %A Biburger, Markus %A Altmann, Leon %A Schneider, Martin %A Amon, Lukas %A Lehmann, Christian H.K %A Lee, Sooyeon %A Kunz, Meik %A Dudziak, Diana %A Schett, Georg %A Bäuerle, Tobias %A Lux, Anja %A Tuckermann, Jan %A Vögtle, Timo %A Nieswandt, Bernhardt %A Sauer, Markus %A Böckmann, Rainer A. %A Nimmerjahn, Falk %D 2023 %J Immunity %N 5 %P 1046--1063.e7 %R https://doi.org/10.1016/j.immuni.2023.02.019 %T Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1 %U https://www.sciencedirect.com/science/article/pii/S1074761323000936 %V 56 %X Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.
• Current Progress in Expansion Microscopy: Chemical Strategies and Applications. Wen, Gang; Leen, Volker; Rohand, Taoufik; Sauer, Markus; Hofkens, Johan. In Chem. Rev., 123(6), S. 3299–3323. American Chemical Society, 2023.
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  Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.

  @article{wen2023current, abstract = {Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.}, author = {Wen, Gang and Leen, Volker and Rohand, Taoufik and Sauer, Markus and Hofkens, Johan}, booktitle = {Chemical Reviews}, journal = {Chem. Rev.}, keywords = {sauer}, month = {03}, number = 6, pages = {3299–3323}, publisher = {American Chemical Society}, title = {Current Progress in Expansion Microscopy: Chemical Strategies and Applications}, volume = 123, year = 2023 }

  %0 Journal Article %1 wen2023current %A Wen, Gang %A Leen, Volker %A Rohand, Taoufik %A Sauer, Markus %A Hofkens, Johan %B Chemical Reviews %D 2023 %I American Chemical Society %J Chem. Rev. %N 6 %P 3299--3323 %R 10.1021/acs.chemrev.2c00711 %T Current Progress in Expansion Microscopy: Chemical Strategies and Applications %U https://doi.org/10.1021/acs.chemrev.2c00711 %V 123 %X Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.
• Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment. Haake, Markus; Haack, Beatrice; Schäfer, Tina; Harter, Patrick N.; Mattavelli, Greta; Eiring, Patrick; Vashist, Neha; Wedekink, Florian; Genssler, Sabrina; Fischer, Birgitt; Dahlhoff, Julia; Mokhtari, Fatemeh; Kuzkina, Anastasia; Welters, Marij J. P.; Benz, Tamara M.; Sorger, Lena; Thiemann, Vincent; Almanzar, Giovanni; Selle, Martina; Thein, Klara; Späth, Jacob; Gonzalez, Maria Cecilia; Reitinger, Carmen; Ipsen-Escobedo, Andrea; Wistuba-Hamprecht, Kilian; Eichler, Kristin; Filipski, Katharina; Zeiner, Pia S.; Beschorner, Rudi; Goedemans, Renske; Gogolla, Falk Hagen; Hackl, Hubert; Rooswinkel, Rogier W.; Thiem, Alexander; Roche, Paula Romer; Joshi, Hemant; Pühringer, Dirk; Wöckel, Achim; Diessner, Joachim E.; Rüdiger, Manfred; Leo, Eugen; Cheng, Phil F.; Levesque, Mitchell P.; Goebeler, Matthias; Sauer, Markus; Nimmerjahn, Falk; Schuberth-Wagner, Christine; von Felten, Stefanie; Mittelbronn, Michel; Mehling, Matthias; Beilhack, Andreas; van der Burg, Sjoerd H.; Riedel, Angela; Weide, Benjamin; Dummer, Reinhard; Wischhusen, Jörg. In Nature Communications, 14(1), S. 4253-. 2023.
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  Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

  @article{haake2023tumorderived, abstract = {Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.}, author = {Haake, Markus and Haack, Beatrice and Schäfer, Tina and Harter, Patrick N. and Mattavelli, Greta and Eiring, Patrick and Vashist, Neha and Wedekink, Florian and Genssler, Sabrina and Fischer, Birgitt and Dahlhoff, Julia and Mokhtari, Fatemeh and Kuzkina, Anastasia and Welters, Marij J. P. and Benz, Tamara M. and Sorger, Lena and Thiemann, Vincent and Almanzar, Giovanni and Selle, Martina and Thein, Klara and Späth, Jacob and Gonzalez, Maria Cecilia and Reitinger, Carmen and Ipsen-Escobedo, Andrea and Wistuba-Hamprecht, Kilian and Eichler, Kristin and Filipski, Katharina and Zeiner, Pia S. and Beschorner, Rudi and Goedemans, Renske and Gogolla, Falk Hagen and Hackl, Hubert and Rooswinkel, Rogier W. and Thiem, Alexander and Roche, Paula Romer and Joshi, Hemant and Pühringer, Dirk and Wöckel, Achim and Diessner, Joachim E. and Rüdiger, Manfred and Leo, Eugen and Cheng, Phil F. and Levesque, Mitchell P. and Goebeler, Matthias and Sauer, Markus and Nimmerjahn, Falk and Schuberth-Wagner, Christine and von Felten, Stefanie and Mittelbronn, Michel and Mehling, Matthias and Beilhack, Andreas and van der Burg, Sjoerd H. and Riedel, Angela and Weide, Benjamin and Dummer, Reinhard and Wischhusen, Jörg}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {4253–}, title = {Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment}, volume = 14, year = 2023 }

  %0 Journal Article %1 haake2023tumorderived %A Haake, Markus %A Haack, Beatrice %A Schäfer, Tina %A Harter, Patrick N. %A Mattavelli, Greta %A Eiring, Patrick %A Vashist, Neha %A Wedekink, Florian %A Genssler, Sabrina %A Fischer, Birgitt %A Dahlhoff, Julia %A Mokhtari, Fatemeh %A Kuzkina, Anastasia %A Welters, Marij J. P. %A Benz, Tamara M. %A Sorger, Lena %A Thiemann, Vincent %A Almanzar, Giovanni %A Selle, Martina %A Thein, Klara %A Späth, Jacob %A Gonzalez, Maria Cecilia %A Reitinger, Carmen %A Ipsen-Escobedo, Andrea %A Wistuba-Hamprecht, Kilian %A Eichler, Kristin %A Filipski, Katharina %A Zeiner, Pia S. %A Beschorner, Rudi %A Goedemans, Renske %A Gogolla, Falk Hagen %A Hackl, Hubert %A Rooswinkel, Rogier W. %A Thiem, Alexander %A Roche, Paula Romer %A Joshi, Hemant %A Pühringer, Dirk %A Wöckel, Achim %A Diessner, Joachim E. %A Rüdiger, Manfred %A Leo, Eugen %A Cheng, Phil F. %A Levesque, Mitchell P. %A Goebeler, Matthias %A Sauer, Markus %A Nimmerjahn, Falk %A Schuberth-Wagner, Christine %A von Felten, Stefanie %A Mittelbronn, Michel %A Mehling, Matthias %A Beilhack, Andreas %A van der Burg, Sjoerd H. %A Riedel, Angela %A Weide, Benjamin %A Dummer, Reinhard %A Wischhusen, Jörg %D 2023 %J Nature Communications %N 1 %P 4253-- %R 10.1038/s41467-023-39817-3 %T Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment %U https://doi.org/10.1038/s41467-023-39817-3 %V 14 %X Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.
• Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy. Wen, Gang; Lycas, Matthew Domenic; Jia, Yuqing; Leen, Volker; Sauer, Markus; Hofkens, Johan. In ACS Nano, 17(20), S. 20589–20600. American Chemical Society, 2023.
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  Expansion microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases in which the concentration of target-bound monomers is insufficient for high-quality ExM.

  @article{wen2023trifunctional, abstract = {Expansion microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases in which the concentration of target-bound monomers is insufficient for high-quality ExM.}, author = {Wen, Gang and Lycas, Matthew Domenic and Jia, Yuqing and Leen, Volker and Sauer, Markus and Hofkens, Johan}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = 10, number = 20, pages = {20589–20600}, publisher = {American Chemical Society}, title = {Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy}, volume = 17, year = 2023 }

  %0 Journal Article %1 wen2023trifunctional %A Wen, Gang %A Lycas, Matthew Domenic %A Jia, Yuqing %A Leen, Volker %A Sauer, Markus %A Hofkens, Johan %B ACS Nano %D 2023 %I American Chemical Society %J ACS Nano %N 20 %P 20589--20600 %R 10.1021/acsnano.3c07510 %T Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy %U https://doi.org/10.1021/acsnano.3c07510 %V 17 %X Expansion microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have a high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of optimized trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10X ExM of actin labeled with optimized TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases in which the concentration of target-bound monomers is insufficient for high-quality ExM.
• Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy. Hennlein, Luisa; Ghanawi, Hanaa; Gerstner, Florian; Palominos García, Eduardo; Yildirim, Ezgi; Saal-Bauernschubert, Lena; Moradi, Mehri; Deng, Chunchu; Klein, Teresa; Appenzeller, Silke; Sauer, Markus; Briese, Michael; Simon, Christian; Sendtner, Michael; Jablonka, Sibylle. In Journal of Cell Biology, 222(3), S. e202204113-. 2023.
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  Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 “cluster-like” formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.

  @article{hennlein2023plastin, abstract = {Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 “cluster-like” formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.}, author = {Hennlein, Luisa and Ghanawi, Hanaa and Gerstner, Florian and Palominos García, Eduardo and Yildirim, Ezgi and Saal-Bauernschubert, Lena and Moradi, Mehri and Deng, Chunchu and Klein, Teresa and Appenzeller, Silke and Sauer, Markus and Briese, Michael and Simon, Christian and Sendtner, Michael and Jablonka, Sibylle}, journal = {Journal of Cell Biology}, keywords = {sauer}, month = {01}, number = 3, pages = {e202204113–}, title = {Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy}, volume = 222, year = 2023 }

  %0 Journal Article %1 hennlein2023plastin %A Hennlein, Luisa %A Ghanawi, Hanaa %A Gerstner, Florian %A Palominos García, Eduardo %A Yildirim, Ezgi %A Saal-Bauernschubert, Lena %A Moradi, Mehri %A Deng, Chunchu %A Klein, Teresa %A Appenzeller, Silke %A Sauer, Markus %A Briese, Michael %A Simon, Christian %A Sendtner, Michael %A Jablonka, Sibylle %D 2023 %J Journal of Cell Biology %N 3 %P e202204113-- %R 10.1083/jcb.202204113 %T Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy %U https://doi.org/10.1083/jcb.202204113 %V 222 %X Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degeneration. Despite the observed beneficial modifying effect of PLS3, the mechanism of how it supports F-actin-mediated cellular processes in motoneurons is not yet well understood. Our data reveal disturbed F-actin-dependent translocation of the Tropomyosin receptor kinase B (TrkB) to the cell surface of Smn-deficient motor axon terminals, resulting in reduced TrkB activation by its ligand brain-derived neurotrophic factor (BDNF). Improved actin dynamics by overexpression of hPLS3 restores membrane recruitment and activation of TrkB and enhances spontaneous calcium transients by increasing Cav2.1/2 “cluster-like” formations in SMA axon terminals. Thus, our study provides a novel role for PLS3 in supporting correct alignment of transmembrane proteins, a key mechanism for (moto)-neuronal development.
• Domain swap facilitates structural transitions of spider silk protein C-terminal domains. Rat, Charlotte; Heindl, Cedric; Neuweiler, Hannes. In Protein Science, 32(11), S. e4783-. John Wiley & Sons, Ltd, 2023.
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  Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.

  @article{rat2023domain, abstract = {Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.}, author = {Rat, Charlotte and Heindl, Cedric and Neuweiler, Hannes}, booktitle = {Protein Science}, journal = {Protein Science}, keywords = {hannes}, month = 11, number = 11, pages = {e4783–}, publisher = {John Wiley & Sons, Ltd}, title = {Domain swap facilitates structural transitions of spider silk protein C-terminal domains}, volume = 32, year = 2023 }

  %0 Journal Article %1 rat2023domain %A Rat, Charlotte %A Heindl, Cedric %A Neuweiler, Hannes %B Protein Science %D 2023 %I John Wiley & Sons, Ltd %J Protein Science %N 11 %P e4783-- %R https://doi.org/10.1002/pro.4783 %T Domain swap facilitates structural transitions of spider silk protein C-terminal domains %U https://doi.org/10.1002/pro.4783 %V 32 %X Domain swap is a mechanism of protein dimerization where the two interacting domains exchange parts of their structure. Web spiders make use of the process in the connection of C-terminal domains (CTDs) of spidroins, the soluble protein building blocks that form tough silk fibers. Besides providing connectivity and solubility, spidroin CTDs are responsible for inducing structural transitions during passage through an acidified assembly zone within spinning ducts. The underlying molecular mechanisms are elusive. Here, we studied the folding of five homologous spidroin CTDs from different spider species or glands. Four of these are domain-swapped dimers formed by five-helix bundles from spidroins of major and minor ampullate glands. The fifth is a dimer that lacks domain swap, formed by four-helix bundles from a spidroin of a flagelliform gland. Spidroins from this gland do not undergo structural transitions whereas the others do. We found a three-state mechanism of folding and dimerization that was conserved across homologues. In chemical denaturation experiments the native CTD dimer unfolded to a dimeric, partially structured intermediate, followed by full unfolding to denatured monomers. The energetics of the individual folding steps varied between homologues. Contrary to the common belief that domain swap stabilizes protein assemblies, the non-swapped homologue was most stable and folded four orders of magnitude faster than a swapped variant. Domain swap of spidroin CTDs induces an entropic penalty to the folding of peripheral helices, thus unfastening them for acid-induced unfolding within a spinning duct, which primes them for refolding into alternative structures during silk formation.
• Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy. Eilts, Janna; Reinhard, Sebastian; Michetschläger, Nikolas; Werner, Christian; Sauer, Markus. In Neurophotonics, 10(4), S. 044412-. 2023.
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  SignificanceUnderstanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30 nm spatial resolution.AimWe developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.ApproachWe used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM).ResultsWe demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30 nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites.ConclusionsOur optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.

  @article{eilts2023enhanced, abstract = {SignificanceUnderstanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30 nm spatial resolution.AimWe developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.ApproachWe used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM).ResultsWe demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30 nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites.ConclusionsOur optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.}, author = {Eilts, Janna and Reinhard, Sebastian and Michetschläger, Nikolas and Werner, Christian and Sauer, Markus}, booktitle = {Neurophotonics}, journal = {Neurophotonics}, keywords = {werner}, month = 10, number = 4, pages = {044412–}, title = {Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy}, volume = 10, year = 2023 }

  %0 Journal Article %1 eilts2023enhanced %A Eilts, Janna %A Reinhard, Sebastian %A Michetschläger, Nikolas %A Werner, Christian %A Sauer, Markus %B Neurophotonics %D 2023 %J Neurophotonics %N 4 %P 044412-- %R 10.1117/1.NPh.10.4.044412 %T Enhanced synaptic protein visualization by multicolor super-resolution expansion microscopy %U https://doi.org/10.1117/1.NPh.10.4.044412 %V 10 %X SignificanceUnderstanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30 nm spatial resolution.AimWe developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution.ApproachWe used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM).ResultsWe demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30 nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites.ConclusionsOur optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses.
• Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection. Jungblut, Marvin; Backes, Simone; Streit, Marcel; Gasteiger, Georg; Doose, Sören; Sauer, Markus; Beliu, Gerti. In ACS Nano, 17(21), S. 21822–21828. American Chemical Society, 2023.
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  Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus–cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus–cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus–host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.

  @article{jungblut2023reengineered, abstract = {Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus–cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus–cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus–host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.}, author = {Jungblut, Marvin and Backes, Simone and Streit, Marcel and Gasteiger, Georg and Doose, Sören and Sauer, Markus and Beliu, Gerti}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = 11, number = 21, pages = {21822–21828}, publisher = {American Chemical Society}, title = {Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection}, volume = 17, year = 2023 }

  %0 Journal Article %1 jungblut2023reengineered %A Jungblut, Marvin %A Backes, Simone %A Streit, Marcel %A Gasteiger, Georg %A Doose, Sören %A Sauer, Markus %A Beliu, Gerti %B ACS Nano %D 2023 %I American Chemical Society %J ACS Nano %N 21 %P 21822--21828 %R 10.1021/acsnano.3c07767 %T Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection %U https://doi.org/10.1021/acsnano.3c07767 %V 17 %X Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus–cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus–cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus–host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.
• Coronaviruses Use ACE2 Monomers as Entry-Receptors. Eiring, Patrick; Klein, Teresa; Backes, Simone; Streit, Marcel; Jungblut, Marvin; Doose, Sören; Beliu, Gerti; Sauer, Markus. In Angewandte Chemie International Edition, 62(30), S. e202300821-. John Wiley & Sons, Ltd, 2023.
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  The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1?2 receptors ?m?2. In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.

  @article{eiring2023coronaviruses, abstract = {The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1?2 receptors ?m?2. In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.}, author = {Eiring, Patrick and Klein, Teresa and Backes, Simone and Streit, Marcel and Jungblut, Marvin and Doose, Sören and Beliu, Gerti and Sauer, Markus}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, month = {07}, number = 30, pages = {e202300821–}, publisher = {John Wiley & Sons, Ltd}, title = {Coronaviruses Use ACE2 Monomers as Entry-Receptors}, volume = 62, year = 2023 }

  %0 Journal Article %1 eiring2023coronaviruses %A Eiring, Patrick %A Klein, Teresa %A Backes, Simone %A Streit, Marcel %A Jungblut, Marvin %A Doose, Sören %A Beliu, Gerti %A Sauer, Markus %B Angewandte Chemie International Edition %D 2023 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 30 %P e202300821-- %R https://doi.org/10.1002/anie.202300821 %T Coronaviruses Use ACE2 Monomers as Entry-Receptors %U https://doi.org/10.1002/anie.202300821 %V 62 %X The angiotensin-converting enzyme 2 (ACE2) has been identified as entry receptor on cells enabling binding and infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via trimeric spike (S) proteins protruding from the viral surface. It has been suggested that trimeric S proteins preferably bind to plasma membrane areas with high concentrations of possibly multimeric ACE2 receptors to achieve a higher binding and infection efficiency. Here we used direct stochastic optical reconstruction microscopy (dSTORM) in combination with different labeling approaches to visualize the distribution and quantify the expression of ACE2 on different cells. Our results reveal that endogenous ACE2 receptors are present as monomers in the plasma membrane with densities of only 1?2 receptors ?m?2. In addition, binding of trimeric S proteins does not induce the formation of ACE2 oligomers in the plasma membrane. Supported by infection studies using vesicular stomatitis virus (VSV) particles bearing S proteins our data demonstrate that a single S protein interaction per virus particle with a monomeric ACE2 receptor is sufficient for infection, which provides SARS-CoV-2 a high infectivity.
• Schärfer als das Mikroskop erlaubt. Helmerich, Dominic Andreas; Beliu, Gerti; Sauer, Markus. In Physik in unserer Zeit, 54(3), S. 124–130. John Wiley & Sons, Ltd, 2023.
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  Mit Hilfe der hochauflösenden Fluoreszenzmikroskopie lassen sich Bilder mit einer räumlichen Auflösung von rund 20 nm, weit unterhalb der Beugungsgrenze, aufnehmen. Eine weitere Verbesserung der räumlichen Auflösung bis in den molekularen Bereich ist jedoch durch die Interaktion der Farbstoffe miteinander erschwert. Bei Abständen unterhalb von 10 nm führen verschiedene Energietransferprozesse zu einer schlechteren Lokalisationswahrscheinlichkeit, was die erreichbare strukturelle Auflösung stark einschränkt. Eine genaue zeitaufgelöste Analyse des Fluoreszenzsignals ermöglicht es, die Anzahl der interagierenden Farbstoffe und deren Abstände zu ermitteln. Durch die zeitaufgelöste Analyse des Photoswitching-Fingerprints ist es uns gelungen, Abstände von 5 nm an Membranrezeptoren zu identifizieren.

  @article{helmerich2023schrfer, abstract = {Mit Hilfe der hochauflösenden Fluoreszenzmikroskopie lassen sich Bilder mit einer räumlichen Auflösung von rund 20 nm, weit unterhalb der Beugungsgrenze, aufnehmen. Eine weitere Verbesserung der räumlichen Auflösung bis in den molekularen Bereich ist jedoch durch die Interaktion der Farbstoffe miteinander erschwert. Bei Abständen unterhalb von 10 nm führen verschiedene Energietransferprozesse zu einer schlechteren Lokalisationswahrscheinlichkeit, was die erreichbare strukturelle Auflösung stark einschränkt. Eine genaue zeitaufgelöste Analyse des Fluoreszenzsignals ermöglicht es, die Anzahl der interagierenden Farbstoffe und deren Abstände zu ermitteln. Durch die zeitaufgelöste Analyse des Photoswitching-Fingerprints ist es uns gelungen, Abstände von 5 nm an Membranrezeptoren zu identifizieren.}, author = {Helmerich, Dominic Andreas and Beliu, Gerti and Sauer, Markus}, booktitle = {Physik in unserer Zeit}, journal = {Physik in unserer Zeit}, keywords = {sauer}, month = {05}, number = 3, pages = {124–130}, publisher = {John Wiley & Sons, Ltd}, title = {Schärfer als das Mikroskop erlaubt}, volume = 54, year = 2023 }

  %0 Journal Article %1 helmerich2023schrfer %A Helmerich, Dominic Andreas %A Beliu, Gerti %A Sauer, Markus %B Physik in unserer Zeit %D 2023 %I John Wiley & Sons, Ltd %J Physik in unserer Zeit %N 3 %P 124--130 %R https://doi.org/10.1002/piuz.202301668 %T Schärfer als das Mikroskop erlaubt %U https://doi.org/10.1002/piuz.202301668 %V 54 %X Mit Hilfe der hochauflösenden Fluoreszenzmikroskopie lassen sich Bilder mit einer räumlichen Auflösung von rund 20 nm, weit unterhalb der Beugungsgrenze, aufnehmen. Eine weitere Verbesserung der räumlichen Auflösung bis in den molekularen Bereich ist jedoch durch die Interaktion der Farbstoffe miteinander erschwert. Bei Abständen unterhalb von 10 nm führen verschiedene Energietransferprozesse zu einer schlechteren Lokalisationswahrscheinlichkeit, was die erreichbare strukturelle Auflösung stark einschränkt. Eine genaue zeitaufgelöste Analyse des Fluoreszenzsignals ermöglicht es, die Anzahl der interagierenden Farbstoffe und deren Abstände zu ermitteln. Durch die zeitaufgelöste Analyse des Photoswitching-Fingerprints ist es uns gelungen, Abstände von 5 nm an Membranrezeptoren zu identifizieren.
• Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network. Keller, Thorsten; Trinks, Nora; Brand, Jessica; Trippmacher, Steffen; Stahlhut, Philipp; Albrecht, Krystyna; Papastavrou, Georg; Koepsell, Hermann; Sauer, Markus; Groll, Jürgen. In Advanced Healthcare Materials, 12(13), S. 2201794-. John Wiley & Sons, Ltd, 2023.
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  Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+-D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.

  @article{keller2023design, abstract = {Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+-D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.}, author = {Keller, Thorsten and Trinks, Nora and Brand, Jessica and Trippmacher, Steffen and Stahlhut, Philipp and Albrecht, Krystyna and Papastavrou, Georg and Koepsell, Hermann and Sauer, Markus and Groll, Jürgen}, booktitle = {Advanced Healthcare Materials}, journal = {Advanced Healthcare Materials}, keywords = {sauer}, month = {05}, number = 13, pages = {2201794–}, publisher = {John Wiley & Sons, Ltd}, title = {Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network}, volume = 12, year = 2023 }

  %0 Journal Article %1 keller2023design %A Keller, Thorsten %A Trinks, Nora %A Brand, Jessica %A Trippmacher, Steffen %A Stahlhut, Philipp %A Albrecht, Krystyna %A Papastavrou, Georg %A Koepsell, Hermann %A Sauer, Markus %A Groll, Jürgen %B Advanced Healthcare Materials %D 2023 %I John Wiley & Sons, Ltd %J Advanced Healthcare Materials %N 13 %P 2201794-- %R https://doi.org/10.1002/adhm.202201794 %T Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network %U https://doi.org/10.1002/adhm.202201794 %V 12 %X Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+-D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.
• All-trans retinoic acid works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells. García-Guerrero, Estefanía; Rodríguez-Lobato, Luis G.; Sierro-Martínez, Belén; Danhof, Sophia; Bates, Stephan; Frenz, Silke; Haertle, Larissa; Götz, Ralph; Sauer, Markus; Rasche, Leo; Kortüm, K. Martin; Pérez-Simón, Jose A.; Einsele, Hermann; Hudecek, Michael; Prommersberger, Sabrina R. In Haematologica, 108(2), S. 568–580. 2023.
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  B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

  @article{garciaguerrero2023alltrans, abstract = {B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.}, author = {García-Guerrero, Estefanía and Rodríguez-Lobato, Luis G. and Sierro-Martínez, Belén and Danhof, Sophia and Bates, Stephan and Frenz, Silke and Haertle, Larissa and Götz, Ralph and Sauer, Markus and Rasche, Leo and Kortüm, K. Martin and Pérez-Simón, Jose A. and Einsele, Hermann and Hudecek, Michael and Prommersberger, Sabrina R.}, journal = {Haematologica}, keywords = {sauer}, month = {02}, number = 2, pages = {568-580–}, title = {All-trans retinoic acid works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells}, volume = 108, year = 2023 }

  %0 Journal Article %1 garciaguerrero2023alltrans %A García-Guerrero, Estefanía %A Rodríguez-Lobato, Luis G. %A Sierro-Martínez, Belén %A Danhof, Sophia %A Bates, Stephan %A Frenz, Silke %A Haertle, Larissa %A Götz, Ralph %A Sauer, Markus %A Rasche, Leo %A Kortüm, K. Martin %A Pérez-Simón, Jose A. %A Einsele, Hermann %A Hudecek, Michael %A Prommersberger, Sabrina R. %D 2023 %J Haematologica %N 2 %P 568-580-- %R 10.3324/haematol.2022.281339 %T All-trans retinoic acid works synergistically with the γ-secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells %U https://haematologica.org/article/view/haematol.2022.281339 %V 108 %X B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

• The Acid Ceramidase Is a SARS-CoV-2 Host Factor. Geiger, Nina; Kersting, Louise; Schlegel, Jan; Stelz, Linda; Fähr, Sofie; Diesendorf, Viktoria; Roll, Valeria; Sostmann, Marie; König, Eva-Maria; Reinhard, Sebastian; Brenner, Daniela; Schneider-Schaulies, Sibylle; Sauer, Markus; Seibel, Jürgen; Bodem, Jochen. 2022.
  • [ Abstract ]
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  SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

  @electronic{geiger2022ceramidase, abstract = {SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.}, author = {Geiger, Nina and Kersting, Louise and Schlegel, Jan and Stelz, Linda and Fähr, Sofie and Diesendorf, Viktoria and Roll, Valeria and Sostmann, Marie and König, Eva-Maria and Reinhard, Sebastian and Brenner, Daniela and Schneider-Schaulies, Sibylle and Sauer, Markus and Seibel, Jürgen and Bodem, Jochen}, booktitle = {Cells}, keywords = {sauer}, number = 16, title = {The Acid Ceramidase Is a SARS-CoV-2 Host Factor}, volume = 11, year = 2022 }

  %0 Generic %1 geiger2022ceramidase %A Geiger, Nina %A Kersting, Louise %A Schlegel, Jan %A Stelz, Linda %A Fähr, Sofie %A Diesendorf, Viktoria %A Roll, Valeria %A Sostmann, Marie %A König, Eva-Maria %A Reinhard, Sebastian %A Brenner, Daniela %A Schneider-Schaulies, Sibylle %A Sauer, Markus %A Seibel, Jürgen %A Bodem, Jochen %B Cells %D 2022 %N 16 %R 10.3390/cells11162532 %T The Acid Ceramidase Is a SARS-CoV-2 Host Factor %V 11 %X SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.
• CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models. Seif, Michelle; Kakoschke, Tamara Katharina; Ebel, Frank; Bellet, Marina Maria; Trinks, Nora; Renga, Giorgia; Pariano, Marilena; Romani, Luigina; Tappe, Beeke; Espie, David; Donnadieu, Emmanuel; Hünniger, Kerstin; Häder, Antje; Sauer, Markus; Damotte, Diane; Alifano, Marco; White, P. Lewis; Backx, Matthijs; Nerreter, Thomas; Machwirth, Markus; Kurzai, Oliver; Prommersberger, Sabrina; Einsele, Hermann; Hudecek, Michael; Löffler, Jürgen. In Science Translational Medicine, 14(664), S. eabh1209-. American Association for the Advancement of Science, 2022.
  • [ Abstract ]
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  Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus?specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA. Aspergillus fumigatus?specific CAR T cells confer a direct antifungal effect and engage innate immune cells in preclinical models of aspergillosis. CAR T cells are most commonly thought of as a cancer therapy. Recently, however, investigators have been testing CAR T cells for additional, nonmalignant disease. Here, Seif et al. developed and tested a CAR T cell construct that targeted Aspergillus fumigatus hyphae, a ubiquitous mold that can cause invasive pulmonary aspergillosis in immunocompromised individuals. A. fumigatus?specific CAR T cells had potent and selective antifungal activity in vitro and in preclinical animal models, supporting further clinical development of these CAR T cells to treat invasive pulmonary aspergillosis.

  @article{seifcells, abstract = {Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus?specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA. Aspergillus fumigatus?specific CAR T cells confer a direct antifungal effect and engage innate immune cells in preclinical models of aspergillosis. CAR T cells are most commonly thought of as a cancer therapy. Recently, however, investigators have been testing CAR T cells for additional, nonmalignant disease. Here, Seif et al. developed and tested a CAR T cell construct that targeted Aspergillus fumigatus hyphae, a ubiquitous mold that can cause invasive pulmonary aspergillosis in immunocompromised individuals. A. fumigatus?specific CAR T cells had potent and selective antifungal activity in vitro and in preclinical animal models, supporting further clinical development of these CAR T cells to treat invasive pulmonary aspergillosis.}, author = {Seif, Michelle and Kakoschke, Tamara Katharina and Ebel, Frank and Bellet, Marina Maria and Trinks, Nora and Renga, Giorgia and Pariano, Marilena and Romani, Luigina and Tappe, Beeke and Espie, David and Donnadieu, Emmanuel and Hünniger, Kerstin and Häder, Antje and Sauer, Markus and Damotte, Diane and Alifano, Marco and White, P. Lewis and Backx, Matthijs and Nerreter, Thomas and Machwirth, Markus and Kurzai, Oliver and Prommersberger, Sabrina and Einsele, Hermann and Hudecek, Michael and Löffler, Jürgen}, booktitle = {Science Translational Medicine}, journal = {Science Translational Medicine}, keywords = {sauer}, number = 664, pages = {eabh1209–}, publisher = {American Association for the Advancement of Science}, title = {CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models}, volume = 14, year = 2022 }

  %0 Journal Article %1 seifcells %A Seif, Michelle %A Kakoschke, Tamara Katharina %A Ebel, Frank %A Bellet, Marina Maria %A Trinks, Nora %A Renga, Giorgia %A Pariano, Marilena %A Romani, Luigina %A Tappe, Beeke %A Espie, David %A Donnadieu, Emmanuel %A Hünniger, Kerstin %A Häder, Antje %A Sauer, Markus %A Damotte, Diane %A Alifano, Marco %A White, P. Lewis %A Backx, Matthijs %A Nerreter, Thomas %A Machwirth, Markus %A Kurzai, Oliver %A Prommersberger, Sabrina %A Einsele, Hermann %A Hudecek, Michael %A Löffler, Jürgen %B Science Translational Medicine %D 2022 %I American Association for the Advancement of Science %J Science Translational Medicine %N 664 %P eabh1209-- %R 10.1126/scitranslmed.abh1209 %T CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models %U https://doi.org/10.1126/scitranslmed.abh1209 %V 14 %X Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus?specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA. Aspergillus fumigatus?specific CAR T cells confer a direct antifungal effect and engage innate immune cells in preclinical models of aspergillosis. CAR T cells are most commonly thought of as a cancer therapy. Recently, however, investigators have been testing CAR T cells for additional, nonmalignant disease. Here, Seif et al. developed and tested a CAR T cell construct that targeted Aspergillus fumigatus hyphae, a ubiquitous mold that can cause invasive pulmonary aspergillosis in immunocompromised individuals. A. fumigatus?specific CAR T cells had potent and selective antifungal activity in vitro and in preclinical animal models, supporting further clinical development of these CAR T cells to treat invasive pulmonary aspergillosis.
• Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration. Brosch, Philippa K.; Korsa, Tessa; Taban, Danush; Eiring, Patrick; Kreisz, Philipp; Hildebrand, Sascha; Neubauer, Julia; Zimmermann, Heiko; Sauer, Markus; Shirakashi, Ryo; Djuzenova, Cholpon S.; Sisario, Dmitri; Sukhorukov, Vladimir L. 2022.
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  (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

  @electronic{brosch2022glucose, abstract = {(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.}, author = {Brosch, Philippa K. and Korsa, Tessa and Taban, Danush and Eiring, Patrick and Kreisz, Philipp and Hildebrand, Sascha and Neubauer, Julia and Zimmermann, Heiko and Sauer, Markus and Shirakashi, Ryo and Djuzenova, Cholpon S. and Sisario, Dmitri and Sukhorukov, Vladimir L.}, booktitle = {Cancers}, keywords = {sauer}, number = 23, title = {Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration}, volume = 14, year = 2022 }

  %0 Generic %1 brosch2022glucose %A Brosch, Philippa K. %A Korsa, Tessa %A Taban, Danush %A Eiring, Patrick %A Kreisz, Philipp %A Hildebrand, Sascha %A Neubauer, Julia %A Zimmermann, Heiko %A Sauer, Markus %A Shirakashi, Ryo %A Djuzenova, Cholpon S. %A Sisario, Dmitri %A Sukhorukov, Vladimir L. %B Cancers %D 2022 %N 23 %R 10.3390/cancers14235794 %T Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration %V 14 %X (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.
• Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth. Halder, Luke D.; Babych, Svitlana; Palme, Diana I.; Mansouri-Ghahnavieh, Elham; Ivanov, Lia; Ashonibare, Victory; Langenhorst, Daniela; Prusty, Bhupesh; Rambach, Günter; Wich, Melissa; Trinks, Nora; Blango, Matthew G.; Kornitzer, Daniel; Terpitz, Ulrich; Speth, Cornelia; Jungnickel, Berit; Beyersdorf, Niklas; Zipfel, Peter F.; Brakhage, Axel A.; Skerka, Christine; Lorenz, Michael. In mBio, 13, S. e03563–21. 2022.
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  Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.

  @article{halder2022candida, abstract = {Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.}, author = {Halder, Luke D. and Babych, Svitlana and Palme, Diana I. and Mansouri-Ghahnavieh, Elham and Ivanov, Lia and Ashonibare, Victory and Langenhorst, Daniela and Prusty, Bhupesh and Rambach, Günter and Wich, Melissa and Trinks, Nora and Blango, Matthew G. and Kornitzer, Daniel and Terpitz, Ulrich and Speth, Cornelia and Jungnickel, Berit and Beyersdorf, Niklas and Zipfel, Peter F. and Brakhage, Axel A. and Skerka, Christine and Lorenz, Michael}, journal = {mBio}, keywords = {terpitz}, pages = {e03563-21}, series = 1, title = {Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth}, volume = 13, year = 2022 }

  %0 Journal Article %1 halder2022candida %A Halder, Luke D. %A Babych, Svitlana %A Palme, Diana I. %A Mansouri-Ghahnavieh, Elham %A Ivanov, Lia %A Ashonibare, Victory %A Langenhorst, Daniela %A Prusty, Bhupesh %A Rambach, Günter %A Wich, Melissa %A Trinks, Nora %A Blango, Matthew G. %A Kornitzer, Daniel %A Terpitz, Ulrich %A Speth, Cornelia %A Jungnickel, Berit %A Beyersdorf, Niklas %A Zipfel, Peter F. %A Brakhage, Axel A. %A Skerka, Christine %A Lorenz, Michael %B 1 %D 2022 %J mBio %P e03563-21 %T Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth %U https://journals.asm.org/doi/abs/10.1128/mbio.03563-21 %V 13 %X Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. In response to infections, human immune cells release extracellular vesicles (EVs) that carry a situationally adapted cocktail of proteins and nucleic acids, including microRNAs (miRNAs), to coordinate the immune response. In this study, we identified hsa-miR-21-5p and hsa-miR-24-3p as the most common miRNAs in exosomes released by human monocytes in response to the pathogenic fungus Candida albicans. Functional analysis of miRNAs revealed that hsa-miR-24-3p, but not hsa-miR-21-5p, acted across species and kingdoms, entering C. albicans and inducing fungal cell growth by inhibiting translation of the cyclin-dependent kinase inhibitor Sol1. Packaging of hsa-miR-24-3p into monocyte exosomes required binding of fungal soluble β-glucan to complement receptor 3 (CR3) and binding of mannan to Toll-like receptor 4 (TLR4), resulting in receptor colocalization. Together, our in vitro and in vivo findings reveal a novel cross-species evasion mechanism by which C. albicans exploits a human miRNA to promote fungal growth and survival in the host. IMPORTANCE Over the last decade, communication between immune cells by extracellular vesicle-associated miRNAs has emerged as an important regulator of the coordinated immune response. Therefore, a thorough understanding of the conversation occurring via miRNAs, especially during infection, may provide novel insights into both the host reaction to the microbe as well as the microbial response. This study provides evidence that the pathogenic fungus C. albicans communicates with human monocytes and induces the release of a human miRNA that promotes fungal growth. This mechanism represents an unexpected cross-species interaction and implies that an inhibition of specific miRNAs offers new possibilities for the treatment of human fungal infections.
• Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier. Helmerich, Dominic A.; Beliu, Gerti; Taban, Danush; Meub, Mara; Streit, Marcel; Kuhlemann, Alexander; Doose, Sören; Sauer, Markus. In Nature Methods, 19(8), S. 986–994. 2022.
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  Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.

  @article{helmerich2022photoswitching, abstract = {Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Taban, Danush and Meub, Mara and Streit, Marcel and Kuhlemann, Alexander and Doose, Sören and Sauer, Markus}, journal = {Nature Methods}, keywords = {sauer}, number = 8, pages = {986–994}, title = {Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier}, volume = 19, year = 2022 }

  %0 Journal Article %1 helmerich2022photoswitching %A Helmerich, Dominic A. %A Beliu, Gerti %A Taban, Danush %A Meub, Mara %A Streit, Marcel %A Kuhlemann, Alexander %A Doose, Sören %A Sauer, Markus %D 2022 %J Nature Methods %N 8 %P 986--994 %R 10.1038/s41592-022-01548-6 %T Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier %U https://doi.org/10.1038/s41592-022-01548-6 %V 19 %X Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging. We demonstrate that time-resolved fluorescence detection in combination with photoswitching fingerprint analysis can be used to determine the number and distance even of spatially unresolvable fluorophores in the sub-10-nm range. In combination with genetic code expansion with unnatural amino acids and bioorthogonal click labeling with small fluorophores, photoswitching fingerprint analysis can be used advantageously to reveal information about the number of fluorophores present and their distances in the sub-10-nm range in cells.
• Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection. Endres, Leo M.; Jungblut, Marvin; Divyapicigil, Mustafa; Sauer, Markus; Stigloher, Christian; Christodoulides, Myron; Kim, Brandon J.; Schubert-Unkmeir, Alexandra. In Fluids and Barriers of the CNS, 19(1), S. 81-. 2022.
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  Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.

  @article{endres2022development, abstract = {Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.}, author = {Endres, Leo M. and Jungblut, Marvin and Divyapicigil, Mustafa and Sauer, Markus and Stigloher, Christian and Christodoulides, Myron and Kim, Brandon J. and Schubert-Unkmeir, Alexandra}, journal = {Fluids and Barriers of the CNS}, keywords = {sauer}, number = 1, pages = {81–}, title = {Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection}, volume = 19, year = 2022 }

  %0 Journal Article %1 endres2022development %A Endres, Leo M. %A Jungblut, Marvin %A Divyapicigil, Mustafa %A Sauer, Markus %A Stigloher, Christian %A Christodoulides, Myron %A Kim, Brandon J. %A Schubert-Unkmeir, Alexandra %D 2022 %J Fluids and Barriers of the CNS %N 1 %P 81-- %R 10.1186/s12987-022-00379-z %T Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection %U https://doi.org/10.1186/s12987-022-00379-z %V 19 %X Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction.
• Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes. Shirakashi, Ryo; Sisario, Dmitri; Taban, Danush; Korsa, Tessa; Wanner, Sophia B.; Neubauer, Julia; Djuzenova, Cholpon S.; Zimmermann, Heiko; Sukhorukov, Vladimir L. In Biomechanics and Modeling in Mechanobiology. 2022.
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  Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.

  @article{shirakashi2022contraction, abstract = {Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.}, author = {Shirakashi, Ryo and Sisario, Dmitri and Taban, Danush and Korsa, Tessa and Wanner, Sophia B. and Neubauer, Julia and Djuzenova, Cholpon S. and Zimmermann, Heiko and Sukhorukov, Vladimir L.}, journal = {Biomechanics and Modeling in Mechanobiology}, keywords = {vladimir}, title = {Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes}, year = 2022 }

  %0 Journal Article %1 shirakashi2022contraction %A Shirakashi, Ryo %A Sisario, Dmitri %A Taban, Danush %A Korsa, Tessa %A Wanner, Sophia B. %A Neubauer, Julia %A Djuzenova, Cholpon S. %A Zimmermann, Heiko %A Sukhorukov, Vladimir L. %D 2022 %J Biomechanics and Modeling in Mechanobiology %R 10.1007/s10237-022-01654-6 %T Contraction of the rigor actomyosin complex drives bulk hemoglobin expulsion from hemolyzing erythrocytes %U https://doi.org/10.1007/s10237-022-01654-6 %X Erythrocyte ghost formation via hemolysis is a key event in the physiological clearance of senescent red blood cells (RBCs) in the spleen. The turnover rate of millions of RBCs per second necessitates a rapid efflux of hemoglobin (Hb) from RBCs by a not yet identified mechanism. Using high-speed video-microscopy of isolated RBCs, we show that electroporation-induced efflux of cytosolic ATP and other small solutes leads to transient cell shrinkage and echinocytosis, followed by osmotic swelling to the critical hemolytic volume. The onset of hemolysis coincided with a sudden self-propelled cell motion, accompanied by cell contraction and Hb-jet ejection. Our biomechanical model, which relates the Hb-jet-driven cell motion to the cytosolic pressure generation via elastic contraction of the RBC membrane, showed that the contributions of the bilayer and the bilayer-anchored spectrin cytoskeleton to the hemolytic cell motion are negligible. Consistent with the biomechanical analysis, our biochemical experiments, involving extracellular ATP and the myosin inhibitor blebbistatin, identify the low abundant non-muscle myosin 2A (NM2A) as the key contributor to the Hb-jet emission and fast hemolytic cell motion. Thus, our data reveal a rapid myosin-based mechanism of hemolysis, as opposed to a much slower diffusive Hb efflux.
• Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer. Thiele, Jan Christoph; Jungblut, Marvin; Helmerich, Dominic A.; Tsukanov, Roman; Chizhik, Anna; Chizhik, Alexey I.; Schnermann, Martin J.; Sauer, Markus; Nevskyi, Oleksii; Enderlein, Jörg. In Science Advances, 8(23), S. eabo2506-. American Association for the Advancement of Science, 2022.
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  Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution, which is by a factor of 2 to 3 worse. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging, which achieves an axial resolution down to nanometers. It exploits the distance-dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously. MIET-SMLM is a fluorescence lifetime?based approach to image subcellular structures with isotropic 3D super-resolution.

  @article{thieleisotropic, abstract = {Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution, which is by a factor of 2 to 3 worse. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging, which achieves an axial resolution down to nanometers. It exploits the distance-dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously. MIET-SMLM is a fluorescence lifetime?based approach to image subcellular structures with isotropic 3D super-resolution.}, author = {Thiele, Jan Christoph and Jungblut, Marvin and Helmerich, Dominic A. and Tsukanov, Roman and Chizhik, Anna and Chizhik, Alexey I. and Schnermann, Martin J. and Sauer, Markus and Nevskyi, Oleksii and Enderlein, Jörg}, booktitle = {Science Advances}, journal = {Science Advances}, keywords = {sauer}, number = 23, pages = {eabo2506–}, publisher = {American Association for the Advancement of Science}, title = {Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer}, volume = 8, year = 2022 }

  %0 Journal Article %1 thieleisotropic %A Thiele, Jan Christoph %A Jungblut, Marvin %A Helmerich, Dominic A. %A Tsukanov, Roman %A Chizhik, Anna %A Chizhik, Alexey I. %A Schnermann, Martin J. %A Sauer, Markus %A Nevskyi, Oleksii %A Enderlein, Jörg %B Science Advances %D 2022 %I American Association for the Advancement of Science %J Science Advances %N 23 %P eabo2506-- %R 10.1126/sciadv.abo2506 %T Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer %U https://doi.org/10.1126/sciadv.abo2506 %V 8 %X Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists a remarkable gap between lateral and axial resolution, which is by a factor of 2 to 3 worse. One recently developed method to close this gap is metal-induced energy transfer (MIET) imaging, which achieves an axial resolution down to nanometers. It exploits the distance-dependent quenching of fluorescence when a fluorescent molecule is brought close to a metal surface. In the present manuscript, we combine the extreme axial resolution of MIET imaging with the extraordinary lateral resolution of single-molecule localization microscopy, in particular with direct stochastic optical reconstruction microscopy (dSTORM). This combination allows us to achieve isotropic three-dimensional super-resolution imaging of subcellular structures. Moreover, we used spectral demixing for implementing dual-color MIET-dSTORM that allows us to image and colocalize, in three dimensions, two different cellular structures simultaneously. MIET-SMLM is a fluorescence lifetime?based approach to image subcellular structures with isotropic 3D super-resolution.
• Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA. Hennig, Thomas; Prusty, Archana B.; Kaufer, Benedikt B.; Whisnant, Adam W.; Lodha, Manivel; Enders, Antje; Thomas, Julius; Kasimir, Francesca; Grothey, Arnhild; Klein, Teresa; Herb, Stefanie; Jürges, Christopher; Sauer, Markus; Fischer, Utz; Rudel, Thomas; Meister, Gunter; Erhard, Florian; Dölken, Lars; Prusty, Bhupesh K. In Nature, 605(7910), S. 539–544. 2022.
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  Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic–latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic–latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.

  @article{hennig2022selective, abstract = {Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic–latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic–latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.}, author = {Hennig, Thomas and Prusty, Archana B. and Kaufer, Benedikt B. and Whisnant, Adam W. and Lodha, Manivel and Enders, Antje and Thomas, Julius and Kasimir, Francesca and Grothey, Arnhild and Klein, Teresa and Herb, Stefanie and Jürges, Christopher and Sauer, Markus and Fischer, Utz and Rudel, Thomas and Meister, Gunter and Erhard, Florian and Dölken, Lars and Prusty, Bhupesh K.}, journal = {Nature}, keywords = {sauer}, number = 7910, pages = {539–544}, title = {Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA}, volume = 605, year = 2022 }

  %0 Journal Article %1 hennig2022selective %A Hennig, Thomas %A Prusty, Archana B. %A Kaufer, Benedikt B. %A Whisnant, Adam W. %A Lodha, Manivel %A Enders, Antje %A Thomas, Julius %A Kasimir, Francesca %A Grothey, Arnhild %A Klein, Teresa %A Herb, Stefanie %A Jürges, Christopher %A Sauer, Markus %A Fischer, Utz %A Rudel, Thomas %A Meister, Gunter %A Erhard, Florian %A Dölken, Lars %A Prusty, Bhupesh K. %D 2022 %J Nature %N 7910 %P 539--544 %R 10.1038/s41586-022-04667-4 %T Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA %U https://doi.org/10.1038/s41586-022-04667-4 %V 605 %X Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic–latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30–p53–DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic–latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.
• The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis. La Greca, Mariafrancesca; Chen, Jheng-Liang; Schubert, Luiz; Kozuch, Jacek; Berneiser, Tim; Terpitz, Ulrich; Heberle, Joachim; Schlesinger, Ramona. In Frontiers in Molecular Biosciences, 9. 2022.
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  Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.

  @article{lagreca2022photoreaction, abstract = {Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.}, author = {La Greca, Mariafrancesca and Chen, Jheng-Liang and Schubert, Luiz and Kozuch, Jacek and Berneiser, Tim and Terpitz, Ulrich and Heberle, Joachim and Schlesinger, Ramona}, journal = {Frontiers in Molecular Biosciences}, keywords = {terpitz}, title = {The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis}, volume = 9, year = 2022 }

  %0 Journal Article %1 lagreca2022photoreaction %A La Greca, Mariafrancesca %A Chen, Jheng-Liang %A Schubert, Luiz %A Kozuch, Jacek %A Berneiser, Tim %A Terpitz, Ulrich %A Heberle, Joachim %A Schlesinger, Ramona %D 2022 %J Frontiers in Molecular Biosciences %T The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis %U https://www.frontiersin.org/article/10.3389/fmolb.2022.826990 %V 9 %X Microbial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.
• Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration. Brosch, Philippa K.; Korsa, Tessa; Taban, Danush; Eiring, Patrick; Hildebrand, Sascha; Neubauer, Julia; Zimmermann, Heiko; Sauer, Markus; Shirakashi, Ryo; Djuzenova, Cholpon S.; Sisario, Dmitri; Sukhorukov, Vladimir L. In Cancers, 14, S. 5794. 2022.
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  (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

  @article{brosch2022glucose, abstract = {(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.}, author = {Brosch, Philippa K. and Korsa, Tessa and Taban, Danush and Eiring, Patrick and Hildebrand, Sascha and Neubauer, Julia and Zimmermann, Heiko and Sauer, Markus and Shirakashi, Ryo and Djuzenova, Cholpon S. and Sisario, Dmitri and Sukhorukov, Vladimir L.}, journal = {Cancers}, keywords = {sauer}, pages = 5794, series = 23, title = {Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration}, volume = 14, year = 2022 }

  %0 Journal Article %1 brosch2022glucose %A Brosch, Philippa K. %A Korsa, Tessa %A Taban, Danush %A Eiring, Patrick %A Hildebrand, Sascha %A Neubauer, Julia %A Zimmermann, Heiko %A Sauer, Markus %A Shirakashi, Ryo %A Djuzenova, Cholpon S. %A Sisario, Dmitri %A Sukhorukov, Vladimir L. %B 23 %D 2022 %J Cancers %P 5794 %T Glucose and Inositol Transporters, SLC5A1 and SLC5A3, in Glioblastoma Cell Migration %U https://www.mdpi.com/2072-6694/14/23/5794 %V 14 %X (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.
• Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models. Prieto Huarcaya, Susy; Drobny, Alice; Marques, André R. A.; Di Spiezio, Alessandro; Dobert, Jan Philipp; Balta, Denise; Werner, Christian; Rizo, Tania; Gallwitz, Lisa; Bub, Simon; Stojkovska, Iva; Belur, Nandkishore R.; Fogh, Jens; Mazzulli, Joseph R; Xiang, Wei; Fulzele, Amitkumar; Dejung, Mario; Sauer, Markus; Winner, Beate; Rose-John, Stefan; Arnold, Philipp; Saftig, Paul; Zunke, Friederike. In Autophagy, 18(5), S. 1127–1151. Taylor & Francis, 2022.
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  Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden. Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/?-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus

  @article{prietohuarcaya2022recombinant, abstract = {Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden. Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/?-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus}, author = {Prieto Huarcaya, Susy and Drobny, Alice and Marques, André R. A. and Di Spiezio, Alessandro and Dobert, Jan Philipp and Balta, Denise and Werner, Christian and Rizo, Tania and Gallwitz, Lisa and Bub, Simon and Stojkovska, Iva and Belur, Nandkishore R. and Fogh, Jens and Mazzulli, Joseph R and Xiang, Wei and Fulzele, Amitkumar and Dejung, Mario and Sauer, Markus and Winner, Beate and Rose-John, Stefan and Arnold, Philipp and Saftig, Paul and Zunke, Friederike}, booktitle = {Autophagy}, journal = {Autophagy}, keywords = {werner}, month = {05}, number = 5, pages = {1127–1151}, publisher = {Taylor & Francis}, title = {Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models}, volume = 18, year = 2022 }

  %0 Journal Article %1 prietohuarcaya2022recombinant %A Prieto Huarcaya, Susy %A Drobny, Alice %A Marques, André R. A. %A Di Spiezio, Alessandro %A Dobert, Jan Philipp %A Balta, Denise %A Werner, Christian %A Rizo, Tania %A Gallwitz, Lisa %A Bub, Simon %A Stojkovska, Iva %A Belur, Nandkishore R. %A Fogh, Jens %A Mazzulli, Joseph R %A Xiang, Wei %A Fulzele, Amitkumar %A Dejung, Mario %A Sauer, Markus %A Winner, Beate %A Rose-John, Stefan %A Arnold, Philipp %A Saftig, Paul %A Zunke, Friederike %B Autophagy %D 2022 %I Taylor & Francis %J Autophagy %N 5 %P 1127--1151 %R 10.1080/15548627.2022.2045534 %T Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models %U https://doi.org/10.1080/15548627.2022.2045534 %V 18 %X Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden. Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/?-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus
• Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation. Dannhäuser, Sven; Mrestani, Achmed; Gundelach, Florian; Pauli, Martin; Komma, Fabian; Kollmannsberger, Philip; Sauer, Markus; Heckmann, Manfred; Paul, Mila M. In Frontiers in Cellular Neuroscience, 16. 2022.
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  Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13 GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13 GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

  @article{dannhauser2022endogenous, abstract = {Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13 GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13 GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.}, author = {Dannhäuser, Sven and Mrestani, Achmed and Gundelach, Florian and Pauli, Martin and Komma, Fabian and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Paul, Mila M.}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, title = {Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation}, volume = 16, year = 2022 }

  %0 Journal Article %1 dannhauser2022endogenous %A Dannhäuser, Sven %A Mrestani, Achmed %A Gundelach, Florian %A Pauli, Martin %A Komma, Fabian %A Kollmannsberger, Philip %A Sauer, Markus %A Heckmann, Manfred %A Paul, Mila M. %D 2022 %J Frontiers in Cellular Neuroscience %T Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation %U https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2022.1074304 %V 16 %X Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13 GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13 GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.
• Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable. Lu, Jinping; Dreyer, Ingo; Dickinson, Miles Sasha; Panzer, Sabine; Jaslan, Dawid; Navarro-Retamal, Carlos; Geiger, Dietmar; Terpitz, Ulrich; Becker, Dirk; Stroud, Robert M.; Marten, Irene; Hedrich, Rainer. In bioRxiv, S. 2021.12.22.473873-. 2022.
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  To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.

  @article{lu2022ltemgtvicia, abstract = {To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.}, author = {Lu, Jinping and Dreyer, Ingo and Dickinson, Miles Sasha and Panzer, Sabine and Jaslan, Dawid and Navarro-Retamal, Carlos and Geiger, Dietmar and Terpitz, Ulrich and Becker, Dirk and Stroud, Robert M. and Marten, Irene and Hedrich, Rainer}, journal = {bioRxiv}, keywords = {terpitz}, month = {01}, pages = {2021.12.22.473873–}, title = {Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable}, year = 2022 }

  %0 Journal Article %1 lu2022ltemgtvicia %A Lu, Jinping %A Dreyer, Ingo %A Dickinson, Miles Sasha %A Panzer, Sabine %A Jaslan, Dawid %A Navarro-Retamal, Carlos %A Geiger, Dietmar %A Terpitz, Ulrich %A Becker, Dirk %A Stroud, Robert M. %A Marten, Irene %A Hedrich, Rainer %D 2022 %J bioRxiv %P 2021.12.22.473873-- %R 10.1101/2021.12.22.473873 %T Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable %U http://biorxiv.org/content/early/2022/03/04/2021.12.22.473873.abstract %X To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.Significance statement Vacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.Competing Interest StatementThe authors have declared no competing interest.
• ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy. Reinhard, Sebastian; Helmerich, Dominic A.; Boras, Dominik; Sauer, Markus; Kollmannsberger, Philip. In BMC Bioinformatics, 23(1), S. 530-. 2022.
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  Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method.

  @article{reinhard2022recsai, abstract = {Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method.}, author = {Reinhard, Sebastian and Helmerich, Dominic A. and Boras, Dominik and Sauer, Markus and Kollmannsberger, Philip}, journal = {BMC Bioinformatics}, keywords = {sauer}, number = 1, pages = {530–}, title = {ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy}, volume = 23, year = 2022 }

  %0 Journal Article %1 reinhard2022recsai %A Reinhard, Sebastian %A Helmerich, Dominic A. %A Boras, Dominik %A Sauer, Markus %A Kollmannsberger, Philip %D 2022 %J BMC Bioinformatics %N 1 %P 530-- %R 10.1186/s12859-022-05071-5 %T ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy %U https://doi.org/10.1186/s12859-022-05071-5 %V 23 %X Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method.
• Convex hull as diagnostic tool in single-molecule localization microscopy. Ebert, Vincent; Eiring, Patrick; Helmerich, Dominic A; Seifert, Rick; Sauer, Markus; Doose, Sören. In Bioinformatics, 38(24), S. 5421–5429. 2022.
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  Single-molecule localization microscopy resolves individual fluorophores or fluorescence-labeled biomolecules. Data are provided as a set of localizations that distribute normally around the true fluorophore position with a variance determined by the localization precision. Characterizing the spatial fluorophore distribution to differentiate between resolution-limited localization clusters, which resemble individual biomolecules, and extended structures, which represent aggregated molecular complexes, is a common challenge.We demonstrate the use of the convex hull and related hull properties of localization clusters for diagnostic purposes, as a parameter for cluster selection or as a tool to determine localization precision.https://github.com/super-resolution/Ebert-et-al-2022-supplement.Supplementary data are available at Bioinformatics online.

  @article{ebert2022convex, abstract = {Single-molecule localization microscopy resolves individual fluorophores or fluorescence-labeled biomolecules. Data are provided as a set of localizations that distribute normally around the true fluorophore position with a variance determined by the localization precision. Characterizing the spatial fluorophore distribution to differentiate between resolution-limited localization clusters, which resemble individual biomolecules, and extended structures, which represent aggregated molecular complexes, is a common challenge.We demonstrate the use of the convex hull and related hull properties of localization clusters for diagnostic purposes, as a parameter for cluster selection or as a tool to determine localization precision.https://github.com/super-resolution/Ebert-et-al-2022-supplement.Supplementary data are available at Bioinformatics online.}, author = {Ebert, Vincent and Eiring, Patrick and Helmerich, Dominic A and Seifert, Rick and Sauer, Markus and Doose, Sören}, journal = {Bioinformatics}, keywords = {sauer}, month = 12, number = 24, pages = {5421–5429}, title = {Convex hull as diagnostic tool in single-molecule localization microscopy}, volume = 38, year = 2022 }

  %0 Journal Article %1 ebert2022convex %A Ebert, Vincent %A Eiring, Patrick %A Helmerich, Dominic A %A Seifert, Rick %A Sauer, Markus %A Doose, Sören %D 2022 %J Bioinformatics %N 24 %P 5421--5429 %R 10.1093/bioinformatics/btac700 %T Convex hull as diagnostic tool in single-molecule localization microscopy %U https://doi.org/10.1093/bioinformatics/btac700 %V 38 %X Single-molecule localization microscopy resolves individual fluorophores or fluorescence-labeled biomolecules. Data are provided as a set of localizations that distribute normally around the true fluorophore position with a variance determined by the localization precision. Characterizing the spatial fluorophore distribution to differentiate between resolution-limited localization clusters, which resemble individual biomolecules, and extended structures, which represent aggregated molecular complexes, is a common challenge.We demonstrate the use of the convex hull and related hull properties of localization clusters for diagnostic purposes, as a parameter for cluster selection or as a tool to determine localization precision.https://github.com/super-resolution/Ebert-et-al-2022-supplement.Supplementary data are available at Bioinformatics online.
• The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release. Paul, Mila M; Dannhäuser, Sven; Morris, Lydia; Mrestani, Achmed; Hübsch, Martha; Gehring, Jennifer; Hatzopoulos, Georgios N; Pauli, Martin; Auger, Genevieve M; Bornschein, Grit; Scholz, Nicole; Ljaschenko, Dmitrij; Müller, Martin; Sauer, Markus; Schmidt, Hartmut; Kittel, Robert J; DiAntonio, Aaron; Vakonakis, Ioannis; Heckmann, Manfred; Langenhan, Tobias. In Brain, 145(11), S. 3787–3802. 2022.
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  Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far.Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST.We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

  @article{paul2022human, abstract = {Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far.Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST.We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.}, author = {Paul, Mila M and Dannhäuser, Sven and Morris, Lydia and Mrestani, Achmed and Hübsch, Martha and Gehring, Jennifer and Hatzopoulos, Georgios N and Pauli, Martin and Auger, Genevieve M and Bornschein, Grit and Scholz, Nicole and Ljaschenko, Dmitrij and Müller, Martin and Sauer, Markus and Schmidt, Hartmut and Kittel, Robert J and DiAntonio, Aaron and Vakonakis, Ioannis and Heckmann, Manfred and Langenhan, Tobias}, journal = {Brain}, keywords = {sauer}, month = 11, number = 11, pages = {3787–3802}, title = {The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release}, volume = 145, year = 2022 }

  %0 Journal Article %1 paul2022human %A Paul, Mila M %A Dannhäuser, Sven %A Morris, Lydia %A Mrestani, Achmed %A Hübsch, Martha %A Gehring, Jennifer %A Hatzopoulos, Georgios N %A Pauli, Martin %A Auger, Genevieve M %A Bornschein, Grit %A Scholz, Nicole %A Ljaschenko, Dmitrij %A Müller, Martin %A Sauer, Markus %A Schmidt, Hartmut %A Kittel, Robert J %A DiAntonio, Aaron %A Vakonakis, Ioannis %A Heckmann, Manfred %A Langenhan, Tobias %D 2022 %J Brain %N 11 %P 3787--3802 %R 10.1093/brain/awac011 %T The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release %U https://doi.org/10.1093/brain/awac011 %V 145 %X Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far.Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST.We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.
• CXCR4 expression of multiple myeloma as a dynamic process: influence of therapeutic agents. Bögelein, Anna; Stolzenburg, Antje; Eiring, Patrick; Lückerath, Katharina; Munawar, Umair; Werner, Rudolf; Schirbel, Andreas; Samnick, Samuel; Kortüm, Klaus Martin; Sauer, Markus; Lapa, Constantin; Buck, Andreas K. In Leukemia & Lymphoma, 63(10), S. 2393–2402. Taylor & Francis, 2022.
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  Chemokine receptors represent novel targets for treatment of multiple myeloma (MM). However, CXCR4 expression appears to be highly dynamic. This in vitro study investigated the impact of commonly used anti-myeloma agents on CXCR4 expression. Established human myeloma cell lines as well as patient-derived CD138+ plasma cells were exposed to antineoplastic drugs. Cells were analyzed for CXCR4 expression by flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM). In addition, cellular uptake of 68Ga-Pentixafor, a PET radiotracer for noninvasive assessment of CXCR4 expression in vivo, was assessed. CXCR4 expression was highly variable and turned out to be substance, dose and time dependent. Treatment with bortezomib was associated with reduced expression, while dexamethasone and doxorubicin significantly increased expression of CXCR4. Combination of these compounds further increased CXCR4 expression. In conclusion, drugs or combination of drugs can induce CXCR4 expression in myeloma cells. Hence, pretreatment may impact on response to CXCR4-based therapies.

  @article{bogelein2022cxcr4, abstract = {Chemokine receptors represent novel targets for treatment of multiple myeloma (MM). However, CXCR4 expression appears to be highly dynamic. This in vitro study investigated the impact of commonly used anti-myeloma agents on CXCR4 expression. Established human myeloma cell lines as well as patient-derived CD138+ plasma cells were exposed to antineoplastic drugs. Cells were analyzed for CXCR4 expression by flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM). In addition, cellular uptake of 68Ga-Pentixafor, a PET radiotracer for noninvasive assessment of CXCR4 expression in vivo, was assessed. CXCR4 expression was highly variable and turned out to be substance, dose and time dependent. Treatment with bortezomib was associated with reduced expression, while dexamethasone and doxorubicin significantly increased expression of CXCR4. Combination of these compounds further increased CXCR4 expression. In conclusion, drugs or combination of drugs can induce CXCR4 expression in myeloma cells. Hence, pretreatment may impact on response to CXCR4-based therapies.}, author = {Bögelein, Anna and Stolzenburg, Antje and Eiring, Patrick and Lückerath, Katharina and Munawar, Umair and Werner, Rudolf and Schirbel, Andreas and Samnick, Samuel and Kortüm, Klaus Martin and Sauer, Markus and Lapa, Constantin and Buck, Andreas K.}, booktitle = {Leukemia & Lymphoma}, journal = {Leukemia & Lymphoma}, keywords = {sauer}, month = {08}, number = 10, pages = {2393–2402}, publisher = {Taylor & Francis}, title = {CXCR4 expression of multiple myeloma as a dynamic process: influence of therapeutic agents}, volume = 63, year = 2022 }

  %0 Journal Article %1 bogelein2022cxcr4 %A Bögelein, Anna %A Stolzenburg, Antje %A Eiring, Patrick %A Lückerath, Katharina %A Munawar, Umair %A Werner, Rudolf %A Schirbel, Andreas %A Samnick, Samuel %A Kortüm, Klaus Martin %A Sauer, Markus %A Lapa, Constantin %A Buck, Andreas K. %B Leukemia & Lymphoma %D 2022 %I Taylor & Francis %J Leukemia & Lymphoma %N 10 %P 2393--2402 %R 10.1080/10428194.2022.2074986 %T CXCR4 expression of multiple myeloma as a dynamic process: influence of therapeutic agents %U https://doi.org/10.1080/10428194.2022.2074986 %V 63 %X Chemokine receptors represent novel targets for treatment of multiple myeloma (MM). However, CXCR4 expression appears to be highly dynamic. This in vitro study investigated the impact of commonly used anti-myeloma agents on CXCR4 expression. Established human myeloma cell lines as well as patient-derived CD138+ plasma cells were exposed to antineoplastic drugs. Cells were analyzed for CXCR4 expression by flow cytometry and direct stochastic optical reconstruction microscopy (dSTORM). In addition, cellular uptake of 68Ga-Pentixafor, a PET radiotracer for noninvasive assessment of CXCR4 expression in vivo, was assessed. CXCR4 expression was highly variable and turned out to be substance, dose and time dependent. Treatment with bortezomib was associated with reduced expression, while dexamethasone and doxorubicin significantly increased expression of CXCR4. Combination of these compounds further increased CXCR4 expression. In conclusion, drugs or combination of drugs can induce CXCR4 expression in myeloma cells. Hence, pretreatment may impact on response to CXCR4-based therapies.
• Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity. Lycas, Matthew D.; Ejdrup, Aske L.; Sørensen, Andreas T.; Haahr, Nicolai O.; Jørgensen, Søren H.; Guthrie, Daryl A.; Støier, Jonatan F.; Werner, Christian; Newman, Amy Hauck; Sauer, Markus; Herborg, Freja; Gether, Ulrik. In Cell Reports, 40(13), S. 111431-. 2022.
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  The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.

  @article{lycas2022nanoscopic, abstract = {The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.}, author = {Lycas, Matthew D. and Ejdrup, Aske L. and Sørensen, Andreas T. and Haahr, Nicolai O. and Jørgensen, Søren H. and Guthrie, Daryl A. and Støier, Jonatan F. and Werner, Christian and Newman, Amy Hauck and Sauer, Markus and Herborg, Freja and Gether, Ulrik}, journal = {Cell Reports}, keywords = {werner}, number = 13, pages = {111431–}, title = {Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity}, volume = 40, year = 2022 }

  %0 Journal Article %1 lycas2022nanoscopic %A Lycas, Matthew D. %A Ejdrup, Aske L. %A Sørensen, Andreas T. %A Haahr, Nicolai O. %A Jørgensen, Søren H. %A Guthrie, Daryl A. %A Støier, Jonatan F. %A Werner, Christian %A Newman, Amy Hauck %A Sauer, Markus %A Herborg, Freja %A Gether, Ulrik %D 2022 %J Cell Reports %N 13 %P 111431-- %R https://doi.org/10.1016/j.celrep.2022.111431 %T Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity %U https://www.sciencedirect.com/science/article/pii/S2211124722012724 %V 40 %X The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.
• Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence. Balakrishnan, Ashwin; Hemmen, Katherina; Choudhury, Susobhan; Krohn, Jan-Hagen; Jansen, Kerstin; Friedrich, Mike; Beliu, Gerti; Sauer, Markus; Lohse, Martin J.; Heinze, Katrin G. In Communications Biology, 5(1), S. 176-. 2022.
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  G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β2-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.

  @article{balakrishnan2022unraveling, abstract = {G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β2-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.}, author = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {176–}, title = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence}, volume = 5, year = 2022 }

  %0 Journal Article %1 balakrishnan2022unraveling %A Balakrishnan, Ashwin %A Hemmen, Katherina %A Choudhury, Susobhan %A Krohn, Jan-Hagen %A Jansen, Kerstin %A Friedrich, Mike %A Beliu, Gerti %A Sauer, Markus %A Lohse, Martin J. %A Heinze, Katrin G. %D 2022 %J Communications Biology %N 1 %P 176-- %R 10.1038/s42003-022-03106-4 %T Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence %U https://doi.org/10.1038/s42003-022-03106-4 %V 5 %X G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β2-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.
• ShareLoc — an open platform for sharing localization microscopy data. Ouyang, Wei; Bai, Jiachuan; Singh, Manish Kumar; Leterrier, Christophe; Barthelemy, Paul; Barnett, Samuel F. H.; Klein, Teresa; Sauer, Markus; Kanchanawong, Pakorn; Bourg, Nicolas; Cohen, Mickael M.; Lelandais, Benoît; Zimmer, Christophe. In Nature Methods, 19(11), S. 1331–1333. 2022.
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  @article{ouyang2022shareloc, author = {Ouyang, Wei and Bai, Jiachuan and Singh, Manish Kumar and Leterrier, Christophe and Barthelemy, Paul and Barnett, Samuel F. H. and Klein, Teresa and Sauer, Markus and Kanchanawong, Pakorn and Bourg, Nicolas and Cohen, Mickael M. and Lelandais, Benoît and Zimmer, Christophe}, journal = {Nature Methods}, keywords = {sauer}, number = 11, pages = {1331–1333}, title = {ShareLoc — an open platform for sharing localization microscopy data}, volume = 19, year = 2022 }

  %0 Journal Article %1 ouyang2022shareloc %A Ouyang, Wei %A Bai, Jiachuan %A Singh, Manish Kumar %A Leterrier, Christophe %A Barthelemy, Paul %A Barnett, Samuel F. H. %A Klein, Teresa %A Sauer, Markus %A Kanchanawong, Pakorn %A Bourg, Nicolas %A Cohen, Mickael M. %A Lelandais, Benoît %A Zimmer, Christophe %D 2022 %J Nature Methods %N 11 %P 1331--1333 %R 10.1038/s41592-022-01659-0 %T ShareLoc — an open platform for sharing localization microscopy data %U https://doi.org/10.1038/s41592-022-01659-0 %V 19
• Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus–response in spinal muscular atrophy. Deng, Chunchu; Reinhard, Sebastian; Hennlein, Luisa; Eilts, Janna; Sachs, Stefan; Doose, Sören; Jablonka, Sibylle; Sauer, Markus; Moradi, Mehri; Sendtner, Michael. In Translational Neurodegeneration, 11(1), S. 31-. 2022.
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  Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA.

  @article{deng2022impaired, abstract = {Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA.}, author = {Deng, Chunchu and Reinhard, Sebastian and Hennlein, Luisa and Eilts, Janna and Sachs, Stefan and Doose, Sören and Jablonka, Sibylle and Sauer, Markus and Moradi, Mehri and Sendtner, Michael}, journal = {Translational Neurodegeneration}, keywords = {sauer}, number = 1, pages = {31–}, title = {Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus–response in spinal muscular atrophy}, volume = 11, year = 2022 }

  %0 Journal Article %1 deng2022impaired %A Deng, Chunchu %A Reinhard, Sebastian %A Hennlein, Luisa %A Eilts, Janna %A Sachs, Stefan %A Doose, Sören %A Jablonka, Sibylle %A Sauer, Markus %A Moradi, Mehri %A Sendtner, Michael %D 2022 %J Translational Neurodegeneration %N 1 %P 31-- %R 10.1186/s40035-022-00304-2 %T Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus–response in spinal muscular atrophy %U https://doi.org/10.1186/s40035-022-00304-2 %V 11 %X Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA.
• MYC multimers shield stalled replication forks from RNA polymerase. Solvie, Daniel; Baluapuri, Apoorva; Uhl, Leonie; Fleischhauer, Daniel; Endres, Theresa; Papadopoulos, Dimitrios; Aziba, Amel; Gaballa, Abdallah; Mikicic, Ivan; Isaakova, Ekaterina; Giansanti, Celeste; Jansen, Jennifer; Jungblut, Marvin; Klein, Teresa; Schülein-Völk, Christina; Maric, Hans; Doose, Sören; Sauer, Markus; Beli, Petra; Rosenwald, Andreas; Dobbelstein, Matthias; Wolf, Elmar; Eilers, Martin. In Nature, 612(7938), S. 148–155. 2022.
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  Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

  @article{solvie2022multimers, abstract = {Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.}, author = {Solvie, Daniel and Baluapuri, Apoorva and Uhl, Leonie and Fleischhauer, Daniel and Endres, Theresa and Papadopoulos, Dimitrios and Aziba, Amel and Gaballa, Abdallah and Mikicic, Ivan and Isaakova, Ekaterina and Giansanti, Celeste and Jansen, Jennifer and Jungblut, Marvin and Klein, Teresa and Schülein-Völk, Christina and Maric, Hans and Doose, Sören and Sauer, Markus and Beli, Petra and Rosenwald, Andreas and Dobbelstein, Matthias and Wolf, Elmar and Eilers, Martin}, journal = {Nature}, keywords = {sauer}, number = 7938, pages = {148–155}, title = {MYC multimers shield stalled replication forks from RNA polymerase}, volume = 612, year = 2022 }

  %0 Journal Article %1 solvie2022multimers %A Solvie, Daniel %A Baluapuri, Apoorva %A Uhl, Leonie %A Fleischhauer, Daniel %A Endres, Theresa %A Papadopoulos, Dimitrios %A Aziba, Amel %A Gaballa, Abdallah %A Mikicic, Ivan %A Isaakova, Ekaterina %A Giansanti, Celeste %A Jansen, Jennifer %A Jungblut, Marvin %A Klein, Teresa %A Schülein-Völk, Christina %A Maric, Hans %A Doose, Sören %A Sauer, Markus %A Beli, Petra %A Rosenwald, Andreas %A Dobbelstein, Matthias %A Wolf, Elmar %A Eilers, Martin %D 2022 %J Nature %N 7938 %P 148--155 %R 10.1038/s41586-022-05469-4 %T MYC multimers shield stalled replication forks from RNA polymerase %U https://doi.org/10.1038/s41586-022-05469-4 %V 612 %X Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.

• Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs. Djuzenova, Cholpon S.; Fischer, Thomas; Katzer, Astrid; Sisario, Dmitri; Korsa, Tessa; Steussloff, Gudrun; Sukhorukov, Vladimir L.; Flentje, Michael. In BMC Cancer, 21(1), S. 1201-. 2021.
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  Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.

  @article{djuzenova2021opposite, abstract = {Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.}, author = {Djuzenova, Cholpon S. and Fischer, Thomas and Katzer, Astrid and Sisario, Dmitri and Korsa, Tessa and Steussloff, Gudrun and Sukhorukov, Vladimir L. and Flentje, Michael}, journal = {BMC Cancer}, keywords = {vladimir}, number = 1, pages = {1201–}, title = {Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs}, volume = 21, year = 2021 }

  %0 Journal Article %1 djuzenova2021opposite %A Djuzenova, Cholpon S. %A Fischer, Thomas %A Katzer, Astrid %A Sisario, Dmitri %A Korsa, Tessa %A Steussloff, Gudrun %A Sukhorukov, Vladimir L. %A Flentje, Michael %D 2021 %J BMC Cancer %N 1 %P 1201-- %R 10.1186/s12885-021-08930-1 %T Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs %U https://doi.org/10.1186/s12885-021-08930-1 %V 21 %X Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells.
• Superaufgelöste Mikroskopie: Pilze unter Beobachtung. Sputh, Sebastian; Panzer, Sabine; Stigloher, Christian; Terpitz, Ulrich. In BIOspektrum, 27(4), S. 380–382. 2021.
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  The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).

  @article{sputh2021superaufgelste, abstract = {The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).}, author = {Sputh, Sebastian and Panzer, Sabine and Stigloher, Christian and Terpitz, Ulrich}, journal = {BIOspektrum}, keywords = {terpitz}, number = 4, pages = {380–382}, title = {Superaufgelöste Mikroskopie: Pilze unter Beobachtung}, volume = 27, year = 2021 }

  %0 Journal Article %1 sputh2021superaufgelste %A Sputh, Sebastian %A Panzer, Sabine %A Stigloher, Christian %A Terpitz, Ulrich %D 2021 %J BIOspektrum %N 4 %P 380--382 %R 10.1007/s12268-021-1592-6 %T Superaufgelöste Mikroskopie: Pilze unter Beobachtung %U https://doi.org/10.1007/s12268-021-1592-6 %V 27 %X The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).
• Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia. Garitano-Trojaola, Andoni; Sancho, Ana; Götz, Ralph; Eiring, Patrick; Walz, Susanne; Jetani, Hardikkumar; Gil-Pulido, Jesus; Da Via, Matteo Claudio; Teufel, Eva; Rhodes, Nadine; Haertle, Larissa; Arellano-Viera, Estibaliz; Tibes, Raoul; Rosenwald, Andreas; Rasche, Leo; Hudecek, Michael; Sauer, Markus; Groll, Jürgen; Einsele, Hermann; Kraus, Sabrina; Kortüm, Martin K. In Communications Biology, 4(1), S. 799-. 2021.
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  The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.

  @article{garitanotrojaola2021actin, abstract = {The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.}, author = {Garitano-Trojaola, Andoni and Sancho, Ana and Götz, Ralph and Eiring, Patrick and Walz, Susanne and Jetani, Hardikkumar and Gil-Pulido, Jesus and Da Via, Matteo Claudio and Teufel, Eva and Rhodes, Nadine and Haertle, Larissa and Arellano-Viera, Estibaliz and Tibes, Raoul and Rosenwald, Andreas and Rasche, Leo and Hudecek, Michael and Sauer, Markus and Groll, Jürgen and Einsele, Hermann and Kraus, Sabrina and Kortüm, Martin K.}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {799–}, title = {Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia}, volume = 4, year = 2021 }

  %0 Journal Article %1 garitanotrojaola2021actin %A Garitano-Trojaola, Andoni %A Sancho, Ana %A Götz, Ralph %A Eiring, Patrick %A Walz, Susanne %A Jetani, Hardikkumar %A Gil-Pulido, Jesus %A Da Via, Matteo Claudio %A Teufel, Eva %A Rhodes, Nadine %A Haertle, Larissa %A Arellano-Viera, Estibaliz %A Tibes, Raoul %A Rosenwald, Andreas %A Rasche, Leo %A Hudecek, Michael %A Sauer, Markus %A Groll, Jürgen %A Einsele, Hermann %A Kraus, Sabrina %A Kortüm, Martin K. %D 2021 %J Communications Biology %N 1 %P 799-- %R 10.1038/s42003-021-02215-w %T Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia %U https://doi.org/10.1038/s42003-021-02215-w %V 4 %X The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.
• Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis. Lehmann, Julian; Jørgensen, Morten E.; Fratz, Stefanie; Müller, Heike M.; Kusch, Jana; Scherzer, Sönke; Navarro-Retamal, Carlos; Mayer, Dominik; Böhm, Jennifer; Konrad, Kai R.; Terpitz, Ulrich; Dreyer, Ingo; Mueller, Thomas D.; Sauer, Markus; Hedrich, Rainer; Geiger, Dietmar; Maierhofer, Tobias. In Current Biology, 31(16), S. 3575–3585.e9. 2021.
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  Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

  @article{lehmann2021acidosisinduced, abstract = {Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.}, author = {Lehmann, Julian and Jørgensen, Morten E. and Fratz, Stefanie and Müller, Heike M. and Kusch, Jana and Scherzer, Sönke and Navarro-Retamal, Carlos and Mayer, Dominik and Böhm, Jennifer and Konrad, Kai R. and Terpitz, Ulrich and Dreyer, Ingo and Mueller, Thomas D. and Sauer, Markus and Hedrich, Rainer and Geiger, Dietmar and Maierhofer, Tobias}, journal = {Current Biology}, keywords = {terpitz}, number = 16, pages = {3575–3585.e9}, title = {Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis}, volume = 31, year = 2021 }

  %0 Journal Article %1 lehmann2021acidosisinduced %A Lehmann, Julian %A Jørgensen, Morten E. %A Fratz, Stefanie %A Müller, Heike M. %A Kusch, Jana %A Scherzer, Sönke %A Navarro-Retamal, Carlos %A Mayer, Dominik %A Böhm, Jennifer %A Konrad, Kai R. %A Terpitz, Ulrich %A Dreyer, Ingo %A Mueller, Thomas D. %A Sauer, Markus %A Hedrich, Rainer %A Geiger, Dietmar %A Maierhofer, Tobias %D 2021 %J Current Biology %N 16 %P 3575--3585.e9 %R https://doi.org/10.1016/j.cub.2021.06.018 %T Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis %U https://www.sciencedirect.com/science/article/pii/S0960982221008137 %V 31 %X Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.
• Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain. Beliu, Gerti; Altrichter, Steffen; Guixà-González, Ramon; Hemberger, Mareike; Brauer, Ina; Dahse, Anne-Kristin; Scholz, Nicole; Wieduwild, Robert; Kuhlemann, Alexander; Batebi, Hossein; Seufert, Florian; Pérez-Hernández, Guillermo; Hildebrand, Peter W.; Sauer, Markus; Langenhan, Tobias. In Molecular Cell, 81(5), S. 905–921.e5. 2021.
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  Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.

  @article{beliu2021tethered, abstract = {Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.}, author = {Beliu, Gerti and Altrichter, Steffen and Guixà-González, Ramon and Hemberger, Mareike and Brauer, Ina and Dahse, Anne-Kristin and Scholz, Nicole and Wieduwild, Robert and Kuhlemann, Alexander and Batebi, Hossein and Seufert, Florian and Pérez-Hernández, Guillermo and Hildebrand, Peter W. and Sauer, Markus and Langenhan, Tobias}, journal = {Molecular Cell}, keywords = {sauer}, number = 5, pages = {905–921.e5}, title = {Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain}, volume = 81, year = 2021 }

  %0 Journal Article %1 beliu2021tethered %A Beliu, Gerti %A Altrichter, Steffen %A Guixà-González, Ramon %A Hemberger, Mareike %A Brauer, Ina %A Dahse, Anne-Kristin %A Scholz, Nicole %A Wieduwild, Robert %A Kuhlemann, Alexander %A Batebi, Hossein %A Seufert, Florian %A Pérez-Hernández, Guillermo %A Hildebrand, Peter W. %A Sauer, Markus %A Langenhan, Tobias %D 2021 %J Molecular Cell %N 5 %P 905--921.e5 %R https://doi.org/10.1016/j.molcel.2020.12.042 %T Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain %U https://www.sciencedirect.com/science/article/pii/S1097276520309631 %V 81 %X Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.
• Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates. Panzer, Sabine; Zhang, Chong; Konte, Tilen; Bräuer, Celine; Diemar, Anne; Yogendran, Parathy; Yu-Strzelczyk, Jing; Nagel, Georg; Gao, Shiqiang; Terpitz, Ulrich. In Frontiers in Molecular Biosciences, 8. 2021.
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  Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

  @article{panzer2021modified, abstract = {Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.}, author = {Panzer, Sabine and Zhang, Chong and Konte, Tilen and Bräuer, Celine and Diemar, Anne and Yogendran, Parathy and Yu-Strzelczyk, Jing and Nagel, Georg and Gao, Shiqiang and Terpitz, Ulrich}, journal = {Frontiers in Molecular Biosciences}, keywords = {terpitz}, series = 941, title = {Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates}, volume = 8, year = 2021 }

  %0 Journal Article %1 panzer2021modified %A Panzer, Sabine %A Zhang, Chong %A Konte, Tilen %A Bräuer, Celine %A Diemar, Anne %A Yogendran, Parathy %A Yu-Strzelczyk, Jing %A Nagel, Georg %A Gao, Shiqiang %A Terpitz, Ulrich %B 941 %D 2021 %J Frontiers in Molecular Biosciences %T Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates %U https://www.frontiersin.org/article/10.3389/fmolb.2021.750528 %V 8 %X Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.
• Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains. Rajab, Suhaila; Bismin, Leah; Schwarze, Simone; Pinggera, Alexandra; Greger, Ingo H.; Neuweiler, Hannes. In Communications Biology, 4(1), S. 1056-. 2021.
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  Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.

  @article{rajab2021allosteric, abstract = {Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.}, author = {Rajab, Suhaila and Bismin, Leah and Schwarze, Simone and Pinggera, Alexandra and Greger, Ingo H. and Neuweiler, Hannes}, journal = {Communications Biology}, keywords = {hannes}, number = 1, pages = {1056–}, title = {Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains}, volume = 4, year = 2021 }

  %0 Journal Article %1 rajab2021allosteric %A Rajab, Suhaila %A Bismin, Leah %A Schwarze, Simone %A Pinggera, Alexandra %A Greger, Ingo H. %A Neuweiler, Hannes %D 2021 %J Communications Biology %N 1 %P 1056-- %R 10.1038/s42003-021-02605-0 %T Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains %U https://doi.org/10.1038/s42003-021-02605-0 %V 4 %X Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.
• Photoblueing of organic dyes can cause artifacts in super-resolution microscopy. Helmerich, Dominic A.; Beliu, Gerti; Matikonda, Siddharth S.; Schnermann, Martin J.; Sauer, Markus. In Nature Methods, 18(3), S. 253–257. 2021.
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  Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.

  @article{helmerich2021photoblueing, abstract = {Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Matikonda, Siddharth S. and Schnermann, Martin J. and Sauer, Markus}, journal = {Nature Methods}, keywords = {sauer}, number = 3, pages = {253–257}, title = {Photoblueing of organic dyes can cause artifacts in super-resolution microscopy}, volume = 18, year = 2021 }

  %0 Journal Article %1 helmerich2021photoblueing %A Helmerich, Dominic A. %A Beliu, Gerti %A Matikonda, Siddharth S. %A Schnermann, Martin J. %A Sauer, Markus %D 2021 %J Nature Methods %N 3 %P 253--257 %R 10.1038/s41592-021-01061-2 %T Photoblueing of organic dyes can cause artifacts in super-resolution microscopy %U https://doi.org/10.1038/s41592-021-01061-2 %V 18 %X Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.
• Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures. Zwettler, Fabian U.; Reinhard, Sebastian; Sauer, Markus. In Expansion Microscopy for Cell Biology, Bd. 161, P. Guichard, V. Hamel (Hrsg.), S. 317–340. Academic Press, 2021.
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  This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.

  @inbook{zwettler2021chapter, abstract = {This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.}, author = {Zwettler, Fabian U. and Reinhard, Sebastian and Sauer, Markus}, booktitle = {Expansion Microscopy for Cell Biology}, editor = {Guichard, Paul and Hamel, Virginie}, keywords = {sauer}, pages = {317–340}, publisher = {Academic Press}, title = {Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures}, volume = 161, year = 2021 }

  %0 Book Section %1 zwettler2021chapter %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Sauer, Markus %B Expansion Microscopy for Cell Biology %D 2021 %E Guichard, Paul %E Hamel, Virginie %I Academic Press %P 317--340 %R https://doi.org/10.1016/bs.mcb.2020.05.004 %T Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures %U https://www.sciencedirect.com/science/article/pii/S0091679X20301126 %V 161 %X This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.
• Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis. Brych, Annika; Haas, Fabian B.; Parzefall, Katharina; Panzer, Sabine; Schermuly, Jeanette; Altmüller, Janine; Engelsdorf, Timo; Terpitz, Ulrich; Rensing, Stefan A.; Kiontke, Stephan; Batschauer, Alfred. In Fungal Genetics and Biology, S. 103570-. 2021.
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  Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.

  @article{brych2021coregulation, abstract = {Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.}, author = {Brych, Annika and Haas, Fabian B. and Parzefall, Katharina and Panzer, Sabine and Schermuly, Jeanette and Altmüller, Janine and Engelsdorf, Timo and Terpitz, Ulrich and Rensing, Stefan A. and Kiontke, Stephan and Batschauer, Alfred}, journal = {Fungal Genetics and Biology}, keywords = {terpitz}, pages = {103570–}, title = {Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis}, year = 2021 }

  %0 Journal Article %1 brych2021coregulation %A Brych, Annika %A Haas, Fabian B. %A Parzefall, Katharina %A Panzer, Sabine %A Schermuly, Jeanette %A Altmüller, Janine %A Engelsdorf, Timo %A Terpitz, Ulrich %A Rensing, Stefan A. %A Kiontke, Stephan %A Batschauer, Alfred %D 2021 %J Fungal Genetics and Biology %P 103570-- %R https://doi.org/10.1016/j.fgb.2021.103570 %T Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis %U https://www.sciencedirect.com/science/article/pii/S1087184521000542 %X Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes.
• Active zone compaction correlates with presynaptic homeostatic potentiation. Mrestani, Achmed; Pauli, Martin; Kollmannsberger, Philip; Repp, Felix; Kittel, Robert J.; Eilers, Jens; Doose, Sören; Sauer, Markus; Sirén, Anna-Leena; Heckmann, Manfred; Paul, Mila M. In Cell Reports, 37(1), S. 109770-. 2021.
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  Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.

  @article{mrestani2021active, abstract = {Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.}, author = {Mrestani, Achmed and Pauli, Martin and Kollmannsberger, Philip and Repp, Felix and Kittel, Robert J. and Eilers, Jens and Doose, Sören and Sauer, Markus and Sirén, Anna-Leena and Heckmann, Manfred and Paul, Mila M.}, journal = {Cell Reports}, keywords = {sauer}, number = 1, pages = {109770–}, title = {Active zone compaction correlates with presynaptic homeostatic potentiation}, volume = 37, year = 2021 }

  %0 Journal Article %1 mrestani2021active %A Mrestani, Achmed %A Pauli, Martin %A Kollmannsberger, Philip %A Repp, Felix %A Kittel, Robert J. %A Eilers, Jens %A Doose, Sören %A Sauer, Markus %A Sirén, Anna-Leena %A Heckmann, Manfred %A Paul, Mila M. %D 2021 %J Cell Reports %N 1 %P 109770-- %R https://doi.org/10.1016/j.celrep.2021.109770 %T Active zone compaction correlates with presynaptic homeostatic potentiation %U https://www.sciencedirect.com/science/article/pii/S2211124721012249 %V 37 %X Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.
• RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes. Heib, Tobias; Hermanns, Heike M.; Manukjan, Georgi; Englert, Maximilian; Kusch, Charly; Becker, Isabelle Carlotta; Gerber, Annika; Wackerbarth, Lou Martha; Burkard, Philipp; Dandekar, Thomas; Balkenhol, Johannes; Jahn, Daniel; Beck, Sarah; Meub, Mara; Dütting, Sebastian; Stigloher, Christian; Sauer, Markus; Cherpokova, Deya; Schulze, Harald; Brakebusch, Cord; Nieswandt, Bernhard; Nagy, Zoltan; Pleines, Irina. In Cell Reports, 35(6), S. 109102-. 2021.
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  Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.

  @article{heib2021rhoacdc42, abstract = {Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.}, author = {Heib, Tobias and Hermanns, Heike M. and Manukjan, Georgi and Englert, Maximilian and Kusch, Charly and Becker, Isabelle Carlotta and Gerber, Annika and Wackerbarth, Lou Martha and Burkard, Philipp and Dandekar, Thomas and Balkenhol, Johannes and Jahn, Daniel and Beck, Sarah and Meub, Mara and Dütting, Sebastian and Stigloher, Christian and Sauer, Markus and Cherpokova, Deya and Schulze, Harald and Brakebusch, Cord and Nieswandt, Bernhard and Nagy, Zoltan and Pleines, Irina}, journal = {Cell Reports}, keywords = {sauer}, number = 6, pages = {109102–}, title = {RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes}, volume = 35, year = 2021 }

  %0 Journal Article %1 heib2021rhoacdc42 %A Heib, Tobias %A Hermanns, Heike M. %A Manukjan, Georgi %A Englert, Maximilian %A Kusch, Charly %A Becker, Isabelle Carlotta %A Gerber, Annika %A Wackerbarth, Lou Martha %A Burkard, Philipp %A Dandekar, Thomas %A Balkenhol, Johannes %A Jahn, Daniel %A Beck, Sarah %A Meub, Mara %A Dütting, Sebastian %A Stigloher, Christian %A Sauer, Markus %A Cherpokova, Deya %A Schulze, Harald %A Brakebusch, Cord %A Nieswandt, Bernhard %A Nagy, Zoltan %A Pleines, Irina %D 2021 %J Cell Reports %N 6 %P 109102-- %R https://doi.org/10.1016/j.celrep.2021.109102 %T RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes %U https://www.sciencedirect.com/science/article/pii/S2211124721004368 %V 35 %X Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.
• Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy. Thiele, Jan Christoph; Nevskyi, Oleksii; Helmerich, Dominic A.; Sauer, Markus; Enderlein, Jörg. In Frontiers in Bioinformatics, 1. 2021.
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  Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package “Fluorescence-Lifetime TrackNTrace,” which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.

  @article{thiele2021advanced, abstract = {Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package “Fluorescence-Lifetime TrackNTrace,” which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.}, author = {Thiele, Jan Christoph and Nevskyi, Oleksii and Helmerich, Dominic A. and Sauer, Markus and Enderlein, Jörg}, journal = {Frontiers in Bioinformatics}, keywords = {sauer}, title = {Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy}, volume = 1, year = 2021 }

  %0 Journal Article %1 thiele2021advanced %A Thiele, Jan Christoph %A Nevskyi, Oleksii %A Helmerich, Dominic A. %A Sauer, Markus %A Enderlein, Jörg %D 2021 %J Frontiers in Bioinformatics %T Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy %U https://www.frontiersin.org/journals/bioinformatics/articles/10.3389/fbinf.2021.740281 %V 1 %X Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package “Fluorescence-Lifetime TrackNTrace,” which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.
• Expanding GABAAR pharmacology via receptor-associated proteins. Schulte, Clemens; Maric, Hans Michael. In Current Opinion in Pharmacology, 57, S. 98–106. 2021.
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  Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.

  @article{schulte2021expanding, abstract = {Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.}, author = {Schulte, Clemens and Maric, Hans Michael}, journal = {Current Opinion in Pharmacology}, keywords = {maric}, pages = {98–106}, title = {Expanding GABAAR pharmacology via receptor-associated proteins}, volume = 57, year = 2021 }

  %0 Journal Article %1 schulte2021expanding %A Schulte, Clemens %A Maric, Hans Michael %D 2021 %J Current Opinion in Pharmacology %P 98--106 %R https://doi.org/10.1016/j.coph.2021.01.004 %T Expanding GABAAR pharmacology via receptor-associated proteins %U https://www.sciencedirect.com/science/article/pii/S1471489221000059 %V 57 %X Drugs directly targeting γ-aminobutyric acid type A receptors (GABAARs), the major mediators of fast synaptic inhibition, contribute significantly to today's neuropharmacology. Emerging evidence establishes intracellularly GABAAR-associated proteins as the central players in determining cellular and subcellular GABAergic input sites, thereby providing pharmacological opportunities to affect distinct receptor populations and address discrete neuronal dysfunctions. Here, we report on recently studied GABAAR-associated proteins and highlight challenges and newly available methods for their functional and physical mapping. We anticipate these efforts to contribute to decipher the complexity of GABAergic signalling in the brain and eventually enable therapeutic avenues for, so far, untreatable neuronal disorders and diseases.
• Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons. Bessa-Neto, Diogo; Beliu, Gerti; Kuhlemann, Alexander; Pecoraro, Valeria; Doose, Sören; Retailleau, Natacha; Chevrier, Nicolas; Perrais, David; Sauer, Markus; Choquet, Daniel. In Nature Communications, 12(1), S. 6715-. 2021.
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  Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

  @article{bessaneto2021bioorthogonal, abstract = {Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.}, author = {Bessa-Neto, Diogo and Beliu, Gerti and Kuhlemann, Alexander and Pecoraro, Valeria and Doose, Sören and Retailleau, Natacha and Chevrier, Nicolas and Perrais, David and Sauer, Markus and Choquet, Daniel}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {6715–}, title = {Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons}, volume = 12, year = 2021 }

  %0 Journal Article %1 bessaneto2021bioorthogonal %A Bessa-Neto, Diogo %A Beliu, Gerti %A Kuhlemann, Alexander %A Pecoraro, Valeria %A Doose, Sören %A Retailleau, Natacha %A Chevrier, Nicolas %A Perrais, David %A Sauer, Markus %A Choquet, Daniel %D 2021 %J Nature Communications %N 1 %P 6715-- %R 10.1038/s41467-021-27025-w %T Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons %U https://doi.org/10.1038/s41467-021-27025-w %V 12 %X Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.
• Single-molecule localization microscopy. Lelek, Mickaël; Gyparaki, Melina T.; Beliu, Gerti; Schueder, Florian; Griffié, Juliette; Manley, Suliana; Jungmann, Ralf; Sauer, Markus; Lakadamyali, Melike; Zimmer, Christophe. In Nature Reviews Methods Primers, 1(1), S. 39-. 2021.
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  Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.

  @article{lelek2021singlemolecule, abstract = {Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.}, author = {Lelek, Mickaël and Gyparaki, Melina T. and Beliu, Gerti and Schueder, Florian and Griffié, Juliette and Manley, Suliana and Jungmann, Ralf and Sauer, Markus and Lakadamyali, Melike and Zimmer, Christophe}, journal = {Nature Reviews Methods Primers}, keywords = {sauer}, number = 1, pages = {39–}, title = {Single-molecule localization microscopy}, volume = 1, year = 2021 }

  %0 Journal Article %1 lelek2021singlemolecule %A Lelek, Mickaël %A Gyparaki, Melina T. %A Beliu, Gerti %A Schueder, Florian %A Griffié, Juliette %A Manley, Suliana %A Jungmann, Ralf %A Sauer, Markus %A Lakadamyali, Melike %A Zimmer, Christophe %D 2021 %J Nature Reviews Methods Primers %N 1 %P 39-- %R 10.1038/s43586-021-00038-x %T Single-molecule localization microscopy %U https://doi.org/10.1038/s43586-021-00038-x %V 1 %X Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM.
• Anti–contactin-1 Antibodies Affect Surface Expression and Sodium Currents in Dorsal Root Ganglia. Grüner, Julia; Stengel, Helena; Werner, Christian; Appeltshauser, Luise; Sommer, Claudia; Villmann, Carmen; Doppler, Kathrin. In Neurology Neuroimmunology &amp; Neuroinflammation, 8(5), S. e1056-. Wolters Kluwer, 2021.
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  Background and Objectives: As autoantibodies to contactin-1 from patients with chronic inflammatory demyelinating polyradiculoneuropathy not only bind to the paranodes where they are supposed to cause conduction failure but also bind to other neuronal cell types, we aimed to investigate the effect of anti–contactin-1 autoantibodies on contactin-1 surface expression in cerebellar granule neurons, dorsal root ganglion neurons, and contactin-1–transfected human embryonic kidney 293 cells. Methods: Immunocytochemistry including structured illumination microscopy and immunoblotting was used to determine expression levels of contactin-1 and/or sodium channels after long-term exposure to autoantibodies from 3 seropositive patients. For functional analysis of sodium channels, whole-cell recordings of sodium currents were performed on dorsal root ganglion neurons incubated with anti–contactin-1 autoantibodies. Results: We found a reduction in contactin-1 expression levels on dorsal root ganglion neurons, cerebellar granule neurons, and contactin-1–transfected human embryonic kidney 293 cells and decreased dorsal root ganglion sodium currents after long-term exposure to anti–contactin-1 autoantibodies. Sodium channel density did not decrease. Discussion: Our results demonstrate a direct effect of anti–contactin-1 autoantibodies on the surface expression of contactin-1 and sodium currents in dorsal root ganglion neurons. This may be the pathophysiologic correlate of sensory ataxia reported in these patients.

  @article{gruneranticontactin1, abstract = {Background and Objectives: As autoantibodies to contactin-1 from patients with chronic inflammatory demyelinating polyradiculoneuropathy not only bind to the paranodes where they are supposed to cause conduction failure but also bind to other neuronal cell types, we aimed to investigate the effect of anti–contactin-1 autoantibodies on contactin-1 surface expression in cerebellar granule neurons, dorsal root ganglion neurons, and contactin-1–transfected human embryonic kidney 293 cells. Methods: Immunocytochemistry including structured illumination microscopy and immunoblotting was used to determine expression levels of contactin-1 and/or sodium channels after long-term exposure to autoantibodies from 3 seropositive patients. For functional analysis of sodium channels, whole-cell recordings of sodium currents were performed on dorsal root ganglion neurons incubated with anti–contactin-1 autoantibodies. Results: We found a reduction in contactin-1 expression levels on dorsal root ganglion neurons, cerebellar granule neurons, and contactin-1–transfected human embryonic kidney 293 cells and decreased dorsal root ganglion sodium currents after long-term exposure to anti–contactin-1 autoantibodies. Sodium channel density did not decrease. Discussion: Our results demonstrate a direct effect of anti–contactin-1 autoantibodies on the surface expression of contactin-1 and sodium currents in dorsal root ganglion neurons. This may be the pathophysiologic correlate of sensory ataxia reported in these patients.}, author = {Grüner, Julia and Stengel, Helena and Werner, Christian and Appeltshauser, Luise and Sommer, Claudia and Villmann, Carmen and Doppler, Kathrin}, booktitle = {Neurology Neuroimmunology & Neuroinflammation}, journal = {Neurology Neuroimmunology & Neuroinflammation}, keywords = {werner}, number = 5, pages = {e1056–}, publisher = {Wolters Kluwer}, title = {Anti–contactin-1 Antibodies Affect Surface Expression and Sodium Currents in Dorsal Root Ganglia}, volume = 8, year = 2021 }

  %0 Journal Article %1 gruneranticontactin1 %A Grüner, Julia %A Stengel, Helena %A Werner, Christian %A Appeltshauser, Luise %A Sommer, Claudia %A Villmann, Carmen %A Doppler, Kathrin %B Neurology Neuroimmunology & Neuroinflammation %D 2021 %I Wolters Kluwer %J Neurology Neuroimmunology & Neuroinflammation %N 5 %P e1056-- %R 10.1212/NXI.0000000000001056 %T Anti–contactin-1 Antibodies Affect Surface Expression and Sodium Currents in Dorsal Root Ganglia %U https://doi.org/10.1212/NXI.0000000000001056 %V 8 %X Background and Objectives: As autoantibodies to contactin-1 from patients with chronic inflammatory demyelinating polyradiculoneuropathy not only bind to the paranodes where they are supposed to cause conduction failure but also bind to other neuronal cell types, we aimed to investigate the effect of anti–contactin-1 autoantibodies on contactin-1 surface expression in cerebellar granule neurons, dorsal root ganglion neurons, and contactin-1–transfected human embryonic kidney 293 cells. Methods: Immunocytochemistry including structured illumination microscopy and immunoblotting was used to determine expression levels of contactin-1 and/or sodium channels after long-term exposure to autoantibodies from 3 seropositive patients. For functional analysis of sodium channels, whole-cell recordings of sodium currents were performed on dorsal root ganglion neurons incubated with anti–contactin-1 autoantibodies. Results: We found a reduction in contactin-1 expression levels on dorsal root ganglion neurons, cerebellar granule neurons, and contactin-1–transfected human embryonic kidney 293 cells and decreased dorsal root ganglion sodium currents after long-term exposure to anti–contactin-1 autoantibodies. Sodium channel density did not decrease. Discussion: Our results demonstrate a direct effect of anti–contactin-1 autoantibodies on the surface expression of contactin-1 and sodium currents in dorsal root ganglion neurons. This may be the pathophysiologic correlate of sensory ataxia reported in these patients.
• Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms. Yu, Yidong; Wolf, Ann-Katrin; Thusek, Sina; Heinekamp, Thorsten; Bromley, Michael; Krappmann, Sven; Terpitz, Ulrich; Voigt, Kerstin; Brakhage, Axel A.; Beilhack, Andreas. 2021.
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  Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.

  @misc{yu2021direct, abstract = {Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.}, author = {Yu, Yidong and Wolf, Ann-Katrin and Thusek, Sina and Heinekamp, Thorsten and Bromley, Michael and Krappmann, Sven and Terpitz, Ulrich and Voigt, Kerstin and Brakhage, Axel A. and Beilhack, Andreas}, booktitle = {Journal of Fungi}, keywords = {terpitz}, number = 2, title = {Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms}, volume = 7, year = 2021 }

  %0 Generic %1 yu2021direct %A Yu, Yidong %A Wolf, Ann-Katrin %A Thusek, Sina %A Heinekamp, Thorsten %A Bromley, Michael %A Krappmann, Sven %A Terpitz, Ulrich %A Voigt, Kerstin %A Brakhage, Axel A. %A Beilhack, Andreas %B Journal of Fungi %D 2021 %N 2 %R 10.3390/jof7020136 %T Direct Visualization of Fungal Burden in Filamentous Fungus-Infected Silkworms %V 7 %X Invasive fungal infections (IFIs) are difficult to diagnose and to treat and, despite several available antifungal drugs, cause high mortality rates. In the past decades, the incidence of IFIs has continuously increased. More recently, SARS-CoV-2-associated lethal IFIs have been reported worldwide in critically ill patients. Combating IFIs requires a more profound understanding of fungal pathogenicity to facilitate the development of novel antifungal strategies. Animal models are indispensable for studying fungal infections and to develop new antifungals. However, using mammalian animal models faces various hurdles including ethical issues and high costs, which makes large-scale infection experiments extremely challenging. To overcome these limitations, we optimized an invertebrate model and introduced a simple calcofluor white (CW) staining protocol to macroscopically and microscopically monitor disease progression in silkworms (Bombyx mori) infected with the human pathogenic filamentous fungi Aspergillus fumigatus and Lichtheimia corymbifera. This advanced silkworm A. fumigatus infection model could validate knockout mutants with either attenuated, strongly attenuated or unchanged virulence. Finally, CW staining allowed us to efficiently visualize antifungal treatment outcomes in infected silkworms. Conclusively, we here present a powerful animal model combined with a straightforward staining protocol to expedite large-scale in vivo research of fungal pathogenicity and to investigate novel antifungal candidates.
• High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale. Schulte, Clemens; Khayenko, Vladimir; Nordblom, Noah Frieder; Tippel, Franziska; Peck, Violetta; Gupta, Amit Jean; Maric, Hans Michael. In iScience, 24(1), S. 101898-. 2021.
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  Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.

  @article{schulte2021highthroughput, abstract = {Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.}, author = {Schulte, Clemens and Khayenko, Vladimir and Nordblom, Noah Frieder and Tippel, Franziska and Peck, Violetta and Gupta, Amit Jean and Maric, Hans Michael}, journal = {iScience}, keywords = {maric}, number = 1, pages = {101898–}, title = {High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale}, volume = 24, year = 2021 }

  %0 Journal Article %1 schulte2021highthroughput %A Schulte, Clemens %A Khayenko, Vladimir %A Nordblom, Noah Frieder %A Tippel, Franziska %A Peck, Violetta %A Gupta, Amit Jean %A Maric, Hans Michael %D 2021 %J iScience %N 1 %P 101898-- %R https://doi.org/10.1016/j.isci.2020.101898 %T High-throughput determination of protein affinities using unmodified peptide libraries in nanomolar scale %U https://www.sciencedirect.com/science/article/pii/S2589004220310956 %V 24 %X Protein-protein interactions (PPIs) are of fundamental importance for our understanding of physiology and pathology. PPIs involving short, linear motifs play a major role in immunological recognition, signaling, and regulation and provide attractive starting points for pharmaceutical intervention. Yet, state-of-the-art protein-peptide affinity determination approaches exhibit limited throughput and sensitivity, often resulting from ligand immobilization, labeling, or synthesis. Here, we introduce a high-throughput method for in-solution analysis of protein-peptide interactions using a phenomenon called temperature related intensity change (TRIC). We use TRIC for the identification and fine-mapping of low- and high-affinity protein interaction sites and the definition of sequence binding requirements. Validation is achieved by microarray-based studies using wild-type and mutated recombinant protein and the native protein within tissue lysates. On-chip neutralization and strong correlation with structural data establish TRIC as a quasi-label-free method to determine binding affinities of unmodified peptide libraries with large dynamic range.
• A role for TASK2 channels in the human immunological synapse. Fernández-Orth, Juncal; Rolfes, Leoni; Gola, Lukas; Bittner, Stefan; Andronic, Joseph; Sukhorukov, Vladimir L.; Sisario, Dmitri; Landgraf, Peter; Dieterich, Daniela C.; Cerina, Manuela; Smalla, Karl-Heinz; Kähne, Thilo; Budde, Thomas; Kovac, Stjepana; Ruck, Tobias; Sauer, Markus; Meuth, Sven G. In European Journal of Immunology, 51(2), S. 342–353. John Wiley & Sons, Ltd, 2021.
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  The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.

  @article{fernandezorth2021task2, abstract = {The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.}, author = {Fernández-Orth, Juncal and Rolfes, Leoni and Gola, Lukas and Bittner, Stefan and Andronic, Joseph and Sukhorukov, Vladimir L. and Sisario, Dmitri and Landgraf, Peter and Dieterich, Daniela C. and Cerina, Manuela and Smalla, Karl-Heinz and Kähne, Thilo and Budde, Thomas and Kovac, Stjepana and Ruck, Tobias and Sauer, Markus and Meuth, Sven G.}, booktitle = {European Journal of Immunology}, journal = {European Journal of Immunology}, keywords = {sauer}, month = {02}, number = 2, pages = {342–353}, publisher = {John Wiley & Sons, Ltd}, title = {A role for TASK2 channels in the human immunological synapse}, volume = 51, year = 2021 }

  %0 Journal Article %1 fernandezorth2021task2 %A Fernández-Orth, Juncal %A Rolfes, Leoni %A Gola, Lukas %A Bittner, Stefan %A Andronic, Joseph %A Sukhorukov, Vladimir L. %A Sisario, Dmitri %A Landgraf, Peter %A Dieterich, Daniela C. %A Cerina, Manuela %A Smalla, Karl-Heinz %A Kähne, Thilo %A Budde, Thomas %A Kovac, Stjepana %A Ruck, Tobias %A Sauer, Markus %A Meuth, Sven G. %B European Journal of Immunology %D 2021 %I John Wiley & Sons, Ltd %J European Journal of Immunology %N 2 %P 342--353 %R https://doi.org/10.1002/eji.201948269 %T A role for TASK2 channels in the human immunological synapse %U https://doi.org/10.1002/eji.201948269 %V 51 %X The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.
• Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria. Peters, Simon; Kaiser, Lena; Fink, Julian; Schumacher, Fabian; Perschin, Veronika; Schlegel, Jan; Sauer, Markus; Stigloher, Christian; Kleuser, Burkhard; Seibel, Jürgen; Schubert-Unkmeir, Alexandra. In Scientific Reports, 11(1), S. 4300-. 2021.
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  Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.

  @article{peters2021clickcorrelative, abstract = {Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.}, author = {Peters, Simon and Kaiser, Lena and Fink, Julian and Schumacher, Fabian and Perschin, Veronika and Schlegel, Jan and Sauer, Markus and Stigloher, Christian and Kleuser, Burkhard and Seibel, Jürgen and Schubert-Unkmeir, Alexandra}, journal = {Scientific Reports}, keywords = {sauer}, number = 1, pages = {4300–}, title = {Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria}, volume = 11, year = 2021 }

  %0 Journal Article %1 peters2021clickcorrelative %A Peters, Simon %A Kaiser, Lena %A Fink, Julian %A Schumacher, Fabian %A Perschin, Veronika %A Schlegel, Jan %A Sauer, Markus %A Stigloher, Christian %A Kleuser, Burkhard %A Seibel, Jürgen %A Schubert-Unkmeir, Alexandra %D 2021 %J Scientific Reports %N 1 %P 4300-- %R 10.1038/s41598-021-83813-w %T Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria %U https://doi.org/10.1038/s41598-021-83813-w %V 11 %X Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
• The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes. Hassan, Mohamed I. Abdelwahab; Keller, Monique; Hillger, Michael; Binder, Ulrike; Reuter, Stefanie; Herold, Kristina; Telagathoti, Anusha; Dahse, Hans-Martin; Wicht, Saiedeh; Trinks, Nora; Nietzsche, Sandor; Deckert-Gaudig, Tanja; Deckert, Volker; Mrowka, Ralf; Terpitz, Ulrich; {Peter Saluz}, Hans; Voigt, Kerstin. In Computational and Structural Biotechnology Journal, 19, S. 880–896. 2021.
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  Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.

  @article{HASSAN2021880, abstract = {Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.}, author = {Hassan, Mohamed I. Abdelwahab and Keller, Monique and Hillger, Michael and Binder, Ulrike and Reuter, Stefanie and Herold, Kristina and Telagathoti, Anusha and Dahse, Hans-Martin and Wicht, Saiedeh and Trinks, Nora and Nietzsche, Sandor and Deckert-Gaudig, Tanja and Deckert, Volker and Mrowka, Ralf and Terpitz, Ulrich and {Peter Saluz}, Hans and Voigt, Kerstin}, journal = {Computational and Structural Biotechnology Journal}, keywords = {terpitz}, pages = {880 - 896}, title = {The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes}, volume = 19, year = 2021 }

  %0 Journal Article %1 HASSAN2021880 %A Hassan, Mohamed I. Abdelwahab %A Keller, Monique %A Hillger, Michael %A Binder, Ulrike %A Reuter, Stefanie %A Herold, Kristina %A Telagathoti, Anusha %A Dahse, Hans-Martin %A Wicht, Saiedeh %A Trinks, Nora %A Nietzsche, Sandor %A Deckert-Gaudig, Tanja %A Deckert, Volker %A Mrowka, Ralf %A Terpitz, Ulrich %A {Peter Saluz}, Hans %A Voigt, Kerstin %D 2021 %J Computational and Structural Biotechnology Journal %P 880 - 896 %R https://doi.org/10.1016/j.csbj.2021.01.023 %T The impact of episporic modification of Lichtheimia corymbifera on virulence and interaction with phagocytes %U https://doi.org/10.1016/j.csbj.2021.01.023 %V 19 %X Fungal infections caused by the ancient lineage Mucorales are emerging and increasingly reported in humans. Comprehensive surveys on promising attributes from a multitude of possible virulence factors are limited and so far, focused on Mucor and Rhizopus. This study addresses a systematic approach to monitor phagocytosis after physical and enzymatic modification of the outer spore wall of Lichtheimia corymbifera, one of the major causative agents of mucormycosis. Episporic modifications were performed and their consequences on phagocytosis, intracellular survival and virulence by murine alveolar macrophages and in an invertebrate infection model were elucidated. While depletion of lipids did not affect the phagocytosis of both strains, delipidation led to attenuation of LCA strain but appears to be dispensable for infection with LCV strain in the settings used in this study. Combined glucano-proteolytic treatment was necessary to achieve a significant decrease of virulence of the LCV strain in Galleria mellonella during maintenance of the full potential for spore germination as shown by a novel automated germination assay. Proteolytic and glucanolytic treatments largely increased phagocytosis compared to alive resting and swollen spores. Whilst resting spores barely (1–2%) fuse to lysosomes after invagination in to phagosomes, spore trypsinization led to a 10-fold increase of phagolysosomal fusion as measured by intracellular acidification. This is the first report of a polyphasic measurement of the consequences of episporic modification of a mucormycotic pathogen in spore germination, spore surface ultrastructure, phagocytosis, stimulation of Toll-like receptors (TLRs), phagolysosomal fusion and intracellular acidification, apoptosis, generation of reactive oxygen species (ROS) and virulence.
• Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly. Jansch, Charline; Ziegler, Georg C.; Forero, Andrea; Gredy, Sina; Wäldchen, Sina; Vitale, Maria Rosaria; Svirin, Evgeniy; Zöller, Johanna E. M.; Waider, Jonas; Günther, Katharina; Edenhofer, Frank; Sauer, Markus; Wischmeyer, Erhard; Lesch, Klaus-Peter. In Journal of Neural Transmission, 128(2), S. 225–241. 2021.
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  Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.

  @article{jansch2021serotoninspecific, abstract = {Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (  42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.}, author = {Jansch, Charline and Ziegler, Georg C. and Forero, Andrea and Gredy, Sina and Wäldchen, Sina and Vitale, Maria Rosaria and Svirin, Evgeniy and Zöller, Johanna E. M. and Waider, Jonas and Günther, Katharina and Edenhofer, Frank and Sauer, Markus and Wischmeyer, Erhard and Lesch, Klaus-Peter}, journal = {Journal of Neural Transmission}, keywords = {sauer}, number = 2, pages = {225–241}, title = {Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly}, volume = 128, year = 2021 }

  %0 Journal Article %1 jansch2021serotoninspecific %A Jansch, Charline %A Ziegler, Georg C. %A Forero, Andrea %A Gredy, Sina %A Wäldchen, Sina %A Vitale, Maria Rosaria %A Svirin, Evgeniy %A Zöller, Johanna E. M. %A Waider, Jonas %A Günther, Katharina %A Edenhofer, Frank %A Sauer, Markus %A Wischmeyer, Erhard %A Lesch, Klaus-Peter %D 2021 %J Journal of Neural Transmission %N 2 %P 225--241 %R 10.1007/s00702-021-02303-5 %T Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly %U https://doi.org/10.1007/s00702-021-02303-5 %V 128 %X Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
• Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia. Jetani, Hardikkumar; Navarro-Bailón, Almudena; Maucher, Marius; Frenz, Silke; Verbruggen, Christina; Yeguas, Ana; Vidriales, María Belén; González, Marcos; Rial Saborido, Judit; Kraus, Sabrina; Mestermann, Katrin; Thomas, Simone; Bonig, Halvard; Luu, Maik; Monjezi, Razieh; Mougiakakos, Dimitrios; Sauer, Markus; Einsele, Hermann; Hudecek, Michael. In Blood, 138(19), S. 1830–1842. 2021.
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  Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.

  @article{jetani2021siglec6, abstract = {Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.}, author = {Jetani, Hardikkumar and Navarro-Bailón, Almudena and Maucher, Marius and Frenz, Silke and Verbruggen, Christina and Yeguas, Ana and Vidriales, María Belén and González, Marcos and Rial Saborido, Judit and Kraus, Sabrina and Mestermann, Katrin and Thomas, Simone and Bonig, Halvard and Luu, Maik and Monjezi, Razieh and Mougiakakos, Dimitrios and Sauer, Markus and Einsele, Hermann and Hudecek, Michael}, journal = {Blood}, keywords = {sauer}, month = 11, number = 19, pages = {1830–1842}, title = {Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia}, volume = 138, year = 2021 }

  %0 Journal Article %1 jetani2021siglec6 %A Jetani, Hardikkumar %A Navarro-Bailón, Almudena %A Maucher, Marius %A Frenz, Silke %A Verbruggen, Christina %A Yeguas, Ana %A Vidriales, María Belén %A González, Marcos %A Rial Saborido, Judit %A Kraus, Sabrina %A Mestermann, Katrin %A Thomas, Simone %A Bonig, Halvard %A Luu, Maik %A Monjezi, Razieh %A Mougiakakos, Dimitrios %A Sauer, Markus %A Einsele, Hermann %A Hudecek, Michael %D 2021 %J Blood %N 19 %P 1830--1842 %R 10.1182/blood.2020009192 %T Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia %U https://doi.org/10.1182/blood.2020009192 %V 138 %X Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.
• Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons. Deng, Chunchu; Moradi, Mehri; Reinhard, Sebastian; Ji, Changhe; Jablonka, Sibylle; Hennlein, Luisa; Lüningschrör, Patrick; Doose, Sören; Sauer, Markus; Sendtner, Michael. In Journal of Cell Science, 134(22), S. jcs258785-. 2021.
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  In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation.This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.

  @article{deng2021dynamic, abstract = {In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation.This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.}, author = {Deng, Chunchu and Moradi, Mehri and Reinhard, Sebastian and Ji, Changhe and Jablonka, Sibylle and Hennlein, Luisa and Lüningschrör, Patrick and Doose, Sören and Sauer, Markus and Sendtner, Michael}, journal = {Journal of Cell Science}, keywords = {sauer}, month = 11, number = 22, pages = {jcs258785–}, title = {Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons}, volume = 134, year = 2021 }

  %0 Journal Article %1 deng2021dynamic %A Deng, Chunchu %A Moradi, Mehri %A Reinhard, Sebastian %A Ji, Changhe %A Jablonka, Sibylle %A Hennlein, Luisa %A Lüningschrör, Patrick %A Doose, Sören %A Sauer, Markus %A Sendtner, Michael %D 2021 %J Journal of Cell Science %N 22 %P jcs258785-- %R 10.1242/jcs.258785 %T Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons %U https://doi.org/10.1242/jcs.258785 %V 134 %X In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation.This article has an associated First Person interview with Chunchu Deng, joint first author of the paper.
• Super-resolving Microscopy in Neuroscience. Werner, Christian; Sauer, Markus; Geis, Christian. In Chem. Rev., 121(19), S. 11971–12015. American Chemical Society, 2021.
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  Fluorescence imaging techniques play a pivotal role in our understanding of the nervous system. The emergence of various super-resolution microscopy methods and specialized fluorescent probes enables direct insight into neuronal structure and protein arrangements in cellular subcompartments with so far unmatched resolution. Super-resolving visualization techniques in neurons unveil a novel understanding of cytoskeletal composition, distribution, motility, and signaling of membrane proteins, subsynaptic structure and function, and neuron–glia interaction. Well-defined molecular targets in autoimmune and neurodegenerative disease models provide excellent starting points for in-depth investigation of disease pathophysiology using novel and innovative imaging methodology. Application of super-resolution microscopy in human brain samples and for testing clinical biomarkers is still in its infancy but opens new opportunities for translational research in neurology and neuroscience. In this review, we describe how super-resolving microscopy has improved our understanding of neuronal and brain function and dysfunction in the last two decades.

  @article{werner2021superresolving, abstract = {Fluorescence imaging techniques play a pivotal role in our understanding of the nervous system. The emergence of various super-resolution microscopy methods and specialized fluorescent probes enables direct insight into neuronal structure and protein arrangements in cellular subcompartments with so far unmatched resolution. Super-resolving visualization techniques in neurons unveil a novel understanding of cytoskeletal composition, distribution, motility, and signaling of membrane proteins, subsynaptic structure and function, and neuron–glia interaction. Well-defined molecular targets in autoimmune and neurodegenerative disease models provide excellent starting points for in-depth investigation of disease pathophysiology using novel and innovative imaging methodology. Application of super-resolution microscopy in human brain samples and for testing clinical biomarkers is still in its infancy but opens new opportunities for translational research in neurology and neuroscience. In this review, we describe how super-resolving microscopy has improved our understanding of neuronal and brain function and dysfunction in the last two decades.}, author = {Werner, Christian and Sauer, Markus and Geis, Christian}, booktitle = {Chemical Reviews}, journal = {Chem. Rev.}, keywords = {werner}, month = 10, number = 19, pages = {11971–12015}, publisher = {American Chemical Society}, title = {Super-resolving Microscopy in Neuroscience}, volume = 121, year = 2021 }

  %0 Journal Article %1 werner2021superresolving %A Werner, Christian %A Sauer, Markus %A Geis, Christian %B Chemical Reviews %D 2021 %I American Chemical Society %J Chem. Rev. %N 19 %P 11971--12015 %R 10.1021/acs.chemrev.0c01174 %T Super-resolving Microscopy in Neuroscience %U https://doi.org/10.1021/acs.chemrev.0c01174 %V 121 %X Fluorescence imaging techniques play a pivotal role in our understanding of the nervous system. The emergence of various super-resolution microscopy methods and specialized fluorescent probes enables direct insight into neuronal structure and protein arrangements in cellular subcompartments with so far unmatched resolution. Super-resolving visualization techniques in neurons unveil a novel understanding of cytoskeletal composition, distribution, motility, and signaling of membrane proteins, subsynaptic structure and function, and neuron–glia interaction. Well-defined molecular targets in autoimmune and neurodegenerative disease models provide excellent starting points for in-depth investigation of disease pathophysiology using novel and innovative imaging methodology. Application of super-resolution microscopy in human brain samples and for testing clinical biomarkers is still in its infancy but opens new opportunities for translational research in neurology and neuroscience. In this review, we describe how super-resolving microscopy has improved our understanding of neuronal and brain function and dysfunction in the last two decades.
• Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy. Matikonda, Siddharth S.; Helmerich, Dominic A.; Meub, Mara; Beliu, Gerti; Kollmannsberger, Philip; Greer, Alexander; Sauer, Markus; Schnermann, Martin J. In ACS Cent. Sci., 7(7), S. 1144–1155. American Chemical Society, 2021.
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  The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.

  @article{matikonda2021defining, abstract = {The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.}, author = {Matikonda, Siddharth S. and Helmerich, Dominic A. and Meub, Mara and Beliu, Gerti and Kollmannsberger, Philip and Greer, Alexander and Sauer, Markus and Schnermann, Martin J.}, booktitle = {ACS Central Science}, journal = {ACS Cent. Sci.}, keywords = {sauer}, month = {07}, number = 7, pages = {1144–1155}, publisher = {American Chemical Society}, title = {Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy}, volume = 7, year = 2021 }

  %0 Journal Article %1 matikonda2021defining %A Matikonda, Siddharth S. %A Helmerich, Dominic A. %A Meub, Mara %A Beliu, Gerti %A Kollmannsberger, Philip %A Greer, Alexander %A Sauer, Markus %A Schnermann, Martin J. %B ACS Central Science %D 2021 %I American Chemical Society %J ACS Cent. Sci. %N 7 %P 1144--1155 %R 10.1021/acscentsci.1c00483 %T Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy %U https://doi.org/10.1021/acscentsci.1c00483 %V 7 %X The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multistep sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.
• Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo. Haack, Stephanie; Baiker, Sarah; Schlegel, Jan; Sauer, Markus; Sparwasser, Tim; Langenhorst, Daniela; Beyersdorf, Niklas. In European Journal of Immunology, 51(3), S. 738–741. John Wiley & Sons, Ltd, 2021.
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  Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.

  @article{haack2021superagonistic, abstract = {Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.}, author = {Haack, Stephanie and Baiker, Sarah and Schlegel, Jan and Sauer, Markus and Sparwasser, Tim and Langenhorst, Daniela and Beyersdorf, Niklas}, booktitle = {European Journal of Immunology}, journal = {European Journal of Immunology}, keywords = {sauer}, month = {03}, number = 3, pages = {738–741}, publisher = {John Wiley & Sons, Ltd}, title = {Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo}, volume = 51, year = 2021 }

  %0 Journal Article %1 haack2021superagonistic %A Haack, Stephanie %A Baiker, Sarah %A Schlegel, Jan %A Sauer, Markus %A Sparwasser, Tim %A Langenhorst, Daniela %A Beyersdorf, Niklas %B European Journal of Immunology %D 2021 %I John Wiley & Sons, Ltd %J European Journal of Immunology %N 3 %P 738--741 %R https://doi.org/10.1002/eji.202048803 %T Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo %U https://doi.org/10.1002/eji.202048803 %V 51 %X Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.
• Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications. Eiring, Patrick; McLaughlin, Ryan; Matikonda, Siddharth S.; Han, Zhongying; Grabenhorst, Lennart; Helmerich, Dominic A.; Meub, Mara; Beliu, Gerti; Luciano, Michael; Bandi, Venu; Zijlstra, Niels; Shi, Zhen-Dan; Tarasov, Sergey G.; Swenson, Rolf; Tinnefeld, Philip; Glembockyte, Viktorija; Cordes, Thorben; Sauer, Markus; Schnermann, Martin J. In Angewandte Chemie International Edition, 60(51), S. 26685–26693. John Wiley & Sons, Ltd, 2021.
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  Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

  @article{eiring2021targetable, abstract = {Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.}, author = {Eiring, Patrick and McLaughlin, Ryan and Matikonda, Siddharth S. and Han, Zhongying and Grabenhorst, Lennart and Helmerich, Dominic A. and Meub, Mara and Beliu, Gerti and Luciano, Michael and Bandi, Venu and Zijlstra, Niels and Shi, Zhen-Dan and Tarasov, Sergey G. and Swenson, Rolf and Tinnefeld, Philip and Glembockyte, Viktorija and Cordes, Thorben and Sauer, Markus and Schnermann, Martin J.}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, month = 12, number = 51, pages = {26685–26693}, publisher = {John Wiley & Sons, Ltd}, title = {Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications}, volume = 60, year = 2021 }

  %0 Journal Article %1 eiring2021targetable %A Eiring, Patrick %A McLaughlin, Ryan %A Matikonda, Siddharth S. %A Han, Zhongying %A Grabenhorst, Lennart %A Helmerich, Dominic A. %A Meub, Mara %A Beliu, Gerti %A Luciano, Michael %A Bandi, Venu %A Zijlstra, Niels %A Shi, Zhen-Dan %A Tarasov, Sergey G. %A Swenson, Rolf %A Tinnefeld, Philip %A Glembockyte, Viktorija %A Cordes, Thorben %A Sauer, Markus %A Schnermann, Martin J. %B Angewandte Chemie International Edition %D 2021 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 51 %P 26685--26693 %R https://doi.org/10.1002/anie.202109749 %T Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications %U https://doi.org/10.1002/anie.202109749 %V 60 %X Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
• Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation. Bartling, Christian R. O.; Jensen, Thomas M. T.; Henry, Shawna M.; Colliander, Anna L.; Sereikaite, Vita; Wenzler, Marcella; Jain, Palash; Maric, Hans M.; Harpsøe, Kasper; Pedersen, Søren W.; Clemmensen, Louise S.; Haugaard-Kedström, Linda M.; Gloriam, David E.; Ho, Angela; Strømgaard, Kristian. In J. Am. Chem. Soc., 143(2), S. 891–901. American Chemical Society, 2021.
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  There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.

  @article{bartling2021targeting, abstract = {There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.}, author = {Bartling, Christian R. O. and Jensen, Thomas M. T. and Henry, Shawna M. and Colliander, Anna L. and Sereikaite, Vita and Wenzler, Marcella and Jain, Palash and Maric, Hans M. and Harpsøe, Kasper and Pedersen, Søren W. and Clemmensen, Louise S. and Haugaard-Kedström, Linda M. and Gloriam, David E. and Ho, Angela and Strømgaard, Kristian}, booktitle = {Journal of the American Chemical Society}, journal = {J. Am. Chem. Soc.}, keywords = {maric}, month = {01}, number = 2, pages = {891–901}, publisher = {American Chemical Society}, title = {Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation}, volume = 143, year = 2021 }

  %0 Journal Article %1 bartling2021targeting %A Bartling, Christian R. O. %A Jensen, Thomas M. T. %A Henry, Shawna M. %A Colliander, Anna L. %A Sereikaite, Vita %A Wenzler, Marcella %A Jain, Palash %A Maric, Hans M. %A Harpsøe, Kasper %A Pedersen, Søren W. %A Clemmensen, Louise S. %A Haugaard-Kedström, Linda M. %A Gloriam, David E. %A Ho, Angela %A Strømgaard, Kristian %B Journal of the American Chemical Society %D 2021 %I American Chemical Society %J J. Am. Chem. Soc. %N 2 %P 891--901 %R 10.1021/jacs.0c10696 %T Targeting the APP-Mint2 Protein–Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-β Formation %U https://doi.org/10.1021/jacs.0c10696 %V 143 %X There is an urgent need for novel therapeutic approaches to treat Alzheimer’s disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein–protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.
• Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain. Johe, Patrick; Jung, Sascha; Endres, Erik; Kersten, Christian; Zimmer, Collin; Ye, Weixiang; Sönnichsen, Carsten; Hellmich, Ute A.; Sotriffer, Christoph; Schirmeister, Tanja; Neuweiler, Hannes. In ACS Chem. Biol., 16(4), S. 661–670. American Chemical Society, 2021.
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  Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.

  @article{johe2021warhead, abstract = {Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.}, author = {Johe, Patrick and Jung, Sascha and Endres, Erik and Kersten, Christian and Zimmer, Collin and Ye, Weixiang and Sönnichsen, Carsten and Hellmich, Ute A. and Sotriffer, Christoph and Schirmeister, Tanja and Neuweiler, Hannes}, booktitle = {ACS Chemical Biology}, journal = {ACS Chem. Biol.}, keywords = {hannes}, month = {04}, number = 4, pages = {661–670}, publisher = {American Chemical Society}, title = {Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain}, volume = 16, year = 2021 }

  %0 Journal Article %1 johe2021warhead %A Johe, Patrick %A Jung, Sascha %A Endres, Erik %A Kersten, Christian %A Zimmer, Collin %A Ye, Weixiang %A Sönnichsen, Carsten %A Hellmich, Ute A. %A Sotriffer, Christoph %A Schirmeister, Tanja %A Neuweiler, Hannes %B ACS Chemical Biology %D 2021 %I American Chemical Society %J ACS Chem. Biol. %N 4 %P 661--670 %R 10.1021/acschembio.0c00911 %T Warhead Reactivity Limits the Speed of Inhibition of the Cysteine Protease Rhodesain %U https://doi.org/10.1021/acschembio.0c00911 %V 16 %X Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.
• Superresolution Microscopy of Sphingolipids. Schlegel, Jan; Sauer, Markus. In Lipid Rafts: Methods and Protocols, S. 303–311. Springer US, New York, NY, 2021.
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  This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.

  @inbook{schlegel2021superresolution, abstract = {This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.}, address = {New York, NY}, author = {Schlegel, Jan and Sauer, Markus}, booktitle = {Lipid Rafts: Methods and Protocols}, keywords = {sauer}, pages = {303–311}, publisher = {Springer US}, title = {Superresolution Microscopy of Sphingolipids}, year = 2021 }

  %0 Book Section %1 schlegel2021superresolution %A Schlegel, Jan %A Sauer, Markus %B Lipid Rafts: Methods and Protocols %C New York, NY %D 2021 %I Springer US %P 303--311 %R 10.1007/978-1-0716-0814-2_17 %T Superresolution Microscopy of Sphingolipids %U https://doi.org/10.1007/978-1-0716-0814-2_17 %X This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment. %@ 9781071608142
• Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes. Johé, Patrick; Jaenicke, Elmar; Neuweiler, Hannes; Schirmeister, Tanja; Kersten, Christian; Hellmich, Ute A. In Journal of Biological Chemistry, 296, S. 100565-. 2021.
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  Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.

  @article{johe2021structure, abstract = {Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.}, author = {Johé, Patrick and Jaenicke, Elmar and Neuweiler, Hannes and Schirmeister, Tanja and Kersten, Christian and Hellmich, Ute A.}, journal = {Journal of Biological Chemistry}, keywords = {hannes}, pages = {100565–}, title = {Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes}, volume = 296, year = 2021 }

  %0 Journal Article %1 johe2021structure %A Johé, Patrick %A Jaenicke, Elmar %A Neuweiler, Hannes %A Schirmeister, Tanja %A Kersten, Christian %A Hellmich, Ute A. %D 2021 %J Journal of Biological Chemistry %P 100565-- %R https://doi.org/10.1016/j.jbc.2021.100565 %T Structure, interdomain dynamics, and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes %U https://www.sciencedirect.com/science/article/pii/S0021925821003434 %V 296 %X Rhodesain is the lysosomal cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood–brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating prodomain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in Escherichia coli and determined its crystal structure. The trypanosomal prodomain differs from nonparasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose side-chain interactions resemble those of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations, and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH sensitivity of proenzyme cleavage in (trypanosomal) CathL-like proteases.
• Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy. Trinks, Nora; Reinhard, Sebastian; Drobny, Matthias; Heilig, Linda; Löffler, Jürgen; Sauer, Markus; Terpitz, Ulrich. In Communications Biology, 4(1), S. 1151-. 2021.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

  @article{trinks2021subdiffractionresolution, abstract = {Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.}, author = {Trinks, Nora and Reinhard, Sebastian and Drobny, Matthias and Heilig, Linda and Löffler, Jürgen and Sauer, Markus and Terpitz, Ulrich}, journal = {Communications Biology}, keywords = {terpitz}, number = 1, pages = {1151–}, title = {Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy}, volume = 4, year = 2021 }

  %0 Journal Article %1 trinks2021subdiffractionresolution %A Trinks, Nora %A Reinhard, Sebastian %A Drobny, Matthias %A Heilig, Linda %A Löffler, Jürgen %A Sauer, Markus %A Terpitz, Ulrich %D 2021 %J Communications Biology %N 1 %P 1151-- %R 10.1038/s42003-021-02669-y %T Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy %U https://doi.org/10.1038/s42003-021-02669-y %V 4 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
• Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy. Kuhlemann, Alexander; Beliu, Gerti; Janzen, Dieter; Petrini, Enrica Maria; Taban, Danush; Helmerich, Dominic A.; Doose, Sören; Bruno, Martina; Barberis, Andrea; Villmann, Carmen; Sauer, Markus; Werner, Christian. In Frontiers in Synaptic Neuroscience, 13. 2021.
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  Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.

  @article{kuhlemann2021genetic, abstract = {Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.}, author = {Kuhlemann, Alexander and Beliu, Gerti and Janzen, Dieter and Petrini, Enrica Maria and Taban, Danush and Helmerich, Dominic A. and Doose, Sören and Bruno, Martina and Barberis, Andrea and Villmann, Carmen and Sauer, Markus and Werner, Christian}, journal = {Frontiers in Synaptic Neuroscience}, keywords = {werner}, title = {Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy}, volume = 13, year = 2021 }

  %0 Journal Article %1 kuhlemann2021genetic %A Kuhlemann, Alexander %A Beliu, Gerti %A Janzen, Dieter %A Petrini, Enrica Maria %A Taban, Danush %A Helmerich, Dominic A. %A Doose, Sören %A Bruno, Martina %A Barberis, Andrea %A Villmann, Carmen %A Sauer, Markus %A Werner, Christian %D 2021 %J Frontiers in Synaptic Neuroscience %T Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy %U https://www.frontiersin.org/journals/synaptic-neuroscience/articles/10.3389/fnsyn.2021.727406 %V 13 %X Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
• Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections. Pauli, Martin; Paul, Mila M.; Proppert, Sven; Mrestani, Achmed; Sharifi, Marzieh; Repp, Felix; Kürzinger, Lydia; Kollmannsberger, Philip; Sauer, Markus; Heckmann, Manfred; Sirén, Anna-Leena. In Communications Biology, 4(1), S. 407-. 2021.
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  Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

  @article{pauli2021targeted, abstract = {Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.}, author = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and Kürzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sirén, Anna-Leena}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {407–}, title = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections}, volume = 4, year = 2021 }

  %0 Journal Article %1 pauli2021targeted %A Pauli, Martin %A Paul, Mila M. %A Proppert, Sven %A Mrestani, Achmed %A Sharifi, Marzieh %A Repp, Felix %A Kürzinger, Lydia %A Kollmannsberger, Philip %A Sauer, Markus %A Heckmann, Manfred %A Sirén, Anna-Leena %D 2021 %J Communications Biology %N 1 %P 407-- %R 10.1038/s42003-021-01939-z %T Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections %U https://doi.org/10.1038/s42003-021-01939-z %V 4 %X Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.
• Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab. García-Guerrero, Estefanía; Götz, Ralph; Doose, Sören; Sauer, Markus; Rodríguez-Gil, Alfonso; Nerreter, Thomas; Kortüm, K. Martin; Pérez-Simón, José A.; Einsele, Hermann; Hudecek, Michael; Danhof, Sophia. In Leukemia, 35(1), S. 201–214. 2021.
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  Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.

  @article{garciaguerrero2021upregulation, abstract = {Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.}, author = {García-Guerrero, Estefanía and Götz, Ralph and Doose, Sören and Sauer, Markus and Rodríguez-Gil, Alfonso and Nerreter, Thomas and Kortüm, K. Martin and Pérez-Simón, José A. and Einsele, Hermann and Hudecek, Michael and Danhof, Sophia}, journal = {Leukemia}, keywords = {sauer}, number = 1, pages = {201–214}, title = {Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab}, volume = 35, year = 2021 }

  %0 Journal Article %1 garciaguerrero2021upregulation %A García-Guerrero, Estefanía %A Götz, Ralph %A Doose, Sören %A Sauer, Markus %A Rodríguez-Gil, Alfonso %A Nerreter, Thomas %A Kortüm, K. Martin %A Pérez-Simón, José A. %A Einsele, Hermann %A Hudecek, Michael %A Danhof, Sophia %D 2021 %J Leukemia %N 1 %P 201--214 %R 10.1038/s41375-020-0840-y %T Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab %U https://doi.org/10.1038/s41375-020-0840-y %V 35 %X Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.
• Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics. Schubert, Jonathan; Schulze, Andrea; Prodromou, Chrisostomos; Neuweiler, Hannes. In Nature Communications, 12(1), S. 6964-. 2021.
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  Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

  @article{schubert2021twocolour, abstract = {Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.}, author = {Schubert, Jonathan and Schulze, Andrea and Prodromou, Chrisostomos and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {6964–}, title = {Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics}, volume = 12, year = 2021 }

  %0 Journal Article %1 schubert2021twocolour %A Schubert, Jonathan %A Schulze, Andrea %A Prodromou, Chrisostomos %A Neuweiler, Hannes %D 2021 %J Nature Communications %N 1 %P 6964-- %R 10.1038/s41467-021-27286-5 %T Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics %U https://doi.org/10.1038/s41467-021-27286-5 %V 12 %X Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.
• Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases. Moreno-Yruela, Carlos; Bæk, Michael; Vrsanova, Adela-Eugenie; Schulte, Clemens; Maric, Hans M.; Olsen, Christian A. In Nature Communications, 12(1), S. 62-. 2021.
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  Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.

  @article{morenoyruela2021hydroxamic, abstract = {Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.}, author = {Moreno-Yruela, Carlos and Bæk, Michael and Vrsanova, Adela-Eugenie and Schulte, Clemens and Maric, Hans M. and Olsen, Christian A.}, journal = {Nature Communications}, keywords = {maric}, number = 1, pages = {62–}, title = {Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases}, volume = 12, year = 2021 }

  %0 Journal Article %1 morenoyruela2021hydroxamic %A Moreno-Yruela, Carlos %A Bæk, Michael %A Vrsanova, Adela-Eugenie %A Schulte, Clemens %A Maric, Hans M. %A Olsen, Christian A. %D 2021 %J Nature Communications %N 1 %P 62-- %R 10.1038/s41467-020-20250-9 %T Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases %U https://doi.org/10.1038/s41467-020-20250-9 %V 12 %X Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.
• Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis. Fink, Julian; Schumacher, Fabian; Schlegel, Jan; Stenzel, Philipp; Wigger, Dominik; Sauer, Markus; Kleuser, Burkhard; Seibel, Jürgen. In Org. Biomol. Chem., 19(10), S. 2203–2212. The Royal Society of Chemistry, 2021.
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  Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

  @article{fink2021azidosphinganine, abstract = {Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.}, author = {Fink, Julian and Schumacher, Fabian and Schlegel, Jan and Stenzel, Philipp and Wigger, Dominik and Sauer, Markus and Kleuser, Burkhard and Seibel, Jürgen}, journal = {Org. Biomol. Chem.}, keywords = {sauer}, number = 10, pages = {2203–2212}, publisher = {The Royal Society of Chemistry}, title = {Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis}, volume = 19, year = 2021 }

  %0 Journal Article %1 fink2021azidosphinganine %A Fink, Julian %A Schumacher, Fabian %A Schlegel, Jan %A Stenzel, Philipp %A Wigger, Dominik %A Sauer, Markus %A Kleuser, Burkhard %A Seibel, Jürgen %D 2021 %I The Royal Society of Chemistry %J Org. Biomol. Chem. %N 10 %P 2203--2212 %R 10.1039/D0OB02592E %T Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis %U http://dx.doi.org/10.1039/D0OB02592E %V 19 %X Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

• Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae. Kunz, Tobias C.; Götz, Ralph; Gao, Shiqiang; Sauer, Markus; Kozjak-Pavlovic, Vera. In Frontiers in Cell and Developmental Biology, 8, S. 617-. 2020.
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  Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.

  @article{kunz2020using, abstract = {Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.}, author = {Kunz, Tobias C. and Götz, Ralph and Gao, Shiqiang and Sauer, Markus and Kozjak-Pavlovic, Vera}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {617–}, title = {Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae}, volume = 8, year = 2020 }

  %0 Journal Article %1 kunz2020using %A Kunz, Tobias C. %A Götz, Ralph %A Gao, Shiqiang %A Sauer, Markus %A Kozjak-Pavlovic, Vera %D 2020 %J Frontiers in Cell and Developmental Biology %P 617-- %T Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae %U https://www.frontiersin.org/article/10.3389/fcell.2020.00617 %V 8 %X Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.
• FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy. Memmel, Simon; Sisario, Dmitri; Zimmermann, Heiko; Sauer, Markus; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S.; Flentje, Michael. In BMC Bioinformatics, 21(1), S. 27-. 2020.
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  Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.

  @article{memmel2020focan, abstract = {Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.}, author = {Memmel, Simon and Sisario, Dmitri and Zimmermann, Heiko and Sauer, Markus and Sukhorukov, Vladimir L. and Djuzenova, Cholpon S. and Flentje, Michael}, journal = {BMC Bioinformatics}, keywords = {sauer}, number = 1, pages = {27–}, title = {FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy}, volume = 21, year = 2020 }

  %0 Journal Article %1 memmel2020focan %A Memmel, Simon %A Sisario, Dmitri %A Zimmermann, Heiko %A Sauer, Markus %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %A Flentje, Michael %D 2020 %J BMC Bioinformatics %N 1 %P 27-- %R 10.1186/s12859-020-3370-8 %T FocAn: automated 3D analysis of DNA repair foci in image stacks acquired by confocal fluorescence microscopy %U https://doi.org/10.1186/s12859-020-3370-8 %V 21 %X Phosphorylated histone H2AX, also known as γH2AX, forms μm-sized nuclear foci at the sites of DNA double-strand breaks (DSBs) induced by ionizing radiation and other agents. Due to their specificity and sensitivity, γH2AX immunoassays have become the gold standard for studying DSB induction and repair. One of these assays relies on the immunofluorescent staining of γH2AX followed by microscopic imaging and foci counting. During the last years, semi- and fully automated image analysis, capable of fast detection and quantification of γH2AX foci in large datasets of fluorescence images, are gradually replacing the traditional method of manual foci counting. A major drawback of the non-commercial software for foci counting (available so far) is that they are restricted to 2D-image data. In practice, these algorithms are useful for counting the foci located close to the midsection plane of the nucleus, while the out-of-plane foci are neglected.
• Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function. Forero, Andrea; Ku, Hsing-Ping; Malpartida, Ana Belén; Wäldchen, Sina; Alhama-Riba, Judit; Kulka, Christina; Aboagye, Benjamin; Norton, William H.J; Young, Andrew M.J; Ding, Yu-Qiang; Blum, Robert; Sauer, Markus; Rivero, Olga; Lesch, Klaus-Peter. In Neuropharmacology, 168, S. 108018-. 2020.
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  Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.

  @article{forero2020serotonin, abstract = {Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.}, author = {Forero, Andrea and Ku, Hsing-Ping and Malpartida, Ana Belén and Wäldchen, Sina and Alhama-Riba, Judit and Kulka, Christina and Aboagye, Benjamin and Norton, William H.J and Young, Andrew M.J and Ding, Yu-Qiang and Blum, Robert and Sauer, Markus and Rivero, Olga and Lesch, Klaus-Peter}, journal = {Neuropharmacology}, keywords = {sauer}, pages = {108018–}, title = {Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function}, volume = 168, year = 2020 }

  %0 Journal Article %1 forero2020serotonin %A Forero, Andrea %A Ku, Hsing-Ping %A Malpartida, Ana Belén %A Wäldchen, Sina %A Alhama-Riba, Judit %A Kulka, Christina %A Aboagye, Benjamin %A Norton, William H.J %A Young, Andrew M.J %A Ding, Yu-Qiang %A Blum, Robert %A Sauer, Markus %A Rivero, Olga %A Lesch, Klaus-Peter %D 2020 %J Neuropharmacology %P 108018-- %R https://doi.org/10.1016/j.neuropharm.2020.108018 %T Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function %U https://www.sciencedirect.com/science/article/pii/S0028390820300848 %V 168 %X Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.
• Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy. Götz, Ralph; Kunz, Tobias C.; Fink, Julian; Solger, Franziska; Schlegel, Jan; Seibel, Jürgen; Kozjak-Pavlovic, Vera; Rudel, Thomas; Sauer, Markus. In Nature Communications, 11(1), S. 6173-. 2020.
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  Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

  @article{gotz2020nanoscale, abstract = {Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.}, author = {Götz, Ralph and Kunz, Tobias C. and Fink, Julian and Solger, Franziska and Schlegel, Jan and Seibel, Jürgen and Kozjak-Pavlovic, Vera and Rudel, Thomas and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {6173–}, title = {Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy}, volume = 11, year = 2020 }

  %0 Journal Article %1 gotz2020nanoscale %A Götz, Ralph %A Kunz, Tobias C. %A Fink, Julian %A Solger, Franziska %A Schlegel, Jan %A Seibel, Jürgen %A Kozjak-Pavlovic, Vera %A Rudel, Thomas %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 6173-- %R 10.1038/s41467-020-19897-1 %T Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy %U https://doi.org/10.1038/s41467-020-19897-1 %V 11 %X Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.
• Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy. Zwettler, Fabian U.; Spindler, Marie-Christin; Reinhard, Sebastian; Klein, Teresa; Kurz, Andreas; Benavente, Ricardo; Sauer, Markus. In Nature Communications, 11(1), S. 3222-. 2020.
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  The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

  @article{zwettler2020tracking, abstract = {The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.}, author = {Zwettler, Fabian U. and Spindler, Marie-Christin and Reinhard, Sebastian and Klein, Teresa and Kurz, Andreas and Benavente, Ricardo and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3222–}, title = {Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy}, volume = 11, year = 2020 }

  %0 Journal Article %1 zwettler2020tracking %A Zwettler, Fabian U. %A Spindler, Marie-Christin %A Reinhard, Sebastian %A Klein, Teresa %A Kurz, Andreas %A Benavente, Ricardo %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3222-- %R 10.1038/s41467-020-17017-7 %T Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy %U https://doi.org/10.1038/s41467-020-17017-7 %V 11 %X The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.
• Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus. Weiss, Esther; Schlegel, Jan; Terpitz, Ulrich; Weber, Michael; Linde, Jörg; Schmitt, Anna-Lena; Hünniger, Kerstin; Marischen, Lothar; Gamon, Florian; Bauer, Joachim; Löffler, Claudia; Kurzai, Oliver; Morton, Charles Oliver; Sauer, Markus; Einsele, Hermann; Loeffler, Juergen. In Frontiers in Immunology, 11. 2020.
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  Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

  @article{weiss2020reconstituting, abstract = {Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.}, author = {Weiss, Esther and Schlegel, Jan and Terpitz, Ulrich and Weber, Michael and Linde, Jörg and Schmitt, Anna-Lena and Hünniger, Kerstin and Marischen, Lothar and Gamon, Florian and Bauer, Joachim and Löffler, Claudia and Kurzai, Oliver and Morton, Charles Oliver and Sauer, Markus and Einsele, Hermann and Loeffler, Juergen}, journal = {Frontiers in Immunology}, keywords = {terpitz}, series = 2117, title = {Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus}, volume = 11, year = 2020 }

  %0 Journal Article %1 weiss2020reconstituting %A Weiss, Esther %A Schlegel, Jan %A Terpitz, Ulrich %A Weber, Michael %A Linde, Jörg %A Schmitt, Anna-Lena %A Hünniger, Kerstin %A Marischen, Lothar %A Gamon, Florian %A Bauer, Joachim %A Löffler, Claudia %A Kurzai, Oliver %A Morton, Charles Oliver %A Sauer, Markus %A Einsele, Hermann %A Loeffler, Juergen %B 2117 %D 2020 %J Frontiers in Immunology %T Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus %U https://www.frontiersin.org/article/10.3389/fimmu.2020.02117 %V 11 %X Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
• A Trojan Horse for live-cell super-resolution microscopy. Beliu, Gerti; Sauer, Markus. In Light: Science & Applications, 9(1), S. 2-. 2020.
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  New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.

  @article{beliu2020trojan, abstract = {New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.}, author = {Beliu, Gerti and Sauer, Markus}, journal = {Light: Science & Applications}, keywords = {sauer}, number = 1, pages = {2–}, title = {A Trojan Horse for live-cell super-resolution microscopy}, volume = 9, year = 2020 }

  %0 Journal Article %1 beliu2020trojan %A Beliu, Gerti %A Sauer, Markus %D 2020 %J Light: Science & Applications %N 1 %P 2-- %R 10.1038/s41377-019-0238-7 %T A Trojan Horse for live-cell super-resolution microscopy %U https://doi.org/10.1038/s41377-019-0238-7 %V 9 %X New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.
• ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein. Rozario, Ashley M.; Zwettler, Fabian; Duwé, Sam; Hargreaves, Riley B.; Brice, Aaron; Dedecker, Peter; Sauer, Markus; Moseley, Gregory W.; Whelan, Donna R.; Bell, Toby D. M. In Aust. J. Chem. 2020.
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  The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

  @article{rozario2020large, abstract = {The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.}, author = {Rozario, Ashley M. and Zwettler, Fabian and Duwé, Sam and Hargreaves, Riley B. and Brice, Aaron and Dedecker, Peter and Sauer, Markus and Moseley, Gregory W. and Whelan, Donna R. and Bell, Toby D. M.}, journal = {Aust. J. Chem.}, keywords = {sauer}, title = {‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein}, year = 2020 }

  %0 Journal Article %1 rozario2020large %A Rozario, Ashley M. %A Zwettler, Fabian %A Duwé, Sam %A Hargreaves, Riley B. %A Brice, Aaron %A Dedecker, Peter %A Sauer, Markus %A Moseley, Gregory W. %A Whelan, Donna R. %A Bell, Toby D. M. %D 2020 %J Aust. J. Chem. %T ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein %U https://doi.org/10.1071/CH19571 %X The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.
• Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM). Zwettler, Fabian U.; Reinhard, Sebastian; Gambarotto, Davide; Bell, Toby D. M.; Hamel, Virginie; Guichard, Paul; Sauer, Markus. In Nature Communications, 11(1), S. 3388-. 2020.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

  @article{zwettler2020molecular, abstract = {Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.}, author = {Zwettler, Fabian U. and Reinhard, Sebastian and Gambarotto, Davide and Bell, Toby D. M. and Hamel, Virginie and Guichard, Paul and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3388–}, title = {Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)}, volume = 11, year = 2020 }

  %0 Journal Article %1 zwettler2020molecular %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Gambarotto, Davide %A Bell, Toby D. M. %A Hamel, Virginie %A Guichard, Paul %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3388-- %R 10.1038/s41467-020-17086-8 %T Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM) %U https://doi.org/10.1038/s41467-020-17086-8 %V 11 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.
• Expansion Microscopy for Cell Biology Analysis in Fungi. Götz, Ralph; Panzer, Sabine; Trinks, Nora; Eilts, Janna; Wagener, Johannes; Turrà, David; Di Pietro, Antonio; Sauer, Markus; Terpitz, Ulrich. In Frontiers in Microbiology, 11. 2020.
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  Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

  @article{gotz2020expansion, abstract = {Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.}, author = {Götz, Ralph and Panzer, Sabine and Trinks, Nora and Eilts, Janna and Wagener, Johannes and Turrà, David and Di Pietro, Antonio and Sauer, Markus and Terpitz, Ulrich}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, series = 574, title = {Expansion Microscopy for Cell Biology Analysis in Fungi}, volume = 11, year = 2020 }

  %0 Journal Article %1 gotz2020expansion %A Götz, Ralph %A Panzer, Sabine %A Trinks, Nora %A Eilts, Janna %A Wagener, Johannes %A Turrà, David %A Di Pietro, Antonio %A Sauer, Markus %A Terpitz, Ulrich %B 574 %D 2020 %J Frontiers in Microbiology %T Expansion Microscopy for Cell Biology Analysis in Fungi %U https://www.frontiersin.org/article/10.3389/fmicb.2020.00574 %V 11 %X Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
• Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones. Siddig, Sana; Aufmkolk, Sarah; Doose, Sören; Jobin, Marie-Lise; Werner, Christian; Sauer, Markus; Calebiro, Davide. In Sci Adv, 6(16), S. eaay7193-. 2020.
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  G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.

  @article{siddig2020superresolution, abstract = {G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.}, author = {Siddig, Sana and Aufmkolk, Sarah and Doose, Sören and Jobin, Marie-Lise and Werner, Christian and Sauer, Markus and Calebiro, Davide}, journal = {Sci Adv}, keywords = {home}, month = {04}, number = 16, pages = {eaay7193–}, title = {Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones}, volume = 6, year = 2020 }

  %0 Journal Article %1 siddig2020superresolution %A Siddig, Sana %A Aufmkolk, Sarah %A Doose, Sören %A Jobin, Marie-Lise %A Werner, Christian %A Sauer, Markus %A Calebiro, Davide %D 2020 %J Sci Adv %N 16 %P eaay7193-- %R 10.1126/sciadv.aay7193 %T Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones %U http://advances.sciencemag.org/content/6/16/eaay7193.abstract %V 6 %X G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.
• Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM. Wäldchen, Felix; Schlegel, Jan; Götz, Ralph; Luciano, Michael; Schnermann, Martin; Doose, Sören; Sauer, Markus. In Nature Communications, 11(1), S. 887-. 2020.
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  The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

  @article{waldchen2020wholecell, abstract = {The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.}, author = {Wäldchen, Felix and Schlegel, Jan and Götz, Ralph and Luciano, Michael and Schnermann, Martin and Doose, Sören and Sauer, Markus}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {887–}, title = {Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM}, volume = 11, year = 2020 }

  %0 Journal Article %1 waldchen2020wholecell %A Wäldchen, Felix %A Schlegel, Jan %A Götz, Ralph %A Luciano, Michael %A Schnermann, Martin %A Doose, Sören %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 887-- %R 10.1038/s41467-020-14731-0 %T Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM %U https://doi.org/10.1038/s41467-020-14731-0 %V 11 %X The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.
• Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy. Matikonda, Siddharth S.; Götz, Ralph; McLaughlin, Ryan; Sauer, Markus; Schnermann, Martin J. In Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods, Bd. 641, D. M. Chenoweth (Hrsg.), S. 225–244. Academic Press, 2020.
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  Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).

  @inbook{matikonda2020chapter, abstract = {Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).}, author = {Matikonda, Siddharth S. and Götz, Ralph and McLaughlin, Ryan and Sauer, Markus and Schnermann, Martin J.}, booktitle = {Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods}, editor = {Chenoweth, David M.}, keywords = {sauer}, pages = {225–244}, publisher = {Academic Press}, title = {Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy}, volume = 641, year = 2020 }

  %0 Book Section %1 matikonda2020chapter %A Matikonda, Siddharth S. %A Götz, Ralph %A McLaughlin, Ryan %A Sauer, Markus %A Schnermann, Martin J. %B Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods %D 2020 %E Chenoweth, David M. %I Academic Press %P 225--244 %R https://doi.org/10.1016/bs.mie.2020.04.042 %T Chapter Ten - Conformationally restrained pentamethine cyanines and use in reductive single molecule localization microscopy %U https://www.sciencedirect.com/science/article/pii/S0076687920301701 %V 641 %X Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).
• Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager. Kouhestani, Dina; Geis, Maria; Alsouri, Saed; Bumm, Thomas G. P.; Einsele, Hermann; Sauer, Markus; Stuhler, Gernot. In Cellular & Molecular Immunology. 2020.
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  @article{kouhestani2020variant, author = {Kouhestani, Dina and Geis, Maria and Alsouri, Saed and Bumm, Thomas G. P. and Einsele, Hermann and Sauer, Markus and Stuhler, Gernot}, journal = {Cellular & Molecular Immunology}, keywords = {sauer}, title = {Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager}, year = 2020 }

  %0 Journal Article %1 kouhestani2020variant %A Kouhestani, Dina %A Geis, Maria %A Alsouri, Saed %A Bumm, Thomas G. P. %A Einsele, Hermann %A Sauer, Markus %A Stuhler, Gernot %D 2020 %J Cellular & Molecular Immunology %R 10.1038/s41423-020-0507-7 %T Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager %U https://doi.org/10.1038/s41423-020-0507-7
• Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells. Prieto-Garcia, Cristian; Hartmann, Oliver; Reissland, Michaela; Braun, Fabian; Fischer, Thomas; Walz, Susanne; Schülein-Völk, Christina; Eilers, Ursula; Ade, Carsten P; Calzado, Marco A; Orian, Amir; Maric, Hans M; Münch, Christian; Rosenfeldt, Mathias; Eilers, Martin; Diefenbacher, Markus E. In EMBO Molecular Medicine, 12(4), S. e11101-. John Wiley & Sons, Ltd, 2020.
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  Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.

  @article{prietogarcia2020maintaining, abstract = {Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.}, author = {Prieto-Garcia, Cristian and Hartmann, Oliver and Reissland, Michaela and Braun, Fabian and Fischer, Thomas and Walz, Susanne and Schülein-Völk, Christina and Eilers, Ursula and Ade, Carsten P and Calzado, Marco A and Orian, Amir and Maric, Hans M and Münch, Christian and Rosenfeldt, Mathias and Eilers, Martin and Diefenbacher, Markus E}, booktitle = {EMBO Molecular Medicine}, journal = {EMBO Molecular Medicine}, keywords = {maric}, month = {04}, number = 4, pages = {e11101–}, publisher = {John Wiley & Sons, Ltd}, title = {Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells}, volume = 12, year = 2020 }

  %0 Journal Article %1 prietogarcia2020maintaining %A Prieto-Garcia, Cristian %A Hartmann, Oliver %A Reissland, Michaela %A Braun, Fabian %A Fischer, Thomas %A Walz, Susanne %A Schülein-Völk, Christina %A Eilers, Ursula %A Ade, Carsten P %A Calzado, Marco A %A Orian, Amir %A Maric, Hans M %A Münch, Christian %A Rosenfeldt, Mathias %A Eilers, Martin %A Diefenbacher, Markus E %B EMBO Molecular Medicine %D 2020 %I John Wiley & Sons, Ltd %J EMBO Molecular Medicine %N 4 %P e11101-- %R 10.15252/emmm.201911101 %T Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells %U https://doi.org/10.15252/emmm.201911101 %V 12 %X Abstract The transcription factor ?Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ?Np63 and maintains elevated ?NP63 levels in SCC by counteracting its proteasome-mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9-engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ?Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.
• Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes. Gründl, Marco; Walz, Susanne; Hauf, Laura; Schwab, Melissa; Werner, Kerstin Marcela; Spahr, Susanne; Schulte, Clemens; Maric, Hans Michael; Ade, Carsten P.; Gaubatz, Stefan. In PLOS Genetics, 16(5), S. e1008818-. Public Library of Science, 2020.
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  Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.

  @article{grundl2020interaction, abstract = {Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.}, author = {Gründl, Marco and Walz, Susanne and Hauf, Laura and Schwab, Melissa and Werner, Kerstin Marcela and Spahr, Susanne and Schulte, Clemens and Maric, Hans Michael and Ade, Carsten P. and Gaubatz, Stefan}, journal = {PLOS Genetics}, keywords = {maric}, month = {05}, number = 5, pages = {e1008818–}, publisher = {Public Library of Science}, title = {Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes}, volume = 16, year = 2020 }

  %0 Journal Article %1 grundl2020interaction %A Gründl, Marco %A Walz, Susanne %A Hauf, Laura %A Schwab, Melissa %A Werner, Kerstin Marcela %A Spahr, Susanne %A Schulte, Clemens %A Maric, Hans Michael %A Ade, Carsten P. %A Gaubatz, Stefan %D 2020 %I Public Library of Science %J PLOS Genetics %N 5 %P e1008818-- %T Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes %U https://doi.org/10.1371/journal.pgen.1008818 %V 16 %X Author summary YAP, the major downstream transducer of the Hippo pathway, is a potent inducer of proliferation. Here we show that the Myb-MuvB complex (MMB) mediates cardiomyocyte proliferation by YAP. We find that YAP and MMB regulate an overlapping set of pro-proliferative genes which involves binding of MMB to the promoters of these genes. We also identified a direct interaction between the B-MYB subunit of MMB and YAP. Based on the binding studies, we created a tool called MY-COMP that interferes with the association YAP to B-MYB and strongly inhibits proliferation of cardiomyocytes. Together, our data suggests that the YAP-MMB interaction is essential for division of cardiomyocytes, underscoring the functional relevance of the crosstalk between these two pathways for proper heart development.
• A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition. Langlhofer, Georg; Schaefer, Natascha; Maric, Hans M.; Keramidas, Angelo; Zhang, Yan; Baumann, Peter; Blum, Robert; Breitinger, Ulrike; Strømgaard, Kristian; Schlosser, Andreas; Kessels, Michael M.; Koch, Dennis; Qualmann, Britta; Breitinger, Hans-Georg; Lynch, Joseph W.; Villmann, Carmen. In J. Neurosci., 40(25), S. 4954-. 2020.
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  Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

  @article{langlhofer2020novel, abstract = {Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.}, author = {Langlhofer, Georg and Schaefer, Natascha and Maric, Hans M. and Keramidas, Angelo and Zhang, Yan and Baumann, Peter and Blum, Robert and Breitinger, Ulrike and Strømgaard, Kristian and Schlosser, Andreas and Kessels, Michael M. and Koch, Dennis and Qualmann, Britta and Breitinger, Hans-Georg and Lynch, Joseph W. and Villmann, Carmen}, journal = {J. Neurosci.}, keywords = {maric}, month = {06}, number = 25, pages = {4954–}, title = {A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition}, volume = 40, year = 2020 }

  %0 Journal Article %1 langlhofer2020novel %A Langlhofer, Georg %A Schaefer, Natascha %A Maric, Hans M. %A Keramidas, Angelo %A Zhang, Yan %A Baumann, Peter %A Blum, Robert %A Breitinger, Ulrike %A Strømgaard, Kristian %A Schlosser, Andreas %A Kessels, Michael M. %A Koch, Dennis %A Qualmann, Britta %A Breitinger, Hans-Georg %A Lynch, Joseph W. %A Villmann, Carmen %D 2020 %J J. Neurosci. %N 25 %P 4954-- %R 10.1523/JNEUROSCI.2490-19.2020 %T A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition %U http://www.jneurosci.org/content/40/25/4954.abstract %V 40 %X Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.
• Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer. Helmerich, Dominic A.; Beliu, Gerti; Sauer, Markus. In ACS Nano. American Chemical Society, 2020.
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  The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.

  @article{helmerich2020multiplelabeled, abstract = {The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Sauer, Markus}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = {08}, publisher = {American Chemical Society}, title = {Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer}, year = 2020 }

  %0 Journal Article %1 helmerich2020multiplelabeled %A Helmerich, Dominic A. %A Beliu, Gerti %A Sauer, Markus %B ACS Nano %D 2020 %I American Chemical Society %J ACS Nano %R 10.1021/acsnano.0c06099 %T Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer %U https://doi.org/10.1021/acsnano.0c06099 %X The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.
• Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning. Andreska, Thomas; Rauskolb, Stefanie; Schukraft, Nina; Lüningschrör, Patrick; Sasi, Manju; Signoret-Genest, Jeremy; Behringer, Marcus; Blum, Robert; Sauer, Markus; Tovote, Philip; Sendtner, Michael. In J. Neurosci., 40(33), S. 6289-. 2020.
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  Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.

  @article{andreska2020induction, abstract = {Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.}, author = {Andreska, Thomas and Rauskolb, Stefanie and Schukraft, Nina and Lüningschrör, Patrick and Sasi, Manju and Signoret-Genest, Jeremy and Behringer, Marcus and Blum, Robert and Sauer, Markus and Tovote, Philip and Sendtner, Michael}, journal = {J. Neurosci.}, keywords = {sauer}, month = {08}, number = 33, pages = {6289–}, title = {Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning}, volume = 40, year = 2020 }

  %0 Journal Article %1 andreska2020induction %A Andreska, Thomas %A Rauskolb, Stefanie %A Schukraft, Nina %A Lüningschrör, Patrick %A Sasi, Manju %A Signoret-Genest, Jeremy %A Behringer, Marcus %A Blum, Robert %A Sauer, Markus %A Tovote, Philip %A Sendtner, Michael %D 2020 %J J. Neurosci. %N 33 %P 6289-- %R 10.1523/JNEUROSCI.0288-20.2020 %T Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning %U http://www.jneurosci.org/content/40/33/6289.abstract %V 40 %X Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.
• Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy. Thiele, Jan Christoph; Helmerich, Dominic A.; Oleksiievets, Nazar; Tsukanov, Roman; Butkevich, Eugenia; Sauer, Markus; Nevskyi, Oleksii; Enderlein, Jörg. In ACS Nano, 14(10), S. 14190–14200. American Chemical Society, 2020.
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  Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

  @article{thiele2020confocal, abstract = {Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.}, author = {Thiele, Jan Christoph and Helmerich, Dominic A. and Oleksiievets, Nazar and Tsukanov, Roman and Butkevich, Eugenia and Sauer, Markus and Nevskyi, Oleksii and Enderlein, Jörg}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = 10, number = 10, pages = {14190–14200}, publisher = {American Chemical Society}, title = {Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy}, volume = 14, year = 2020 }

  %0 Journal Article %1 thiele2020confocal %A Thiele, Jan Christoph %A Helmerich, Dominic A. %A Oleksiievets, Nazar %A Tsukanov, Roman %A Butkevich, Eugenia %A Sauer, Markus %A Nevskyi, Oleksii %A Enderlein, Jörg %B ACS Nano %D 2020 %I American Chemical Society %J ACS Nano %N 10 %P 14190--14200 %R 10.1021/acsnano.0c07322 %T Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy %U https://doi.org/10.1021/acsnano.0c07322 %V 14 %X Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.
• BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation. Volz, Julia; Kusch, Charly; Beck, Sarah; Popp, Michael; Vögtle, Timo; Meub, Mara; Scheller, Inga; Heil, Hannah S.; Preu, Julia; Schuhmann, Michael K.; Hemmen, Katherina; Premsler, Thomas; Sickmann, Albert; Heinze, Katrin G.; Stegner, David; Stoll, Guido; Braun, Attila; Sauer, Markus; Nieswandt, Bernhard. In The Journal of Clinical Investigation, 130(11), S. 6064–6079. The American Society for Clinical Investigation, 2020.
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  Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

  @article{volz2020orchestrates, abstract = {Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.}, author = {Volz, Julia and Kusch, Charly and Beck, Sarah and Popp, Michael and Vögtle, Timo and Meub, Mara and Scheller, Inga and Heil, Hannah S. and Preu, Julia and Schuhmann, Michael K. and Hemmen, Katherina and Premsler, Thomas and Sickmann, Albert and Heinze, Katrin G. and Stegner, David and Stoll, Guido and Braun, Attila and Sauer, Markus and Nieswandt, Bernhard}, journal = {The Journal of Clinical Investigation}, keywords = {sauer}, month = 11, number = 11, pages = {6064–6079}, publisher = {The American Society for Clinical Investigation}, title = {BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation}, volume = 130, year = 2020 }

  %0 Journal Article %1 volz2020orchestrates %A Volz, Julia %A Kusch, Charly %A Beck, Sarah %A Popp, Michael %A Vögtle, Timo %A Meub, Mara %A Scheller, Inga %A Heil, Hannah S. %A Preu, Julia %A Schuhmann, Michael K. %A Hemmen, Katherina %A Premsler, Thomas %A Sickmann, Albert %A Heinze, Katrin G. %A Stegner, David %A Stoll, Guido %A Braun, Attila %A Sauer, Markus %A Nieswandt, Bernhard %D 2020 %I The American Society for Clinical Investigation %J The Journal of Clinical Investigation %N 11 %P 6064--6079 %R 10.1172/JCI136457 %T BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation %U https://doi.org/10.1172/JCI136457 %V 130 %X Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.
• NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis. Goretzki, Benedikt; Heiby, Julia C.; Hacker, Carolin; Neuweiler, Hannes; Hellmich, Ute A. In Biomolecular NMR Assignments, 14(1), S. 67–71. 2020.
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  Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

  @article{goretzki2020assignments, abstract = {Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.}, author = {Goretzki, Benedikt and Heiby, Julia C. and Hacker, Carolin and Neuweiler, Hannes and Hellmich, Ute A.}, journal = {Biomolecular NMR Assignments}, keywords = {hannes}, number = 1, pages = {67–71}, title = {NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis}, volume = 14, year = 2020 }

  %0 Journal Article %1 goretzki2020assignments %A Goretzki, Benedikt %A Heiby, Julia C. %A Hacker, Carolin %A Neuweiler, Hannes %A Hellmich, Ute A. %D 2020 %J Biomolecular NMR Assignments %N 1 %P 67--71 %R 10.1007/s12104-019-09922-w %T NMR assignments of a dynamically perturbed and dimerization inhibited N-terminal domain variant of a spider silk protein from E. australis %U https://doi.org/10.1007/s12104-019-09922-w %V 14 %X Web spiders use specialized glands to produce silk proteins, so-called spidroins, which assemble into extraordinarily tough silk fibers through tightly regulated phase and structural transitions. A crucial step in the polymerization of spidroins is the pH-triggered assembly of their N-terminal domains (NTDs) into tight dimers. Major ampullate spidroin NTDs contain an unusually high content of the amino acid methionine. We previously showed that the simultaneous mutation of the six hydrophobic core methionine residues to leucine in the NTD of the major ampullate spidroin 1 from Euprosthenops australis, a nursery web spider, yields a protein (L6-NTD) retaining a three-dimensional fold identical to the wildtype (WT) domain, yet with a significantly increased stability. Further, the dynamics of the L6-NTD are significantly reduced and the ability to dimerize is severely impaired compared to the WT domain. These properties lead to significant changes in the NMR spectra between WT and L6-NTD so that the previously available WT-NTD assignments cannot be transferred to the mutant protein. Here, we thus report the de novo NMR backbone and side chain assignments of the major ampullate spidroin 1 L6-NTD variant from E. australis as a prerequisite for obtaining further insights into protein structure and dynamics.

• Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins. Kasaragod, Vikram Babu; Hausrat, Torben Johann; Schaefer, Natascha; Kuhn, Maximilian; Christensen, Nikolaj Riis; Tessmer, Ingrid; Maric, Hans Michael; Madsen, Kenneth Lindegaard; Sotriffer, Christoph; Villmann, Carmen; Kneussel, Matthias; Schindelin, Hermann. In Neuron, 101(4), S. 673–689.e11. 2019.
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  Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.

  @article{kasaragod2019elucidating, abstract = {Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.}, author = {Kasaragod, Vikram Babu and Hausrat, Torben Johann and Schaefer, Natascha and Kuhn, Maximilian and Christensen, Nikolaj Riis and Tessmer, Ingrid and Maric, Hans Michael and Madsen, Kenneth Lindegaard and Sotriffer, Christoph and Villmann, Carmen and Kneussel, Matthias and Schindelin, Hermann}, journal = {Neuron}, keywords = {maric}, number = 4, pages = {673–689.e11}, title = {Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins}, volume = 101, year = 2019 }

  %0 Journal Article %1 kasaragod2019elucidating %A Kasaragod, Vikram Babu %A Hausrat, Torben Johann %A Schaefer, Natascha %A Kuhn, Maximilian %A Christensen, Nikolaj Riis %A Tessmer, Ingrid %A Maric, Hans Michael %A Madsen, Kenneth Lindegaard %A Sotriffer, Christoph %A Villmann, Carmen %A Kneussel, Matthias %A Schindelin, Hermann %D 2019 %J Neuron %N 4 %P 673--689.e11 %R https://doi.org/10.1016/j.neuron.2019.01.001 %T Elucidating the Molecular Basis for Inhibitory Neurotransmission Regulation by Artemisinins %U http://www.sciencedirect.com/science/article/pii/S0896627319300029 %V 101 %X Summary The frontline anti-malarial drug artemisinin and its derivatives have also been implicated in modulating multiple mammalian cellular pathways, including the recent identification of targeting γ-aminobutyric acid type A receptor (GABAAR) signaling in the pancreas. Their molecular mechanism of action, however, remains elusive. Here, we present crystal structures of gephyrin, the central organizer at inhibitory postsynapses, in complex with artesunate and artemether at 1.5-Å resolution. These artemisinins target the universal inhibitory neurotransmitter receptor-binding epitope of gephyrin, thus inhibiting critical interactions between gephyrin and glycine receptors (GlyRs) as well as GABAARs. Electrophysiological recordings reveal a significant inhibition of gephyrin-mediated neurotransmission by artemisinins. Furthermore, clustering analyses in primary neurons demonstrate a rapid inhibition and a time-dependent regulation of gephyrin and GABAAR cluster parameters. Our data not only provide a comprehensive model for artemisinin-mediated modulation of inhibitory neurotransmission but also establish artemisinins as potential lead compounds to pharmacologically interfere with this process.
• Platelet lamellipodium formation is not required for thrombus formation and stability. Schurr, Yvonne; Sperr, Andreas; Volz, Julia; Beck, Sarah; Reil, Lucy; Kusch, Charly; Eiring, Patrick; Bryson, Sheila; Sauer, Markus; Nieswandt, Bernhard; Machesky, Laura; Bender, Markus. In Blood, 134, S. 2318–2329. 2019.
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  During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

  @article{schurr2019platelet, abstract = {During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.}, author = {Schurr, Yvonne and Sperr, Andreas and Volz, Julia and Beck, Sarah and Reil, Lucy and Kusch, Charly and Eiring, Patrick and Bryson, Sheila and Sauer, Markus and Nieswandt, Bernhard and Machesky, Laura and Bender, Markus}, booktitle = {Blood}, journal = {Blood}, keywords = {sauer}, pages = {2318-2329}, series = 25, title = {Platelet lamellipodium formation is not required for thrombus formation and stability}, volume = 134, year = 2019 }

  %0 Journal Article %1 schurr2019platelet %A Schurr, Yvonne %A Sperr, Andreas %A Volz, Julia %A Beck, Sarah %A Reil, Lucy %A Kusch, Charly %A Eiring, Patrick %A Bryson, Sheila %A Sauer, Markus %A Nieswandt, Bernhard %A Machesky, Laura %A Bender, Markus %B Blood %D 2019 %J Blood %P 2318-2329 %T Platelet lamellipodium formation is not required for thrombus formation and stability %U https://doi.org/10.1182/blood.2019002105 %V 134 %X During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.
• FSP1 is a glutathione-independent ferroptosis suppressor. Doll, Sebastian; Freitas, Florencio Porto; Shah, Ron; Aldrovandi, Maceler; da Silva, Milene Costa; Ingold, Irina; Grocin, Andrea Goya; Xavier da Silva, Thamara Nishida; Panzilius, Elena; Scheel, Christina H.; Mourão, André; Buday, Katalin; Sato, Mami; Wanninger, Jonas; Vignane, Thibaut; Mohana, Vaishnavi; Rehberg, Markus; Flatley, Andrew; Schepers, Aloys; Kurz, Andreas; White, Daniel; Sauer, Markus; Sattler, Michael; Tate, Edward William; Schmitz, Werner; Schulze, Almut; O’Donnell, Valerie; Proneth, Bettina; Popowicz, Grzegorz M.; Pratt, Derek A.; Angeli, José Pedro Friedmann; Conrad, Marcus. In Nature, 575(7784), S. 693–698. 2019.
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  Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

  @article{doll2019glutathioneindependent, abstract = {Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.}, author = {Doll, Sebastian and Freitas, Florencio Porto and Shah, Ron and Aldrovandi, Maceler and da Silva, Milene Costa and Ingold, Irina and Grocin, Andrea Goya and Xavier da Silva, Thamara Nishida and Panzilius, Elena and Scheel, Christina H. and Mourão, André and Buday, Katalin and Sato, Mami and Wanninger, Jonas and Vignane, Thibaut and Mohana, Vaishnavi and Rehberg, Markus and Flatley, Andrew and Schepers, Aloys and Kurz, Andreas and White, Daniel and Sauer, Markus and Sattler, Michael and Tate, Edward William and Schmitz, Werner and Schulze, Almut and O’Donnell, Valerie and Proneth, Bettina and Popowicz, Grzegorz M. and Pratt, Derek A. and Angeli, José Pedro Friedmann and Conrad, Marcus}, journal = {Nature}, keywords = {sauer}, number = 7784, pages = {693–698}, title = {FSP1 is a glutathione-independent ferroptosis suppressor}, volume = 575, year = 2019 }

  %0 Journal Article %1 doll2019glutathioneindependent %A Doll, Sebastian %A Freitas, Florencio Porto %A Shah, Ron %A Aldrovandi, Maceler %A da Silva, Milene Costa %A Ingold, Irina %A Grocin, Andrea Goya %A Xavier da Silva, Thamara Nishida %A Panzilius, Elena %A Scheel, Christina H. %A Mourão, André %A Buday, Katalin %A Sato, Mami %A Wanninger, Jonas %A Vignane, Thibaut %A Mohana, Vaishnavi %A Rehberg, Markus %A Flatley, Andrew %A Schepers, Aloys %A Kurz, Andreas %A White, Daniel %A Sauer, Markus %A Sattler, Michael %A Tate, Edward William %A Schmitz, Werner %A Schulze, Almut %A O’Donnell, Valerie %A Proneth, Bettina %A Popowicz, Grzegorz M. %A Pratt, Derek A. %A Angeli, José Pedro Friedmann %A Conrad, Marcus %D 2019 %J Nature %N 7784 %P 693--698 %R 10.1038/s41586-019-1707-0 %T FSP1 is a glutathione-independent ferroptosis suppressor %U https://doi.org/10.1038/s41586-019-1707-0 %V 575 %X Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.
• MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation. Sdelci, Sara; Rendeiro, André F.; Rathert, Philipp; You, Wanhui; Lin, Jung-Ming G.; Ringler, Anna; Hofstätter, Gerald; Moll, Herwig P.; Gürtl, Bettina; Farlik, Matthias; Schick, Sandra; Klepsch, Freya; Oldach, Matthew; Buphamalai, Pisanu; Schischlik, Fiorella; Májek, Peter; Parapatics, Katja; Schmidl, Christian; Schuster, Michael; Penz, Thomas; Buckley, Dennis L.; Hudecz, Otto; Imre, Richard; Wang, Shuang-Yan; Maric, Hans Michael; Kralovics, Robert; Bennett, Keiryn L.; Müller, Andre C.; Mechtler, Karl; Menche, Jörg; Bradner, James E.; Winter, Georg E.; Klavins, Kristaps; Casanova, Emilio; Bock, Christoph; Zuber, Johannes; Kubicek, Stefan. In Nature Genetics, 51(6), S. 990–998. 2019.
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  The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

  @article{sdelci2019mthfd1, abstract = {The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.}, author = {Sdelci, Sara and Rendeiro, André F. and Rathert, Philipp and You, Wanhui and Lin, Jung-Ming G. and Ringler, Anna and Hofstätter, Gerald and Moll, Herwig P. and Gürtl, Bettina and Farlik, Matthias and Schick, Sandra and Klepsch, Freya and Oldach, Matthew and Buphamalai, Pisanu and Schischlik, Fiorella and Májek, Peter and Parapatics, Katja and Schmidl, Christian and Schuster, Michael and Penz, Thomas and Buckley, Dennis L. and Hudecz, Otto and Imre, Richard and Wang, Shuang-Yan and Maric, Hans Michael and Kralovics, Robert and Bennett, Keiryn L. and Müller, Andre C. and Mechtler, Karl and Menche, Jörg and Bradner, James E. and Winter, Georg E. and Klavins, Kristaps and Casanova, Emilio and Bock, Christoph and Zuber, Johannes and Kubicek, Stefan}, journal = {Nature Genetics}, keywords = {maric}, number = 6, pages = {990–998}, title = {MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation}, volume = 51, year = 2019 }

  %0 Journal Article %1 sdelci2019mthfd1 %A Sdelci, Sara %A Rendeiro, André F. %A Rathert, Philipp %A You, Wanhui %A Lin, Jung-Ming G. %A Ringler, Anna %A Hofstätter, Gerald %A Moll, Herwig P. %A Gürtl, Bettina %A Farlik, Matthias %A Schick, Sandra %A Klepsch, Freya %A Oldach, Matthew %A Buphamalai, Pisanu %A Schischlik, Fiorella %A Májek, Peter %A Parapatics, Katja %A Schmidl, Christian %A Schuster, Michael %A Penz, Thomas %A Buckley, Dennis L. %A Hudecz, Otto %A Imre, Richard %A Wang, Shuang-Yan %A Maric, Hans Michael %A Kralovics, Robert %A Bennett, Keiryn L. %A Müller, Andre C. %A Mechtler, Karl %A Menche, Jörg %A Bradner, James E. %A Winter, Georg E. %A Klavins, Kristaps %A Casanova, Emilio %A Bock, Christoph %A Zuber, Johannes %A Kubicek, Stefan %D 2019 %J Nature Genetics %N 6 %P 990--998 %R 10.1038/s41588-019-0413-z %T MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation %U https://doi.org/10.1038/s41588-019-0413-z %V 51 %X The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.
• Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy. Kunz, Tobias C.; Götz, Ralph; Sauer, Markus; Rudel, Thomas. In Frontiers in Cellular and Infection Microbiology, 9, S. 276-. 2019.
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  Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

  @article{kunz2019detection, abstract = {Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.}, author = {Kunz, Tobias C. and Götz, Ralph and Sauer, Markus and Rudel, Thomas}, journal = {Frontiers in Cellular and Infection Microbiology}, keywords = {sauer}, pages = {276–}, title = {Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy}, volume = 9, year = 2019 }

  %0 Journal Article %1 kunz2019detection %A Kunz, Tobias C. %A Götz, Ralph %A Sauer, Markus %A Rudel, Thomas %D 2019 %J Frontiers in Cellular and Infection Microbiology %P 276-- %T Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy %U https://www.frontiersin.org/article/10.3389/fcimb.2019.00276 %V 9 %X Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.
• Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps. Panzer, Sabine; Brych, Annika; Batschauer, Alfred; Terpitz, Ulrich. In Frontiers in Microbiology, 10. 2019.
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  In fungi green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371) and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of White Collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids, especially by acetic acid and indole-3-acetic acid. In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.

  @article{panzer2019opsin, abstract = {In fungi green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371) and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of White Collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids, especially by acetic acid and indole-3-acetic acid. In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.}, author = {Panzer, Sabine and Brych, Annika and Batschauer, Alfred and Terpitz, Ulrich}, journal = {Frontiers in Microbiology}, keywords = {home}, series = 735, title = {Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps}, volume = 10, year = 2019 }

  %0 Journal Article %1 panzer2019opsin %A Panzer, Sabine %A Brych, Annika %A Batschauer, Alfred %A Terpitz, Ulrich %B 735 %D 2019 %J Frontiers in Microbiology %T Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps %U https://www.frontiersin.org/article/10.3389/fmicb.2019.00735 %V 10 %X In fungi green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371) and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of White Collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids, especially by acetic acid and indole-3-acetic acid. In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.
• Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy. Beliu, Gerti; Kurz, Andreas J.; Kuhlemann, Alexander C.; Behringer-Pliess, Lisa; Meub, Mara; Wolf, Natalia; Seibel, Jürgen; Shi, Zhen-Dan; Schnermann, Martin; Grimm, Jonathan B.; Lavis, Luke D.; Doose, Sören; Sauer, Markus. In Communications Biology, 2(1), S. 261-. 2019.
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  Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

  @article{beliu2019bioorthogonal, abstract = {Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.}, author = {Beliu, Gerti and Kurz, Andreas J. and Kuhlemann, Alexander C. and Behringer-Pliess, Lisa and Meub, Mara and Wolf, Natalia and Seibel, Jürgen and Shi, Zhen-Dan and Schnermann, Martin and Grimm, Jonathan B. and Lavis, Luke D. and Doose, Sören and Sauer, Markus}, journal = {Communications Biology}, keywords = {sauer}, number = 1, pages = {261–}, title = {Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy}, volume = 2, year = 2019 }

  %0 Journal Article %1 beliu2019bioorthogonal %A Beliu, Gerti %A Kurz, Andreas J. %A Kuhlemann, Alexander C. %A Behringer-Pliess, Lisa %A Meub, Mara %A Wolf, Natalia %A Seibel, Jürgen %A Shi, Zhen-Dan %A Schnermann, Martin %A Grimm, Jonathan B. %A Lavis, Luke D. %A Doose, Sören %A Sauer, Markus %D 2019 %J Communications Biology %N 1 %P 261-- %R 10.1038/s42003-019-0518-z %T Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy %U https://doi.org/10.1038/s42003-019-0518-z %V 2 %X Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
• Super-resolution microscopy demystified. Schermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C. In Nature Cell Biology, 21(1), S. 72–84. 2019.
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  Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

  @article{schermelleh2019superresolution, abstract = {Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.}, author = {Schermelleh, Lothar and Ferrand, Alexia and Huser, Thomas and Eggeling, Christian and Sauer, Markus and Biehlmaier, Oliver and Drummen, Gregor P. C.}, journal = {Nature Cell Biology}, keywords = {home}, number = 1, pages = {72–84}, title = {Super-resolution microscopy demystified}, volume = 21, year = 2019 }

  %0 Journal Article %1 schermelleh2019superresolution %A Schermelleh, Lothar %A Ferrand, Alexia %A Huser, Thomas %A Eggeling, Christian %A Sauer, Markus %A Biehlmaier, Oliver %A Drummen, Gregor P. C. %D 2019 %J Nature Cell Biology %N 1 %P 72--84 %R 10.1038/s41556-018-0251-8 %T Super-resolution microscopy demystified %U https://doi.org/10.1038/s41556-018-0251-8 %V 21 %X Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.
• Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T. Nerreter, Thomas; Letschert, Sebastian; Götz, Ralph; Doose, Sören; Danhof, Sophia; Einsele, Hermann; Sauer, Markus; Hudecek, Michael. In Nature Communications, 10(1), S. 3137-. 2019.
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  Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.

  @article{nerreter2019superresolution, abstract = {Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.}, author = {Nerreter, Thomas and Letschert, Sebastian and Götz, Ralph and Doose, Sören and Danhof, Sophia and Einsele, Hermann and Sauer, Markus and Hudecek, Michael}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {3137–}, title = {Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T}, volume = 10, year = 2019 }

  %0 Journal Article %1 nerreter2019superresolution %A Nerreter, Thomas %A Letschert, Sebastian %A Götz, Ralph %A Doose, Sören %A Danhof, Sophia %A Einsele, Hermann %A Sauer, Markus %A Hudecek, Michael %D 2019 %J Nature Communications %N 1 %P 3137-- %R 10.1038/s41467-019-10948-w %T Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T %U https://doi.org/10.1038/s41467-019-10948-w %V 10 %X Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
• The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation. Mercier, Rebecca; Wolmarans, Annemarie; Schubert, Jonathan; Neuweiler, Hannes; Johnson, Jill L.; LaPointe, Paul. In Nature Communications, 10(1), S. 1273-. 2019.
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  Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

  @article{mercier2019conserved, abstract = {Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.}, author = {Mercier, Rebecca and Wolmarans, Annemarie and Schubert, Jonathan and Neuweiler, Hannes and Johnson, Jill L. and LaPointe, Paul}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {1273–}, title = {The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation}, volume = 10, year = 2019 }

  %0 Journal Article %1 mercier2019conserved %A Mercier, Rebecca %A Wolmarans, Annemarie %A Schubert, Jonathan %A Neuweiler, Hannes %A Johnson, Jill L. %A LaPointe, Paul %D 2019 %J Nature Communications %N 1 %P 1273-- %R 10.1038/s41467-019-09299-3 %T The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation %U https://doi.org/10.1038/s41467-019-09299-3 %V 10 %X Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.
• Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model. Doppler, Kathrin; Schuster, Yasmin; Appeltshauser, Luise; Biko, Lydia; Villmann, Carmen; Weishaupt, Andreas; Werner, Christian; Sommer, Claudia. In Journal of Neuroinflammation, 16(1), S. 73-. 2019.
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  Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown.

  @article{doppler2019anticntn1, abstract = {Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown.}, author = {Doppler, Kathrin and Schuster, Yasmin and Appeltshauser, Luise and Biko, Lydia and Villmann, Carmen and Weishaupt, Andreas and Werner, Christian and Sommer, Claudia}, journal = {Journal of Neuroinflammation}, keywords = {werner}, number = 1, pages = {73–}, title = {Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model}, volume = 16, year = 2019 }

  %0 Journal Article %1 doppler2019anticntn1 %A Doppler, Kathrin %A Schuster, Yasmin %A Appeltshauser, Luise %A Biko, Lydia %A Villmann, Carmen %A Weishaupt, Andreas %A Werner, Christian %A Sommer, Claudia %D 2019 %J Journal of Neuroinflammation %N 1 %P 73-- %R 10.1186/s12974-019-1462-z %T Anti-CNTN1 IgG3 induces acute conduction block and motor deficits in a passive transfer rat model %U https://doi.org/10.1186/s12974-019-1462-z %V 16 %X Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown.
• Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells. Djuzenova, Cholpon S.; Fiedler, Vanessa; Memmel, Simon; Katzer, Astrid; Sisario, Dmitri; Brosch, Philippa K.; Göhrung, Alexander; Frister, Svenja; Zimmermann, Heiko; Flentje, Michael; Sukhorukov, Vladimir L. In BMC Cancer, 19(1), S. 299-. 2019.
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  Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.

  @article{djuzenova2019differential, abstract = {Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.}, author = {Djuzenova, Cholpon S. and Fiedler, Vanessa and Memmel, Simon and Katzer, Astrid and Sisario, Dmitri and Brosch, Philippa K. and Göhrung, Alexander and Frister, Svenja and Zimmermann, Heiko and Flentje, Michael and Sukhorukov, Vladimir L.}, journal = {BMC Cancer}, keywords = {vladimir}, number = 1, pages = {299–}, title = {Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells}, volume = 19, year = 2019 }

  %0 Journal Article %1 djuzenova2019differential %A Djuzenova, Cholpon S. %A Fiedler, Vanessa %A Memmel, Simon %A Katzer, Astrid %A Sisario, Dmitri %A Brosch, Philippa K. %A Göhrung, Alexander %A Frister, Svenja %A Zimmermann, Heiko %A Flentje, Michael %A Sukhorukov, Vladimir L. %D 2019 %J BMC Cancer %N 1 %P 299-- %R 10.1186/s12885-019-5517-4 %T Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells %U https://doi.org/10.1186/s12885-019-5517-4 %V 19 %X Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells.
• Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites. Schlegel, Jan; Peters, Simon; Doose, Sören; Schubert-Unkmeir, Alexandra; Sauer, Markus. In Frontiers in Cell and Developmental Biology, 7, S. 194-. 2019.
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  Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.

  @article{schlegel2019superresolution, abstract = {Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.}, author = {Schlegel, Jan and Peters, Simon and Doose, Sören and Schubert-Unkmeir, Alexandra and Sauer, Markus}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {194–}, title = {Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites}, volume = 7, year = 2019 }

  %0 Journal Article %1 schlegel2019superresolution %A Schlegel, Jan %A Peters, Simon %A Doose, Sören %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2019 %J Frontiers in Cell and Developmental Biology %P 194-- %T Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites %U https://www.frontiersin.org/article/10.3389/fcell.2019.00194 %V 7 %X Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.
• Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts. Khayenko, Vladimir; Maric, Hans Michael. In Frontiers in Molecular Neuroscience, 12, S. 162-. 2019.
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  γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.

  @article{khayenko2019targeting, abstract = {γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.}, author = {Khayenko, Vladimir and Maric, Hans Michael}, journal = {Frontiers in Molecular Neuroscience}, keywords = {home}, pages = {162–}, title = {Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts}, volume = 12, year = 2019 }

  %0 Journal Article %1 khayenko2019targeting %A Khayenko, Vladimir %A Maric, Hans Michael %D 2019 %J Frontiers in Molecular Neuroscience %P 162-- %T Targeting GABAAR-Associated Proteins: New Modulators, Labels and Concepts %U https://www.frontiersin.org/article/10.3389/fnmol.2019.00162 %V 12 %X γ-aminobutyric acid type A receptors (GABAARs) are the major mediators of synaptic inhibition in the brain. Aberrant GABAAR activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review we introduce a new strategy to modulate GABAeric signaling: Targeting the intracellular protein interactors of GABAARs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABAAR subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABAAR-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABAAR numbers and clustering, modifying neuronal transmission. Interference with GABAAR trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABAAR to AP2 increase the surface concentration of GABAAR clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with nanometer resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABAAR accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABAAR modulators and following in-vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.
• MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation. Baluapuri, Apoorva; Hofstetter, Julia; Dudvarski Stankovic, Nevenka; Endres, Theresa; Bhandare, Pranjali; Vos, Seychelle Monique; Adhikari, Bikash; Schwarz, Jessica Denise; Narain, Ashwin; Vogt, Markus; Wang, Shuang-Yan; Düster, Robert; Jung, Lisa Anna; Vanselow, Jens Thorsten; Wiegering, Armin; Geyer, Matthias; Maric, Hans Michael; Gallant, Peter; Walz, Susanne; Schlosser, Andreas; Cramer, Patrick; Eilers, Martin; Wolf, Elmar. In Molecular Cell, 74(4), S. 674–687.e11. 2019.
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  Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

  @article{baluapuri2019recruits, abstract = {Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.}, author = {Baluapuri, Apoorva and Hofstetter, Julia and Dudvarski Stankovic, Nevenka and Endres, Theresa and Bhandare, Pranjali and Vos, Seychelle Monique and Adhikari, Bikash and Schwarz, Jessica Denise and Narain, Ashwin and Vogt, Markus and Wang, Shuang-Yan and Düster, Robert and Jung, Lisa Anna and Vanselow, Jens Thorsten and Wiegering, Armin and Geyer, Matthias and Maric, Hans Michael and Gallant, Peter and Walz, Susanne and Schlosser, Andreas and Cramer, Patrick and Eilers, Martin and Wolf, Elmar}, journal = {Molecular Cell}, keywords = {maric}, number = 4, pages = {674–687.e11}, title = {MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation}, volume = 74, year = 2019 }

  %0 Journal Article %1 baluapuri2019recruits %A Baluapuri, Apoorva %A Hofstetter, Julia %A Dudvarski Stankovic, Nevenka %A Endres, Theresa %A Bhandare, Pranjali %A Vos, Seychelle Monique %A Adhikari, Bikash %A Schwarz, Jessica Denise %A Narain, Ashwin %A Vogt, Markus %A Wang, Shuang-Yan %A Düster, Robert %A Jung, Lisa Anna %A Vanselow, Jens Thorsten %A Wiegering, Armin %A Geyer, Matthias %A Maric, Hans Michael %A Gallant, Peter %A Walz, Susanne %A Schlosser, Andreas %A Cramer, Patrick %A Eilers, Martin %A Wolf, Elmar %D 2019 %J Molecular Cell %N 4 %P 674--687.e11 %R https://doi.org/10.1016/j.molcel.2019.02.031 %T MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation %U http://www.sciencedirect.com/science/article/pii/S109727651930142X %V 74 %X Summary The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
• Generation of site-distinct N-glycan variants for in vitro bioactivity testing. Brühlmann, David; Vuillemin, Thomas; Satwekar, Abhijeet; Galano, Eugenio; Palmese, Angelo; D’Angelo, Alessandra; Manco, Zeynep; Souquet, Jonathan; Broly, Hervé; Sauer, Markus; Hemberger, Jürgen; Jordan, Martin. In Biotechnology and Bioengineering, 116(5), S. 1017–1028. John Wiley & Sons, Ltd, 2019.
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  Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

  @article{bruhlmann2019generation, abstract = {Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.}, author = {Brühlmann, David and Vuillemin, Thomas and Satwekar, Abhijeet and Galano, Eugenio and Palmese, Angelo and D'Angelo, Alessandra and Manco, Zeynep and Souquet, Jonathan and Broly, Hervé and Sauer, Markus and Hemberger, Jürgen and Jordan, Martin}, booktitle = {Biotechnology and Bioengineering}, journal = {Biotechnology and Bioengineering}, keywords = {sauer}, month = {05}, number = 5, pages = {1017–1028}, publisher = {John Wiley & Sons, Ltd}, title = {Generation of site-distinct N-glycan variants for in vitro bioactivity testing}, volume = 116, year = 2019 }

  %0 Journal Article %1 bruhlmann2019generation %A Brühlmann, David %A Vuillemin, Thomas %A Satwekar, Abhijeet %A Galano, Eugenio %A Palmese, Angelo %A D'Angelo, Alessandra %A Manco, Zeynep %A Souquet, Jonathan %A Broly, Hervé %A Sauer, Markus %A Hemberger, Jürgen %A Jordan, Martin %B Biotechnology and Bioengineering %D 2019 %I John Wiley & Sons, Ltd %J Biotechnology and Bioengineering %N 5 %P 1017--1028 %R 10.1002/bit.26930 %T Generation of site-distinct N-glycan variants for in vitro bioactivity testing %U https://doi.org/10.1002/bit.26930 %V 116 %X Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.
• Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells. Zilkowski, Ilona; Ziouti, Fani; Schulze, Andres; Hauck, Stefanie; Schmidt, Stefanie; Mainz, Laura; Sauer, Markus; Albrecht, Krystyna; Jundt, Franziska; Groll, Jürgen. In Biomacromolecules, 20(2), S. 916–926. American Chemical Society, 2019.
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  Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.

  @article{zilkowski2019nanogels, abstract = {Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.}, author = {Zilkowski, Ilona and Ziouti, Fani and Schulze, Andres and Hauck, Stefanie and Schmidt, Stefanie and Mainz, Laura and Sauer, Markus and Albrecht, Krystyna and Jundt, Franziska and Groll, Jürgen}, booktitle = {Biomacromolecules}, journal = {Biomacromolecules}, keywords = {sauer}, month = {02}, number = 2, pages = {916–926}, publisher = {American Chemical Society}, title = {Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells}, volume = 20, year = 2019 }

  %0 Journal Article %1 zilkowski2019nanogels %A Zilkowski, Ilona %A Ziouti, Fani %A Schulze, Andres %A Hauck, Stefanie %A Schmidt, Stefanie %A Mainz, Laura %A Sauer, Markus %A Albrecht, Krystyna %A Jundt, Franziska %A Groll, Jürgen %B Biomacromolecules %D 2019 %I American Chemical Society %J Biomacromolecules %N 2 %P 916--926 %R 10.1021/acs.biomac.8b01553 %T Nanogels Enable Efficient miRNA Delivery and Target Gene Downregulation in Transfection-Resistant Multiple Myeloma Cells %U https://doi.org/10.1021/acs.biomac.8b01553 %V 20 %X Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.
• Registration and Visualization of Correlative Super-Resolution Microscopy Data. Reinhard, Sebastian; Aufmkolk, Sarah; Sauer, Markus; Doose, Sören. In Biophysical Journal, 116(11), S. 2073–2078. 2019.
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  We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.

  @article{reinhard2019registration, abstract = {We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.}, author = {Reinhard, Sebastian and Aufmkolk, Sarah and Sauer, Markus and Doose, Sören}, journal = {Biophysical Journal}, keywords = {sauer}, number = 11, pages = {2073–2078}, title = {Registration and Visualization of Correlative Super-Resolution Microscopy Data}, volume = 116, year = 2019 }

  %0 Journal Article %1 reinhard2019registration %A Reinhard, Sebastian %A Aufmkolk, Sarah %A Sauer, Markus %A Doose, Sören %D 2019 %J Biophysical Journal %N 11 %P 2073--2078 %R https://doi.org/10.1016/j.bpj.2019.04.029 %T Registration and Visualization of Correlative Super-Resolution Microscopy Data %U http://www.sciencedirect.com/science/article/pii/S000634951930373X %V 116 %X We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
• Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels. Peters, Simon; Schlegel, Jan; Becam, Jérôme; Avota, Elita; Sauer, Markus; Schubert-Unkmeir, Alexandra. In Infect. Immun., 87(8), A. J. Bäumler (Hrsg.), S. e00410–19. 2019.
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  Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

  @article{peters2019ltspan, abstract = {Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.}, author = {Peters, Simon and Schlegel, Jan and Becam, Jérôme and Avota, Elita and Sauer, Markus and Schubert-Unkmeir, Alexandra}, editor = {Bäumler, Andreas J.}, journal = {Infect. Immun.}, keywords = {sauer}, month = {08}, number = 8, pages = {e00410-19–}, title = {Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels}, volume = 87, year = 2019 }

  %0 Journal Article %1 peters2019ltspan %A Peters, Simon %A Schlegel, Jan %A Becam, Jérôme %A Avota, Elita %A Sauer, Markus %A Schubert-Unkmeir, Alexandra %D 2019 %E Bäumler, Andreas J. %J Infect. Immun. %N 8 %P e00410-19-- %R 10.1128/IAI.00410-19 %T Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels %U http://iai.asm.org/content/87/8/e00410-19.abstract %V 87 %X Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.
• On-target restoration of a split T cell-engaging antibody for precision immunotherapy. Banaszek, Agnes; Bumm, Thomas G. P.; Nowotny, Boris; Geis, Maria; Jacob, Kim; Wölfl, Matthias; Trebing, Johannes; Kucka, Kirstin; Kouhestani, Dina; Gogishvili, Tea; Krenz, Bastian; Lutz, Justina; Rasche, Leo; Hönemann, Dirk; Neuweiler, Hannes; Heiby, Julia C.; Bargou, Ralf C.; Wajant, Harald; Einsele, Hermann; Riethmüller, Gert; Stuhler, Gernot. In Nature Communications, 10(1), S. 5387-. 2019.
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  T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.

  @article{banaszek2019ontarget, abstract = {T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.}, author = {Banaszek, Agnes and Bumm, Thomas G. P. and Nowotny, Boris and Geis, Maria and Jacob, Kim and Wölfl, Matthias and Trebing, Johannes and Kucka, Kirstin and Kouhestani, Dina and Gogishvili, Tea and Krenz, Bastian and Lutz, Justina and Rasche, Leo and Hönemann, Dirk and Neuweiler, Hannes and Heiby, Julia C. and Bargou, Ralf C. and Wajant, Harald and Einsele, Hermann and Riethmüller, Gert and Stuhler, Gernot}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {5387–}, title = {On-target restoration of a split T cell-engaging antibody for precision immunotherapy}, volume = 10, year = 2019 }

  %0 Journal Article %1 banaszek2019ontarget %A Banaszek, Agnes %A Bumm, Thomas G. P. %A Nowotny, Boris %A Geis, Maria %A Jacob, Kim %A Wölfl, Matthias %A Trebing, Johannes %A Kucka, Kirstin %A Kouhestani, Dina %A Gogishvili, Tea %A Krenz, Bastian %A Lutz, Justina %A Rasche, Leo %A Hönemann, Dirk %A Neuweiler, Hannes %A Heiby, Julia C. %A Bargou, Ralf C. %A Wajant, Harald %A Einsele, Hermann %A Riethmüller, Gert %A Stuhler, Gernot %D 2019 %J Nature Communications %N 1 %P 5387-- %R 10.1038/s41467-019-13196-0 %T On-target restoration of a split T cell-engaging antibody for precision immunotherapy %U https://doi.org/10.1038/s41467-019-13196-0 %V 10 %X T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies.
• Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk. Heiby, Julia C.; Goretzki, Benedikt; Johnson, Christopher M.; Hellmich, Ute A.; Neuweiler, Hannes. In Nature Communications, 10(1), S. 4378-. 2019.
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  Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.

  @article{heiby2019methionine, abstract = {Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.}, author = {Heiby, Julia C. and Goretzki, Benedikt and Johnson, Christopher M. and Hellmich, Ute A. and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {4378–}, title = {Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk}, volume = 10, year = 2019 }

  %0 Journal Article %1 heiby2019methionine %A Heiby, Julia C. %A Goretzki, Benedikt %A Johnson, Christopher M. %A Hellmich, Ute A. %A Neuweiler, Hannes %D 2019 %J Nature Communications %N 1 %P 4378-- %R 10.1038/s41467-019-12365-5 %T Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk %U https://doi.org/10.1038/s41467-019-12365-5 %V 10 %X Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.
• Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium. Derakhshani, Shaghayegh; Kurz, Andreas; Japtok, Lukasz; Schumacher, Fabian; Pilgram, Lisa; Steinke, Maria; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle; Avota, Elita. In Frontiers in Immunology, 10, S. 1294-. 2019.
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  Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.

  @article{derakhshani2019measles, abstract = {Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.}, author = {Derakhshani, Shaghayegh and Kurz, Andreas and Japtok, Lukasz and Schumacher, Fabian and Pilgram, Lisa and Steinke, Maria and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, journal = {Frontiers in Immunology}, keywords = {sauer}, pages = {1294–}, title = {Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium}, volume = 10, year = 2019 }

  %0 Journal Article %1 derakhshani2019measles %A Derakhshani, Shaghayegh %A Kurz, Andreas %A Japtok, Lukasz %A Schumacher, Fabian %A Pilgram, Lisa %A Steinke, Maria %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2019 %J Frontiers in Immunology %P 1294-- %T Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium %U https://www.frontiersin.org/article/10.3389/fimmu.2019.01294 %V 10 %X Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.
• Imaging cellular ultrastructures using expansion microscopy (U-ExM). Gambarotto, Davide; Zwettler, Fabian U.; Le Guennec, Maeva; Schmidt-Cernohorska, Marketa; Fortun, Denis; Borgers, Susanne; Heine, Jörn; Schloetel, Jan-Gero; Reuss, Matthias; Unser, Michael; Boyden, Edward S.; Sauer, Markus; Hamel, Virginie; Guichard, Paul. In Nature Methods, 16(1), S. 71–74. 2019.
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  Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

  @article{gambarotto2019imaging, abstract = {Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.}, author = {Gambarotto, Davide and Zwettler, Fabian U. and Le Guennec, Maeva and Schmidt-Cernohorska, Marketa and Fortun, Denis and Borgers, Susanne and Heine, Jörn and Schloetel, Jan-Gero and Reuss, Matthias and Unser, Michael and Boyden, Edward S. and Sauer, Markus and Hamel, Virginie and Guichard, Paul}, journal = {Nature Methods}, keywords = {sauer}, number = 1, pages = {71–74}, title = {Imaging cellular ultrastructures using expansion microscopy (U-ExM)}, volume = 16, year = 2019 }

  %0 Journal Article %1 gambarotto2019imaging %A Gambarotto, Davide %A Zwettler, Fabian U. %A Le Guennec, Maeva %A Schmidt-Cernohorska, Marketa %A Fortun, Denis %A Borgers, Susanne %A Heine, Jörn %A Schloetel, Jan-Gero %A Reuss, Matthias %A Unser, Michael %A Boyden, Edward S. %A Sauer, Markus %A Hamel, Virginie %A Guichard, Paul %D 2019 %J Nature Methods %N 1 %P 71--74 %R 10.1038/s41592-018-0238-1 %T Imaging cellular ultrastructures using expansion microscopy (U-ExM) %U https://doi.org/10.1038/s41592-018-0238-1 %V 16 %X Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

• Sharpening emitter localization in front of a tuned mirror. Heil, Hannah S.; Schreiber, Benjamin; Götz, Ralph; Emmerling, Monika; Dabauvalle, Marie-Christine; Krohne, Georg; Höfling, Sven; Kamp, Martin; Sauer, Markus; Heinze, Katrin G. In Light: Science & Applications, 7(1), S. 99-. 2018.
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  Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.

  @article{heil2018sharpening, abstract = {Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.}, author = {Heil, Hannah S. and Schreiber, Benjamin and Götz, Ralph and Emmerling, Monika and Dabauvalle, Marie-Christine and Krohne, Georg and Höfling, Sven and Kamp, Martin and Sauer, Markus and Heinze, Katrin G.}, journal = {Light: Science & Applications}, keywords = {sauer}, number = 1, pages = {99–}, title = {Sharpening emitter localization in front of a tuned mirror}, volume = 7, year = 2018 }

  %0 Journal Article %1 heil2018sharpening %A Heil, Hannah S. %A Schreiber, Benjamin %A Götz, Ralph %A Emmerling, Monika %A Dabauvalle, Marie-Christine %A Krohne, Georg %A Höfling, Sven %A Kamp, Martin %A Sauer, Markus %A Heinze, Katrin G. %D 2018 %J Light: Science & Applications %N 1 %P 99-- %R 10.1038/s41377-018-0104-z %T Sharpening emitter localization in front of a tuned mirror %U https://doi.org/10.1038/s41377-018-0104-z %V 7 %X Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.
• Two-step self-assembly of a spider silk molecular clamp. Rat, Charlotte; Heiby, Julia C.; Bunz, Jessica P.; Neuweiler, Hannes. In Nature Communications, 9(1), S. 4779-. 2018.
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  Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.

  @article{rat2018twostep, abstract = {Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.}, author = {Rat, Charlotte and Heiby, Julia C. and Bunz, Jessica P. and Neuweiler, Hannes}, journal = {Nature Communications}, keywords = {hannes}, number = 1, pages = {4779–}, title = {Two-step self-assembly of a spider silk molecular clamp}, volume = 9, year = 2018 }

  %0 Journal Article %1 rat2018twostep %A Rat, Charlotte %A Heiby, Julia C. %A Bunz, Jessica P. %A Neuweiler, Hannes %D 2018 %J Nature Communications %N 1 %P 4779-- %R 10.1038/s41467-018-07227-5 %T Two-step self-assembly of a spider silk molecular clamp %U https://doi.org/10.1038/s41467-018-07227-5 %V 9 %X Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.
• The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling. Börtlein, Charlene; Draeger, Annette; Schoenauer, Roman; Kuhlemann, Alexander; Sauer, Markus; Schneider-Schaulies, Sibylle; Avota, Elita. In Frontiers in Immunology, 9, S. 815-. 2018.
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  By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.

  @article{bortlein2018neutral, abstract = {By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.}, author = {Börtlein, Charlene and Draeger, Annette and Schoenauer, Roman and Kuhlemann, Alexander and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, journal = {Frontiers in Immunology}, keywords = {sauer}, pages = {815–}, title = {The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling}, volume = 9, year = 2018 }

  %0 Journal Article %1 bortlein2018neutral %A Börtlein, Charlene %A Draeger, Annette %A Schoenauer, Roman %A Kuhlemann, Alexander %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2018 %J Frontiers in Immunology %P 815-- %T The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling %U https://www.frontiersin.org/article/10.3389/fimmu.2018.00815 %V 9 %X By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.
• Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin. Hines, Rochelle M.; Maric, Hans Michael; Hines, Dustin J.; Modgil, Amit; Panzanelli, Patrizia; Nakamura, Yasuko; Nathanson, Anna J.; Cross, Alan; Deeb, Tarek; Brandon, Nicholas J.; Davies, Paul; Fritschy, Jean-Marc; Schindelin, Hermann; Moss, Stephen J. In Nature Communications, 9(1), S. 3130-. 2018.
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  Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.

  @article{hines2018developmental, abstract = {Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.}, author = {Hines, Rochelle M. and Maric, Hans Michael and Hines, Dustin J. and Modgil, Amit and Panzanelli, Patrizia and Nakamura, Yasuko and Nathanson, Anna J. and Cross, Alan and Deeb, Tarek and Brandon, Nicholas J. and Davies, Paul and Fritschy, Jean-Marc and Schindelin, Hermann and Moss, Stephen J.}, journal = {Nature Communications}, keywords = {maric}, number = 1, pages = {3130–}, title = {Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin}, volume = 9, year = 2018 }

  %0 Journal Article %1 hines2018developmental %A Hines, Rochelle M. %A Maric, Hans Michael %A Hines, Dustin J. %A Modgil, Amit %A Panzanelli, Patrizia %A Nakamura, Yasuko %A Nathanson, Anna J. %A Cross, Alan %A Deeb, Tarek %A Brandon, Nicholas J. %A Davies, Paul %A Fritschy, Jean-Marc %A Schindelin, Hermann %A Moss, Stephen J. %D 2018 %J Nature Communications %N 1 %P 3130-- %R 10.1038/s41467-018-05481-1 %T Developmental seizures and mortality result from reducing GABAA receptor α2-subunit interaction with collybistin %U https://doi.org/10.1038/s41467-018-05481-1 %V 9 %X Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.
• Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms. Bleem, Alissa; Christiansen, Gunna; Madsen, Daniel J.; Maric, Hans; Strømgaard, Kristian; Bryers, James D.; Daggett, Valerie; Meyer, Rikke L.; Otzen, Daniel E. In Journal of Molecular Biology, 430(20), S. 3751–3763. 2018.
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  Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

  @article{bleem2018protein, abstract = {Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.}, author = {Bleem, Alissa and Christiansen, Gunna and Madsen, Daniel J. and Maric, Hans and Strømgaard, Kristian and Bryers, James D. and Daggett, Valerie and Meyer, Rikke L. and Otzen, Daniel E.}, booktitle = {Functional Amyloids in Health and Disease}, journal = {Journal of Molecular Biology}, keywords = {maric}, number = 20, pages = {3751–3763}, title = {Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms}, volume = 430, year = 2018 }

  %0 Journal Article %1 bleem2018protein %A Bleem, Alissa %A Christiansen, Gunna %A Madsen, Daniel J. %A Maric, Hans %A Strømgaard, Kristian %A Bryers, James D. %A Daggett, Valerie %A Meyer, Rikke L. %A Otzen, Daniel E. %B Functional Amyloids in Health and Disease %D 2018 %J Journal of Molecular Biology %N 20 %P 3751--3763 %R https://doi.org/10.1016/j.jmb.2018.06.043 %T Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms %U http://www.sciencedirect.com/science/article/pii/S0022283618307046 %V 430 %X Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.
• Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction. Haselmann, Holger; Mannara, Francesco; Werner, Christian; Planagumà, Jesús; Miguez-Cabello, Federico; Schmidl, Lars; Grünewald, Benedikt; Petit-Pedrol, Mar; Kirmse, Knut; Classen, Joseph; Demir, Fatih; Klöcker, Nikolaj; Soto, David; Doose, Sören; Dalmau, Josep; Hallermann, Stefan; Geis, Christian. In Neuron, 100(1), S. 91–105.e9. 2018.
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  AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.

  @article{haselmann2018human, abstract = {AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.}, author = {Haselmann, Holger and Mannara, Francesco and Werner, Christian and Planagumà, Jesús and Miguez-Cabello, Federico and Schmidl, Lars and Grünewald, Benedikt and Petit-Pedrol, Mar and Kirmse, Knut and Classen, Joseph and Demir, Fatih and Klöcker, Nikolaj and Soto, David and Doose, Sören and Dalmau, Josep and Hallermann, Stefan and Geis, Christian}, journal = {Neuron}, keywords = {werner}, number = 1, pages = {91–105.e9}, title = {Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction}, volume = 100, year = 2018 }

  %0 Journal Article %1 haselmann2018human %A Haselmann, Holger %A Mannara, Francesco %A Werner, Christian %A Planagumà, Jesús %A Miguez-Cabello, Federico %A Schmidl, Lars %A Grünewald, Benedikt %A Petit-Pedrol, Mar %A Kirmse, Knut %A Classen, Joseph %A Demir, Fatih %A Klöcker, Nikolaj %A Soto, David %A Doose, Sören %A Dalmau, Josep %A Hallermann, Stefan %A Geis, Christian %D 2018 %J Neuron %N 1 %P 91--105.e9 %R https://doi.org/10.1016/j.neuron.2018.07.048 %T Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction %U http://www.sciencedirect.com/science/article/pii/S0896627318306469 %V 100 %X AMPA receptors are essential for fast excitatory transmission in the CNS. Autoantibodies to AMPA receptors have been identified in humans with autoimmune encephalitis and severe defects of hippocampal function. Here, combining electrophysiology and high-resolution imaging with neuronal culture preparations and passive-transfer models in wild-type and GluA1-knockout mice, we analyze how specific human autoantibodies against the AMPA receptor subunit GluA2 affect receptor function and composition, synaptic transmission, and plasticity. Anti-GluA2 antibodies induce receptor internalization and a reduction of synaptic GluA2-containing AMPARs followed by compensatory ryanodine receptor-dependent incorporation of synaptic non-GluA2 AMPARs. Furthermore, application of human pathogenic anti-GluA2 antibodies to mice impairs long-term synaptic plasticity in vitro and affects learning and memory in vivo. Our results identify a specific immune-neuronal rearrangement of AMPA receptor subunits, providing a framework to explain disease symptoms.
• HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi. Brunk, Michael; Sputh, Sebastian; Doose, Sören; van de Linde, Sebastian; Terpitz, Ulrich. In Scientific Reports, 8(1), S. 605-. 2018.
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  The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.

  @article{brunk2018hyphatracker, abstract = {The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.}, author = {Brunk, Michael and Sputh, Sebastian and Doose, Sören and van de Linde, Sebastian and Terpitz, Ulrich}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {605–}, title = {HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi}, volume = 8, year = 2018 }

  %0 Journal Article %1 brunk2018hyphatracker %A Brunk, Michael %A Sputh, Sebastian %A Doose, Sören %A van de Linde, Sebastian %A Terpitz, Ulrich %D 2018 %J Scientific Reports %N 1 %P 605-- %R 10.1038/s41598-017-19103-1 %T HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi %U https://doi.org/10.1038/s41598-017-19103-1 %V 8 %X The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
• MEK-inhibitor PD184352 enhances the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922: the role of cell type and drug-irradiation schedule. Grabenbauer, Felix; Katzer, Astrid; Sisario, Dmitri; Memmel, Simon; Flentje, Michael; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S. In Oncotarget, 9, S. 37379–37392. 2018.
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  Targeting MEK protein in cancer cells usually leads to acquired resistance to MEK inhibitors and activation of the prosurvival protein Akt. Since both MEK and Akt are clients of the Hsp90 chaperone system, the present study explores the responses of irradiated lung carcinoma A549 and glioblastoma SNB19 cell lines to combined MEK and Hsp90 inhibition. Unexpectedly, the MEK inhibitor PD184352 administered 24 h prior to irradiation, enhanced cell survival through upregulation of not only MEK and Erk1/2 but also of Akt. In contrast, PD184352 added 1 h before irradiation strongly reduced the expression of Erk and did not upregulate Akt in both cell lines. As a result, the MEK inhibitor increased the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in glioblastoma SNB19 cells. Possible reasons for the enhanced cell killing under this short-term pretreatment schedule may be a down-regulation of Erk during or directly after irradiation, increased DNA damage and/or a strong G2/M arrest 24 h after irradiation. In addition, an 1-h pretreatment with PD184352 and/or NVP-AUY922 under schedule II induced neither G1 arrest nor up-regulation of p-Akt in both cell lines as it did under schedule I. Yet, a long-term treatment with the MEK inhibitor alone caused a strong cytostatical effect. We conclude that the duration of drug pretreatment before irradiation plays a key role in the targeting of MEK in tumor cells. However, due to an aberrant activation of prosurvival proteins, the therapeutic window needs to be carefully defined, or a combination of inhibitors should be considered.

  @article{grabenbauer2018mekinhibitor, abstract = {Targeting MEK protein in cancer cells usually leads to acquired resistance to MEK inhibitors and activation of the prosurvival protein Akt. Since both MEK and Akt are clients of the Hsp90 chaperone system, the present study explores the responses of irradiated lung carcinoma A549 and glioblastoma SNB19 cell lines to combined MEK and Hsp90 inhibition. Unexpectedly, the MEK inhibitor PD184352 administered 24 h prior to irradiation, enhanced cell survival through upregulation of not only MEK and Erk1/2 but also of Akt. In contrast, PD184352 added 1 h before irradiation strongly reduced the expression of Erk and did not upregulate Akt in both cell lines. As a result, the MEK inhibitor increased the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in glioblastoma SNB19 cells. Possible reasons for the enhanced cell killing under this short-term pretreatment schedule may be a down-regulation of Erk during or directly after irradiation, increased DNA damage and/or a strong G2/M arrest 24 h after irradiation. In addition, an 1-h pretreatment with PD184352 and/or NVP-AUY922 under schedule II induced neither G1 arrest nor up-regulation of p-Akt in both cell lines as it did under schedule I. Yet, a long-term treatment with the MEK inhibitor alone caused a strong cytostatical effect. We conclude that the duration of drug pretreatment before irradiation plays a key role in the targeting of MEK in tumor cells. However, due to an aberrant activation of prosurvival proteins, the therapeutic window needs to be carefully defined, or a combination of inhibitors should be considered.}, author = {Grabenbauer, Felix and Katzer, Astrid and Sisario, Dmitri and Memmel, Simon and Flentje, Michael and Sukhorukov, Vladimir L. and Djuzenova, Cholpon S.}, journal = {Oncotarget}, keywords = {vladimir}, pages = {37379-37392}, title = {MEK-inhibitor PD184352 enhances the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922: the role of cell type and drug-irradiation schedule}, volume = 9, year = 2018 }

  %0 Journal Article %1 grabenbauer2018mekinhibitor %A Grabenbauer, Felix %A Katzer, Astrid %A Sisario, Dmitri %A Memmel, Simon %A Flentje, Michael %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %D 2018 %J Oncotarget %P 37379-37392 %T MEK-inhibitor PD184352 enhances the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922: the role of cell type and drug-irradiation schedule %U https://doi.org/10.18632/oncotarget.26436 %V 9 %X Targeting MEK protein in cancer cells usually leads to acquired resistance to MEK inhibitors and activation of the prosurvival protein Akt. Since both MEK and Akt are clients of the Hsp90 chaperone system, the present study explores the responses of irradiated lung carcinoma A549 and glioblastoma SNB19 cell lines to combined MEK and Hsp90 inhibition. Unexpectedly, the MEK inhibitor PD184352 administered 24 h prior to irradiation, enhanced cell survival through upregulation of not only MEK and Erk1/2 but also of Akt. In contrast, PD184352 added 1 h before irradiation strongly reduced the expression of Erk and did not upregulate Akt in both cell lines. As a result, the MEK inhibitor increased the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in glioblastoma SNB19 cells. Possible reasons for the enhanced cell killing under this short-term pretreatment schedule may be a down-regulation of Erk during or directly after irradiation, increased DNA damage and/or a strong G2/M arrest 24 h after irradiation. In addition, an 1-h pretreatment with PD184352 and/or NVP-AUY922 under schedule II induced neither G1 arrest nor up-regulation of p-Akt in both cell lines as it did under schedule I. Yet, a long-term treatment with the MEK inhibitor alone caused a strong cytostatical effect. We conclude that the duration of drug pretreatment before irradiation plays a key role in the targeting of MEK in tumor cells. However, due to an aberrant activation of prosurvival proteins, the therapeutic window needs to be carefully defined, or a combination of inhibitors should be considered.
• Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction. Adam, Alexander; Deimel, Stephan; Pardo-Medina, Javier; García-Martínez, Jorge; Konte, Tilen; Limón, M.; Avalos, Javier; Terpitz, Ulrich. In International Journal of Molecular Sciences, 19, S. 215. 2018.
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  Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.

  @article{adam2018protein, abstract = {Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.}, author = {Adam, Alexander and Deimel, Stephan and Pardo-Medina, Javier and García-Martínez, Jorge and Konte, Tilen and Limón, M. and Avalos, Javier and Terpitz, Ulrich}, journal = {International Journal of Molecular Sciences}, keywords = {terpitz}, pages = 215, series = 1, title = {Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction}, volume = 19, year = 2018 }

  %0 Journal Article %1 adam2018protein %A Adam, Alexander %A Deimel, Stephan %A Pardo-Medina, Javier %A García-Martínez, Jorge %A Konte, Tilen %A Limón, M. %A Avalos, Javier %A Terpitz, Ulrich %B 1 %D 2018 %J International Journal of Molecular Sciences %P 215 %T Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction %U http://www.mdpi.com/1422-0067/19/1/215 %V 19 %X Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.
• Quantification of sweat gland innervation in patients with Fabry disease: A case-control study. Kokotis, Panagiotis; Üçeyler, Nurcan; Werner, Christian; Tsivgoulis, Georgios; Papanikola, Nektaria; Katsanos, Aristeidis H.; Karandreas, Nikos; Sommer, Claudia. In Journal of the Neurological Sciences, 390, S. 135–138. 2018.
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  Hypohidrosis and heat intolerance, frequently reported by men and women with Fabry disease (FD), is thought to be related not only to the deposition of globotriaosylceramide (Gb3) in eccrine sweat glands, but also to reduced sweat gland sympathetic innervation. Methods We performed a case-control study to compare the density of sweat gland innervation between patients with FD and healthy controls by examining lower leg skin punch biopsies. We used a standardized grid of circles superimposed upon the immunofluorescent specimen to create a simple pattern of circles over the sweat gland. Nerve fibers that crossed within the circles were manually counted (“crossed circles”). Nerve fibers that touched the edge of the circle but did not enter were spared (“uncrossed circles”). The percentage of crossed circles from all circles was determined. Results Biopsy specimens were available of 37 FD patients (median age 44 years, 19–67; n = 18 men) and 16 controls (median age 48 years, 24–83, n = 7 men). Totally there were 153 sweat glands from FD patients and 63 from controls, in which innervation was quantified. While mean sweat gland innervation per biopsy did not differ between the entire FD cohort and controls, data stratification for the reported sweating phenotype revealed a stepwise lower innervation in women with FD and hypohidrosis (n.s.) and anhidrosis (p < .05) compared to women reporting normal sweating. Conclusion Sweat gland innervation is reduced in women with FD and anhidrosis compared to female patients without sweating impairment. Loss of sweat gland innervation may play a role in FD associated anhidrosis, at least in women.

  @article{kokotis2018quantification, abstract = {Hypohidrosis and heat intolerance, frequently reported by men and women with Fabry disease (FD), is thought to be related not only to the deposition of globotriaosylceramide (Gb3) in eccrine sweat glands, but also to reduced sweat gland sympathetic innervation. Methods We performed a case-control study to compare the density of sweat gland innervation between patients with FD and healthy controls by examining lower leg skin punch biopsies. We used a standardized grid of circles superimposed upon the immunofluorescent specimen to create a simple pattern of circles over the sweat gland. Nerve fibers that crossed within the circles were manually counted (“crossed circles”). Nerve fibers that touched the edge of the circle but did not enter were spared (“uncrossed circles”). The percentage of crossed circles from all circles was determined. Results Biopsy specimens were available of 37 FD patients (median age 44 years, 19–67; n = 18 men) and 16 controls (median age 48 years, 24–83, n = 7 men). Totally there were 153 sweat glands from FD patients and 63 from controls, in which innervation was quantified. While mean sweat gland innervation per biopsy did not differ between the entire FD cohort and controls, data stratification for the reported sweating phenotype revealed a stepwise lower innervation in women with FD and hypohidrosis (n.s.) and anhidrosis (p < .05) compared to women reporting normal sweating. Conclusion Sweat gland innervation is reduced in women with FD and anhidrosis compared to female patients without sweating impairment. Loss of sweat gland innervation may play a role in FD associated anhidrosis, at least in women.}, author = {Kokotis, Panagiotis and Üçeyler, Nurcan and Werner, Christian and Tsivgoulis, Georgios and Papanikola, Nektaria and Katsanos, Aristeidis H. and Karandreas, Nikos and Sommer, Claudia}, journal = {Journal of the Neurological Sciences}, keywords = {werner}, pages = {135–138}, title = {Quantification of sweat gland innervation in patients with Fabry disease: A case-control study}, volume = 390, year = 2018 }

  %0 Journal Article %1 kokotis2018quantification %A Kokotis, Panagiotis %A Üçeyler, Nurcan %A Werner, Christian %A Tsivgoulis, Georgios %A Papanikola, Nektaria %A Katsanos, Aristeidis H. %A Karandreas, Nikos %A Sommer, Claudia %D 2018 %J Journal of the Neurological Sciences %P 135--138 %R https://doi.org/10.1016/j.jns.2018.04.035 %T Quantification of sweat gland innervation in patients with Fabry disease: A case-control study %U https://www.sciencedirect.com/science/article/pii/S0022510X18302041 %V 390 %X Hypohidrosis and heat intolerance, frequently reported by men and women with Fabry disease (FD), is thought to be related not only to the deposition of globotriaosylceramide (Gb3) in eccrine sweat glands, but also to reduced sweat gland sympathetic innervation. Methods We performed a case-control study to compare the density of sweat gland innervation between patients with FD and healthy controls by examining lower leg skin punch biopsies. We used a standardized grid of circles superimposed upon the immunofluorescent specimen to create a simple pattern of circles over the sweat gland. Nerve fibers that crossed within the circles were manually counted (“crossed circles”). Nerve fibers that touched the edge of the circle but did not enter were spared (“uncrossed circles”). The percentage of crossed circles from all circles was determined. Results Biopsy specimens were available of 37 FD patients (median age 44 years, 19–67; n = 18 men) and 16 controls (median age 48 years, 24–83, n = 7 men). Totally there were 153 sweat glands from FD patients and 63 from controls, in which innervation was quantified. While mean sweat gland innervation per biopsy did not differ between the entire FD cohort and controls, data stratification for the reported sweating phenotype revealed a stepwise lower innervation in women with FD and hypohidrosis (n.s.) and anhidrosis (p < .05) compared to women reporting normal sweating. Conclusion Sweat gland innervation is reduced in women with FD and anhidrosis compared to female patients without sweating impairment. Loss of sweat gland innervation may play a role in FD associated anhidrosis, at least in women.
• β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice. Tauscher, Sabine; Nakagawa, Hitoshi; Völker, Katharina; Werner, Franziska; Krebes, Lisa; Potapenko, Tamara; Doose, Sören; Birkenfeld, Andreas L.; Baba, Hideo A.; Kuhn, Michaela. In Cardiovascular Diabetology, 17, S. 103. 2018.
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  The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.

  @article{tauscher2018cellspecific, abstract = {The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.}, author = {Tauscher, Sabine and Nakagawa, Hitoshi and Völker, Katharina and Werner, Franziska and Krebes, Lisa and Potapenko, Tamara and Doose, Sören and Birkenfeld, Andreas L. and Baba, Hideo A. and Kuhn, Michaela}, journal = {Cardiovascular Diabetology}, keywords = {doose}, pages = 103, series = 1, title = {β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice}, volume = 17, year = 2018 }

  %0 Journal Article %1 tauscher2018cellspecific %A Tauscher, Sabine %A Nakagawa, Hitoshi %A Völker, Katharina %A Werner, Franziska %A Krebes, Lisa %A Potapenko, Tamara %A Doose, Sören %A Birkenfeld, Andreas L. %A Baba, Hideo A. %A Kuhn, Michaela %B 1 %D 2018 %J Cardiovascular Diabetology %P 103 %T β Cell-specific deletion of guanylyl cyclase A, the receptor for atrial natriuretic peptide, accelerates obesity-induced glucose intolerance in mice %U https://doi.org/10.1186/s12933-018-0747-3 %V 17 %X The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
• Nanostructure of DNA repair foci revealed by superresolution microscopy. Sisario, Dmitri; Memmel, Simon; Doose, Sören; Neubauer, Julia; Zimmermann, Heiko; Flentje, Michael; Djuzenova, Cholpon S.; Sauer, Markus; Sukhorukov, Vladimir L. In The FASEB Journal, S. fj.201701435-. Federation of American Societies for Experimental Biology, 2018.
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  Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

  @article{sisario2018nanostructure, abstract = {Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.}, author = {Sisario, Dmitri and Memmel, Simon and Doose, Sören and Neubauer, Julia and Zimmermann, Heiko and Flentje, Michael and Djuzenova, Cholpon S. and Sauer, Markus and Sukhorukov, Vladimir L.}, booktitle = {The FASEB Journal}, journal = {The FASEB Journal}, keywords = {sauer}, month = {06}, pages = {fj.201701435–}, publisher = {Federation of American Societies for Experimental Biology}, title = {Nanostructure of DNA repair foci revealed by superresolution microscopy}, year = 2018 }

  %0 Journal Article %1 sisario2018nanostructure %A Sisario, Dmitri %A Memmel, Simon %A Doose, Sören %A Neubauer, Julia %A Zimmermann, Heiko %A Flentje, Michael %A Djuzenova, Cholpon S. %A Sauer, Markus %A Sukhorukov, Vladimir L. %B The FASEB Journal %D 2018 %I Federation of American Societies for Experimental Biology %J The FASEB Journal %P fj.201701435-- %R 10.1096/fj.201701435 %T Nanostructure of DNA repair foci revealed by superresolution microscopy %U https://doi.org/10.1096/fj.201701435 %X Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.
• Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging. Neubert, Franziska; Beliu, Gerti; Terpitz, Ulrich; Werner, Christian; Geis, Christian; Sauer, Markus; Doose, Sören. In Angewandte Chemie International Edition, 57(50), S. 16364–16369. John Wiley & Sons, Ltd, 2018.
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  Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

  @article{neubert2018bioorthogonal, abstract = {Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.}, author = {Neubert, Franziska and Beliu, Gerti and Terpitz, Ulrich and Werner, Christian and Geis, Christian and Sauer, Markus and Doose, Sören}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {terpitz}, month = 10, number = 50, pages = {16364–16369}, publisher = {John Wiley & Sons, Ltd}, title = {Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging}, volume = 57, year = 2018 }

  %0 Journal Article %1 neubert2018bioorthogonal %A Neubert, Franziska %A Beliu, Gerti %A Terpitz, Ulrich %A Werner, Christian %A Geis, Christian %A Sauer, Markus %A Doose, Sören %B Angewandte Chemie International Edition %D 2018 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 50 %P 16364--16369 %R 10.1002/anie.201808951 %T Bioorthogonal Click Chemistry Enables Site-specific Fluorescence Labeling of Functional NMDA Receptors for Super-Resolution Imaging %U https://doi.org/10.1002/anie.201808951 %V 57 %X Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

• Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species. Markert, Sebastian M.; Bauer, Vivien; Muenz, Thomas S.; Jones, Nicola G.; Helmprobst, Frederik; Britz, Sebastian; Sauer, Markus; Rössler, Wolfgang; Engstler, Markus; Stigloher, Christian. In Correlative Light and Electron Microscopy III, Bd. 140, T. Müller-Reichert, P. Verkade (Hrsg.), S. 21–47. Academic Press, 2017.
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  Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.

  @inbook{markert2017chapter, abstract = {Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.}, author = {Markert, Sebastian M. and Bauer, Vivien and Muenz, Thomas S. and Jones, Nicola G. and Helmprobst, Frederik and Britz, Sebastian and Sauer, Markus and Rössler, Wolfgang and Engstler, Markus and Stigloher, Christian}, booktitle = {Correlative Light and Electron Microscopy III}, editor = {Müller-Reichert, Thomas and Verkade, Paul}, keywords = {sauer}, number = {Supplement C}, pages = {21–47}, publisher = {Academic Press}, title = {Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species}, volume = 140, year = 2017 }

  %0 Book Section %1 markert2017chapter %A Markert, Sebastian M. %A Bauer, Vivien %A Muenz, Thomas S. %A Jones, Nicola G. %A Helmprobst, Frederik %A Britz, Sebastian %A Sauer, Markus %A Rössler, Wolfgang %A Engstler, Markus %A Stigloher, Christian %B Correlative Light and Electron Microscopy III %D 2017 %E Müller-Reichert, Thomas %E Verkade, Paul %I Academic Press %N Supplement C %P 21--47 %R https://doi.org/10.1016/bs.mcb.2017.03.004 %T Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species %U http://www.sciencedirect.com/science/article/pii/S0091679X17300481 %V 140 %X Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.
• Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons. Scholz, Nicole; Guan, Chonglin; Nieberler, Matthias; Grotemeyer, Alexander; Maiellaro, Isabella; Gao, Shiqiang; Beck, Sebastian; Pawlak, Matthias; Sauer, Markus; Asan, Esther; Rothemund, Sven; Winkler, Jana; Prömel, Simone; Nagel, Georg; Langenhan, Tobias; Kittel, Robert J. In eLife, 6, H. J. Bellen (Hrsg.), S. e28360-. eLife Sciences Publications, Ltd, 2017.
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  Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

  @article{scholz2017mechanodependent, abstract = {Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.}, author = {Scholz, Nicole and Guan, Chonglin and Nieberler, Matthias and Grotemeyer, Alexander and Maiellaro, Isabella and Gao, Shiqiang and Beck, Sebastian and Pawlak, Matthias and Sauer, Markus and Asan, Esther and Rothemund, Sven and Winkler, Jana and Prömel, Simone and Nagel, Georg and Langenhan, Tobias and Kittel, Robert J}, editor = {Bellen, Hugo J}, journal = {eLife}, keywords = {sauer}, pages = {e28360–}, publisher = {eLife Sciences Publications, Ltd}, title = {Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons}, volume = 6, year = 2017 }

  %0 Journal Article %1 scholz2017mechanodependent %A Scholz, Nicole %A Guan, Chonglin %A Nieberler, Matthias %A Grotemeyer, Alexander %A Maiellaro, Isabella %A Gao, Shiqiang %A Beck, Sebastian %A Pawlak, Matthias %A Sauer, Markus %A Asan, Esther %A Rothemund, Sven %A Winkler, Jana %A Prömel, Simone %A Nagel, Georg %A Langenhan, Tobias %A Kittel, Robert J %D 2017 %E Bellen, Hugo J %I eLife Sciences Publications, Ltd %J eLife %P e28360-- %R 10.7554/eLife.28360 %T Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons %U https://doi.org/10.7554/eLife.28360 %V 6 %X Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
• CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells. Ziegler, Sabrina; Weiss, Esther; Schmitt, Anna-Lena; Schlegel, Jan; Burgert, Anne; Terpitz, Ulrich; Sauer, Markus; Moretta, Lorenzo; Sivori, Simona; Leonhardt, Ines; Kurzai, Oliver; Einsele, Hermann; Loeffler, Juergen. In Scientific Reports, 7(1), S. 6138-. 2017.
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  Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

  @article{ziegler2017pathogen, abstract = {Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.}, author = {Ziegler, Sabrina and Weiss, Esther and Schmitt, Anna-Lena and Schlegel, Jan and Burgert, Anne and Terpitz, Ulrich and Sauer, Markus and Moretta, Lorenzo and Sivori, Simona and Leonhardt, Ines and Kurzai, Oliver and Einsele, Hermann and Loeffler, Juergen}, journal = {Scientific Reports}, keywords = {terpitz}, number = 1, pages = {6138–}, title = {CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells}, volume = 7, year = 2017 }

  %0 Journal Article %1 ziegler2017pathogen %A Ziegler, Sabrina %A Weiss, Esther %A Schmitt, Anna-Lena %A Schlegel, Jan %A Burgert, Anne %A Terpitz, Ulrich %A Sauer, Markus %A Moretta, Lorenzo %A Sivori, Simona %A Leonhardt, Ines %A Kurzai, Oliver %A Einsele, Hermann %A Loeffler, Juergen %D 2017 %J Scientific Reports %N 1 %P 6138-- %R 10.1038/s41598-017-06238-4 %T CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells %U https://doi.org/10.1038/s41598-017-06238-4 %V 7 %X Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
• Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis. Grünewald, Benedikt; Lange, Maren D; Werner, Christian; O’Leary, Aet; Weishaupt, Andreas; Popp, Sandy; Pearce, David A; Wiendl, Heinz; Reif, Andreas; Pape, Hans C; Toyka, Klaus V; Sommer, Claudia; Geis, Christian. In eLife, 6, C. Rosenmund (Hrsg.), S. e28685-. eLife Sciences Publications, Ltd, 2017.
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  Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

  @article{grunewald2017defective, abstract = {Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.}, author = {Grünewald, Benedikt and Lange, Maren D and Werner, Christian and O'Leary, Aet and Weishaupt, Andreas and Popp, Sandy and Pearce, David A and Wiendl, Heinz and Reif, Andreas and Pape, Hans C and Toyka, Klaus V and Sommer, Claudia and Geis, Christian}, editor = {Rosenmund, Christian}, journal = {eLife}, keywords = {werner}, pages = {e28685–}, publisher = {eLife Sciences Publications, Ltd}, title = {Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis}, volume = 6, year = 2017 }

  %0 Journal Article %1 grunewald2017defective %A Grünewald, Benedikt %A Lange, Maren D %A Werner, Christian %A O'Leary, Aet %A Weishaupt, Andreas %A Popp, Sandy %A Pearce, David A %A Wiendl, Heinz %A Reif, Andreas %A Pape, Hans C %A Toyka, Klaus V %A Sommer, Claudia %A Geis, Christian %D 2017 %E Rosenmund, Christian %I eLife Sciences Publications, Ltd %J eLife %P e28685-- %R 10.7554/eLife.28685 %T Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis %U https://doi.org/10.7554/eLife.28685 %V 6 %X Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.
• Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation. Brühlmann, David; Muhr, Anais; Parker, Rebecca; Vuillemin, Thomas; Bucsella, Blanka; Kalman, Franka; Torre, Serena; La Neve, Fabio; Lembo, Antonio; Haas, Tobias; Sauer, Markus; Souquet, Jonathan; Broly, Hervé; Hemberger, Jürgen; Jordan, Martin. In Journal of Biotechnology, 252(Supplement C), S. 32–42. 2017.
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  Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.

  @article{bruhlmann2017culture, abstract = {Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.}, author = {Brühlmann, David and Muhr, Anais and Parker, Rebecca and Vuillemin, Thomas and Bucsella, Blanka and Kalman, Franka and Torre, Serena and La Neve, Fabio and Lembo, Antonio and Haas, Tobias and Sauer, Markus and Souquet, Jonathan and Broly, Hervé and Hemberger, Jürgen and Jordan, Martin}, journal = {Journal of Biotechnology}, keywords = {sauer}, number = {Supplement C}, pages = {32–42}, title = {Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation}, volume = 252, year = 2017 }

  %0 Journal Article %1 bruhlmann2017culture %A Brühlmann, David %A Muhr, Anais %A Parker, Rebecca %A Vuillemin, Thomas %A Bucsella, Blanka %A Kalman, Franka %A Torre, Serena %A La Neve, Fabio %A Lembo, Antonio %A Haas, Tobias %A Sauer, Markus %A Souquet, Jonathan %A Broly, Hervé %A Hemberger, Jürgen %A Jordan, Martin %D 2017 %J Journal of Biotechnology %N Supplement C %P 32--42 %R https://doi.org/10.1016/j.jbiotec.2017.04.026 %T Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation %U http://www.sciencedirect.com/science/article/pii/S016816561730189X %V 252 %X Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.
• Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality. Brühlmann, David; Sokolov, Michael; Butté, Alessandro; Sauer, Markus; Hemberger, Jürgen; Souquet, Jonathan; Broly, Hervé; Jordan, Martin. In Biotechnology and Bioengineering, 114(7), S. 1448–1458. 2017.
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  Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.

  @article{bruhlmann2017parallel, abstract = {Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.}, author = {Brühlmann, David and Sokolov, Michael and Butté, Alessandro and Sauer, Markus and Hemberger, Jürgen and Souquet, Jonathan and Broly, Hervé and Jordan, Martin}, journal = {Biotechnology and Bioengineering}, keywords = {sauer}, number = 7, pages = {1448–1458}, title = {Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality}, volume = 114, year = 2017 }

  %0 Journal Article %1 bruhlmann2017parallel %A Brühlmann, David %A Sokolov, Michael %A Butté, Alessandro %A Sauer, Markus %A Hemberger, Jürgen %A Souquet, Jonathan %A Broly, Hervé %A Jordan, Martin %D 2017 %J Biotechnology and Bioengineering %N 7 %P 1448--1458 %R 10.1002/bit.26269 %T Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality %U http://dx.doi.org/10.1002/bit.26269 %V 114 %X Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.
• Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle. Büchel, Gabriele; Carstensen, Anne; Mak, Ka-Yan; Roeschert, Isabelle; Leen, Eoin; Sumara, Olga; Hofstetter, Julia; Herold, Steffi; Kalb, Jacqueline; Baluapuri, Apoorva; Poon, Evon; Kwok, Colin; Chesler, Louis; Maric, Hans Michael; Rickman, David S.; Wolf, Elmar; Bayliss, Richard; Walz, Susanne; Eilers, Martin. In Cell Reports, 21(12), S. 3483–3497. 2017.
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  Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

  @article{buchel2017association, abstract = {Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.}, author = {Büchel, Gabriele and Carstensen, Anne and Mak, Ka-Yan and Roeschert, Isabelle and Leen, Eoin and Sumara, Olga and Hofstetter, Julia and Herold, Steffi and Kalb, Jacqueline and Baluapuri, Apoorva and Poon, Evon and Kwok, Colin and Chesler, Louis and Maric, Hans Michael and Rickman, David S. and Wolf, Elmar and Bayliss, Richard and Walz, Susanne and Eilers, Martin}, journal = {Cell Reports}, keywords = {maric}, number = 12, pages = {3483–3497}, title = {Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle}, volume = 21, year = 2017 }

  %0 Journal Article %1 buchel2017association %A Büchel, Gabriele %A Carstensen, Anne %A Mak, Ka-Yan %A Roeschert, Isabelle %A Leen, Eoin %A Sumara, Olga %A Hofstetter, Julia %A Herold, Steffi %A Kalb, Jacqueline %A Baluapuri, Apoorva %A Poon, Evon %A Kwok, Colin %A Chesler, Louis %A Maric, Hans Michael %A Rickman, David S. %A Wolf, Elmar %A Bayliss, Richard %A Walz, Susanne %A Eilers, Martin %D 2017 %J Cell Reports %N 12 %P 3483--3497 %R https://doi.org/10.1016/j.celrep.2017.11.090 %T Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle %U http://www.sciencedirect.com/science/article/pii/S2211124717317576 %V 21 %X Summary MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.
• Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy. Burgert, Anne; Schlegel, Jan; Bécam, Jérôme; Doose, Sören; Bieberich, Erhard; Schubert-Unkmeir, Alexandra; Sauer, Markus. In Angewandte Chemie. 2017.
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  The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

  @article{burgert2017characterization, abstract = {The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.}, author = {Burgert, Anne and Schlegel, Jan and Bécam, Jérôme and Doose, Sören and Bieberich, Erhard and Schubert-Unkmeir, Alexandra and Sauer, Markus}, journal = {Angewandte Chemie}, keywords = {sauer}, title = {Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy}, year = 2017 }

  %0 Journal Article %1 burgert2017characterization %A Burgert, Anne %A Schlegel, Jan %A Bécam, Jérôme %A Doose, Sören %A Bieberich, Erhard %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2017 %J Angewandte Chemie %R 10.1002/ange.201700570 %T Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201700570 %X The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.
• Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain. Forero, Andrea; Rivero, Olga; Wäldchen, Sina; Ku, Hsing-Ping; Kiser, Dominik P.; Gärtner, Yvonne; Pennington, Laura S.; Waider, Jonas; Gaspar, Patricia; Jansch, Charline; Edenhofer, Frank; Resink, Thérèse J.; Blum, Robert; Sauer, Markus; Lesch, Klaus-Peter. In Frontiers in Cellular Neuroscience, 11, S. 307-. 2017.
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  Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.

  @article{forero2017cadherin13, abstract = {Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.}, author = {Forero, Andrea and Rivero, Olga and Wäldchen, Sina and Ku, Hsing-Ping and Kiser, Dominik P. and Gärtner, Yvonne and Pennington, Laura S. and Waider, Jonas and Gaspar, Patricia and Jansch, Charline and Edenhofer, Frank and Resink, Thérèse J. and Blum, Robert and Sauer, Markus and Lesch, Klaus-Peter}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, pages = {307–}, title = {Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain}, volume = 11, year = 2017 }

  %0 Journal Article %1 forero2017cadherin13 %A Forero, Andrea %A Rivero, Olga %A Wäldchen, Sina %A Ku, Hsing-Ping %A Kiser, Dominik P. %A Gärtner, Yvonne %A Pennington, Laura S. %A Waider, Jonas %A Gaspar, Patricia %A Jansch, Charline %A Edenhofer, Frank %A Resink, Thérèse J. %A Blum, Robert %A Sauer, Markus %A Lesch, Klaus-Peter %D 2017 %J Frontiers in Cellular Neuroscience %P 307-- %T Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain %U https://www.frontiersin.org/article/10.3389/fncel.2017.00307 %V 11 %X Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
• Incorporation studies of clickable ceramides in Jurkat cell plasma membranes. Walter, T.; Schlegel, J.; Burgert, A.; Kurz, A.; Seibel, J.; Sauer, M. In Chem. Commun., 53(51), S. 6836–6839. The Royal Society of Chemistry, 2017.
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  The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.

  @article{walter2017incorporation, abstract = {The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.}, author = {Walter, T. and Schlegel, J. and Burgert, A. and Kurz, A. and Seibel, J. and Sauer, M.}, journal = {Chem. Commun.}, keywords = {sauer}, number = 51, pages = {6836–6839}, publisher = {The Royal Society of Chemistry}, title = {Incorporation studies of clickable ceramides in Jurkat cell plasma membranes}, volume = 53, year = 2017 }

  %0 Journal Article %1 walter2017incorporation %A Walter, T. %A Schlegel, J. %A Burgert, A. %A Kurz, A. %A Seibel, J. %A Sauer, M. %D 2017 %I The Royal Society of Chemistry %J Chem. Commun. %N 51 %P 6836--6839 %R 10.1039/C7CC01220A %T Incorporation studies of clickable ceramides in Jurkat cell plasma membranes %U http://dx.doi.org/10.1039/C7CC01220A %V 53 %X The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.
• Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging. Lukeš, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Levet, Florian; Benda, Aleš; Letschert, Sebastian; Sauer, Markus; Brdička, Tomáš; Lasser, Theo; Cebecauer, Marek. In Nature Communications, 8(1), S. 1731-. 2017.
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  Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.

  @article{lukes2017quantifying, abstract = {Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.}, author = {Lukeš, Tomáš and Glatzová, Daniela and Kvíčalová, Zuzana and Levet, Florian and Benda, Aleš and Letschert, Sebastian and Sauer, Markus and Brdička, Tomáš and Lasser, Theo and Cebecauer, Marek}, journal = {Nature Communications}, keywords = {sauer}, number = 1, pages = {1731–}, title = {Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging}, volume = 8, year = 2017 }

  %0 Journal Article %1 lukes2017quantifying %A Lukeš, Tomáš %A Glatzová, Daniela %A Kvíčalová, Zuzana %A Levet, Florian %A Benda, Aleš %A Letschert, Sebastian %A Sauer, Markus %A Brdička, Tomáš %A Lasser, Theo %A Cebecauer, Marek %D 2017 %J Nature Communications %N 1 %P 1731-- %R 10.1038/s41467-017-01857-x %T Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging %U https://doi.org/10.1038/s41467-017-01857-x %V 8 %X Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
• Single-Molecule Localization Microscopy in Eukaryotes. Sauer, Markus; Heilemann, Mike. In Chem. Rev. American Chemical Society, 2017.
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  Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.

  @article{sauer2017singlemolecule, abstract = {Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.}, author = {Sauer, Markus and Heilemann, Mike}, booktitle = {Chemical Reviews}, journal = {Chem. Rev.}, keywords = {mike}, month = {03}, publisher = {American Chemical Society}, title = {Single-Molecule Localization Microscopy in Eukaryotes}, year = 2017 }

  %0 Journal Article %1 sauer2017singlemolecule %A Sauer, Markus %A Heilemann, Mike %B Chemical Reviews %D 2017 %I American Chemical Society %J Chem. Rev. %R 10.1021/acs.chemrev.6b00667 %T Single-Molecule Localization Microscopy in Eukaryotes %U http://dx.doi.org/10.1021/acs.chemrev.6b00667 %X Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.
• Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities. Memmel, Simon; Sisario, Dmitri; Zöller, Caren; Fiedler, Vanessa; Katzer, Astrid; Heiden, Robin; Becker, Nicholas; Eing, Lorenz; Ferreira, Fábio L.R.; Zimmermann, Heiko; Sauer, Markus; Flentje, Michael; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S. In Oncotarget, 8, S. 45298–45310. 2017.
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  High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

  @article{memmel2017migration, abstract = {High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.}, author = {Memmel, Simon and Sisario, Dmitri and Zöller, Caren and Fiedler, Vanessa and Katzer, Astrid and Heiden, Robin and Becker, Nicholas and Eing, Lorenz and Ferreira, Fábio L.R. and Zimmermann, Heiko and Sauer, Markus and Flentje, Michael and Sukhorukov, Vladimir L. and Djuzenova, Cholpon S.}, journal = {Oncotarget}, keywords = {sauer}, month = {04}, pages = {45298–45310}, title = {Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities}, volume = 8, year = 2017 }

  %0 Journal Article %1 memmel2017migration %A Memmel, Simon %A Sisario, Dmitri %A Zöller, Caren %A Fiedler, Vanessa %A Katzer, Astrid %A Heiden, Robin %A Becker, Nicholas %A Eing, Lorenz %A Ferreira, Fábio L.R. %A Zimmermann, Heiko %A Sauer, Markus %A Flentje, Michael %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %D 2017 %J Oncotarget %P 45298–45310 %T Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities %U https://doi.org/10.18632/oncotarget.16847 %V 8 %X High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
• Cyanine Conformational Restraint in the Far-Red Range. Michie, Megan S.; Götz, Ralph; Franke, Christian; Bowler, Matthew; Kumari, Nikita; Magidson, Valentin; Levitus, Marcia; Loncarek, Jadranka; Sauer, Markus; Schnermann, Martin J. In J. Am. Chem. Soc., 139(36), S. 12406–12409. American Chemical Society, 2017.
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  Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

  @article{michie2017cyanine, abstract = {Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.}, author = {Michie, Megan S. and Götz, Ralph and Franke, Christian and Bowler, Matthew and Kumari, Nikita and Magidson, Valentin and Levitus, Marcia and Loncarek, Jadranka and Sauer, Markus and Schnermann, Martin J.}, booktitle = {Journal of the American Chemical Society}, journal = {J. Am. Chem. Soc.}, keywords = {sauer}, month = {09}, number = 36, pages = {12406–12409}, publisher = {American Chemical Society}, title = {Cyanine Conformational Restraint in the Far-Red Range}, volume = 139, year = 2017 }

  %0 Journal Article %1 michie2017cyanine %A Michie, Megan S. %A Götz, Ralph %A Franke, Christian %A Bowler, Matthew %A Kumari, Nikita %A Magidson, Valentin %A Levitus, Marcia %A Loncarek, Jadranka %A Sauer, Markus %A Schnermann, Martin J. %B Journal of the American Chemical Society %D 2017 %I American Chemical Society %J J. Am. Chem. Soc. %N 36 %P 12406--12409 %R 10.1021/jacs.7b07272 %T Cyanine Conformational Restraint in the Far-Red Range %U http://dx.doi.org/10.1021/jacs.7b07272 %V 139 %X Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
• Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission. Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska; Dalby, Nils Ole; Doose, Sören; Sauer, Markus; Kneussel, Matthias; Strømgaard, Kristian. In Nature Chemical Biology, 13, S. 153-. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2017.
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  γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

  @article{maric2016gephyrinbinding, abstract = {γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.}, author = {Maric, Hans Michael and Hausrat, Torben Johann and Neubert, Franziska and Dalby, Nils Ole and Doose, Sören and Sauer, Markus and Kneussel, Matthias and Strømgaard, Kristian}, journal = {Nature Chemical Biology}, keywords = {sauer}, month = 11, pages = {153–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission}, volume = 13, year = 2017 }

  %0 Journal Article %1 maric2016gephyrinbinding %A Maric, Hans Michael %A Hausrat, Torben Johann %A Neubert, Franziska %A Dalby, Nils Ole %A Doose, Sören %A Sauer, Markus %A Kneussel, Matthias %A Strømgaard, Kristian %D 2017 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Chemical Biology %P 153-- %T Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission %U https://doi.org/10.1038/nchembio.2246 %V 13 %X γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.
• Conservation of folding and association within a family of spidroin N-terminal domains. Heiby, Julia C.; Rajab, Suhaila; Rat, Charlotte; Johnson, Christopher M.; Neuweiler, Hannes. In Scientific Reports, 7(1), S. 16789. Nature Publishing Group, 2017.
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  Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.

  @article{heiby2017conservation, abstract = {Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.}, author = {Heiby, Julia C. and Rajab, Suhaila and Rat, Charlotte and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Scientific Reports}, keywords = {hannes}, month = 12, number = 1, pages = 16789, publisher = {Nature Publishing Group}, title = {Conservation of folding and association within a family of spidroin N-terminal domains}, volume = 7, year = 2017 }

  %0 Journal Article %1 heiby2017conservation %A Heiby, Julia C. %A Rajab, Suhaila %A Rat, Charlotte %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2017 %I Nature Publishing Group %J Scientific Reports %N 1 %P 16789 %R 10.1038/s41598-017-16881-6 %T Conservation of folding and association within a family of spidroin N-terminal domains %U https://doi.org/10.1038/s41598-017-16881-6 %V 7 %X Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.

• Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Niehorster, Thomas; Loschberger, Anna; Gregor, Ingo; Kramer, Benedikt; Rahn, Hans-Jurgen; Patting, Matthias; Koberling, Felix; Enderlein, Jorg; Sauer, Markus. In Nat Meth, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
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  We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

  @article{niehorster2016multitarget, abstract = {We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).}, author = {Niehorster, Thomas and Loschberger, Anna and Gregor, Ingo and Kramer, Benedikt and Rahn, Hans-Jurgen and Patting, Matthias and Koberling, Felix and Enderlein, Jorg and Sauer, Markus}, journal = {Nat Meth}, keywords = {sauer}, month = {01}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Multi-target spectrally resolved fluorescence lifetime imaging microscopy}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 niehorster2016multitarget %A Niehorster, Thomas %A Loschberger, Anna %A Gregor, Ingo %A Kramer, Benedikt %A Rahn, Hans-Jurgen %A Patting, Matthias %A Koberling, Felix %A Enderlein, Jorg %A Sauer, Markus %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Multi-target spectrally resolved fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1038/nmeth.3740 %V advance online publication %X We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).
• Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates. Alexander, Mertsch; Sebastian, Letschert; Elisabeth, Memmel; Markus, Sauer; Jürgen, Seibel. S. 347-. 2016.
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  The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.

  @misc{alexander2016synthesis, abstract = {The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.}, author = {Alexander, Mertsch and Sebastian, Letschert and Elisabeth, Memmel and Markus, Sauer and Jürgen, Seibel}, booktitle = {Zeitschrift für Naturforschung C}, keywords = {sauer}, pages = {347–}, title = {Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates}, volume = 71, year = 2016 }

  %0 Generic %1 alexander2016synthesis %A Alexander, Mertsch %A Sebastian, Letschert %A Elisabeth, Memmel %A Markus, Sauer %A Jürgen, Seibel %B Zeitschrift für Naturforschung C %D 2016 %P 347-- %R 10.1515/znc-2016-0123 %T Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates %U https://www.degruyter.com/view/j/znc.2016.71.issue-9-10/znc-2016-0123/znc-2016-0123.xml %V 71 %X The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.
• Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling. Cheng, Jin; Sahani, Sadhna; Hausrat, Torben Johann; Yang, Jenq-Wei; Ji, Haichao; Schmarowski, Nikolai; Endle, Heiko; Liu, Xinfeng; Li, Yunbo; Böttche, Rahel; Radyushkin, Konstantin; Maric, Hans M; Hoerder-Suabedissen, Anna; Molnár, Zoltán; Prouvot, Pierre-Hugues; Trimbuch, Thorsten; Ninnemann, Olaf; Huai, Jisen; Fan, Wei; Visentin, Barbara; Sabbadini, Roger; Strømgaard, Kristian; Stroh, Albrecht; Luhmann, Heiko J; Kneussel, Matthias; Nitsch, Robert; Vogt, Johannes. In Neuron, 92(1), S. 126–142. 2016.
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  Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.

  @article{cheng2016precise, abstract = {Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.}, author = {Cheng, Jin and Sahani, Sadhna and Hausrat, Torben Johann and Yang, Jenq-Wei and Ji, Haichao and Schmarowski, Nikolai and Endle, Heiko and Liu, Xinfeng and Li, Yunbo and Böttche, Rahel and Radyushkin, Konstantin and Maric, Hans M and Hoerder-Suabedissen, Anna and Molnár, Zoltán and Prouvot, Pierre-Hugues and Trimbuch, Thorsten and Ninnemann, Olaf and Huai, Jisen and Fan, Wei and Visentin, Barbara and Sabbadini, Roger and Strømgaard, Kristian and Stroh, Albrecht and Luhmann, Heiko J and Kneussel, Matthias and Nitsch, Robert and Vogt, Johannes}, journal = {Neuron}, keywords = {maric}, number = 1, pages = {126–142}, title = {Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling}, volume = 92, year = 2016 }

  %0 Journal Article %1 cheng2016precise %A Cheng, Jin %A Sahani, Sadhna %A Hausrat, Torben Johann %A Yang, Jenq-Wei %A Ji, Haichao %A Schmarowski, Nikolai %A Endle, Heiko %A Liu, Xinfeng %A Li, Yunbo %A Böttche, Rahel %A Radyushkin, Konstantin %A Maric, Hans M %A Hoerder-Suabedissen, Anna %A Molnár, Zoltán %A Prouvot, Pierre-Hugues %A Trimbuch, Thorsten %A Ninnemann, Olaf %A Huai, Jisen %A Fan, Wei %A Visentin, Barbara %A Sabbadini, Roger %A Strømgaard, Kristian %A Stroh, Albrecht %A Luhmann, Heiko J %A Kneussel, Matthias %A Nitsch, Robert %A Vogt, Johannes %D 2016 %J Neuron %N 1 %P 126--142 %R https://doi.org/10.1016/j.neuron.2016.08.035 %T Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling %U http://www.sciencedirect.com/science/article/pii/S0896627316305281 %V 92 %X Summary Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2−/− thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2−/− animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.
• Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae. Konte, Tilen; Terpitz, Ulrich; Plemenitas, Ana. In Frontiers in Microbiology, 7(901). 2016.
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  The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.

  @article{10.3389/fmicb.2016.00901, abstract = {The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.}, author = {Konte, Tilen and Terpitz, Ulrich and Plemenitas, Ana}, journal = {Frontiers in Microbiology}, keywords = {terpitz}, number = 901, title = {Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae}, volume = 7, year = 2016 }

  %0 Journal Article %1 10.3389/fmicb.2016.00901 %A Konte, Tilen %A Terpitz, Ulrich %A Plemenitas, Ana %D 2016 %J Frontiers in Microbiology %N 901 %R 10.3389/fmicb.2016.00901 %T Reconstruction of the high-osmolarity glycerol (HOG) signaling pathway from the halophilic fungus Wallemia ichthyophaga in Saccharomyces cerevisiae %U http://www.frontiersin.org/extreme_microbiology/10.3389/fmicb.2016.00901/abstract %V 7 %X The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 M and 5.1 M NaCl and is the most halophilic eukaryote described to date. Like in other fungi, in W. ichthyophaga, changes in environmental salinity are detected mainly by the evolutionarily conserved high-osmolarity glycerol (HOG) signaling pathway. In Saccharomyces cerevisiae, the HOG pathway has been extensively studied in connection to osmotic regulation, with a valuable knock-out strain collection established. In the present study, we reconstructed the architecture of the HOG pathway of W. ichthyophaga in suitable S. cerevisiae knock-out strains, through heterologous expression of the W. ichthyophaga HOG pathway proteins. Compared to S. cerevisiae, where the Pbs2 (ScPbs2) kinase of the HOG pathway is activated via the SHO1 and SLN1 branches, the interactions between the W. ichthyophaga Pbs2 (WiPbs2) kinase and the W. ichthyophaga SHO1 branch orthologs are not conserved: as well as evidence of poor interactions between the WiSho1 Src-homology 3 (SH3) domain and the WiPbs2 proline-rich motif, the absence of a considerable part of the osmosensing apparatus in the genome of W. ichthyophaga suggests that the SHO1 branch components are not involved in HOG signaling in this halophilic fungus. In contrast, the conserved activation of WiPbs2 by the S. cerevisiae ScSsk2/ScSsk22 kinase and the sensitivity of W. ichthyophaga cells to fludioxonil, emphasize the significance of two-component (SLN1-like) signaling via Group III histidine kinase. Combined with the protein modeling data, our study reveals conserved and nonconserved protein interactions in the HOG signaling pathway of W. ichthyophaga and therefore significantly improves the knowledge of hyperosmotic signal processing in this halophilic fungus.
• Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome. Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian. In Neurophotonics, 3(4), S. 041802–041802. 2016.
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  Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.

  @article{markert2016filling, abstract = {Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.}, author = {Markert, Sebastian Matthias and Britz, Sebastian and Proppert, Sven and Lang, Marietta and Witvliet, Daniel and Mulcahy, Ben and Sauer, Markus and Zhen, Mei and Bessereau, Jean-Louis and Stigloher, Christian}, journal = {Neurophotonics}, keywords = {sauer}, number = 4, pages = {041802–041802}, title = {Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome}, volume = 3, year = 2016 }

  %0 Journal Article %1 markert2016filling %A Markert, Sebastian Matthias %A Britz, Sebastian %A Proppert, Sven %A Lang, Marietta %A Witvliet, Daniel %A Mulcahy, Ben %A Sauer, Markus %A Zhen, Mei %A Bessereau, Jean-Louis %A Stigloher, Christian %D 2016 %J Neurophotonics %N 4 %P 041802--041802 %T Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome %U http://dx.doi.org/10.1117/1.NPh.3.4.041802 %V 3 %X Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.
• Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling. Yadav, Preeti; Selvaraj, Bhuvaneish T.; Bender, Florian L. P.; Behringer, Marcus; Moradi, Mehri; Sivadasan, Rajeeve; Dombert, Benjamin; Blum, Robert; Asan, Esther; Sauer, Markus; Julien, Jean-Pierre; Sendtner, Michael. In Acta Neuropathologica, 132(1), S. 93–110. 2016.
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  In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

  @article{yadav2016neurofilament, abstract = {In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.}, author = {Yadav, Preeti and Selvaraj, Bhuvaneish T. and Bender, Florian L. P. and Behringer, Marcus and Moradi, Mehri and Sivadasan, Rajeeve and Dombert, Benjamin and Blum, Robert and Asan, Esther and Sauer, Markus and Julien, Jean-Pierre and Sendtner, Michael}, journal = {Acta Neuropathologica}, keywords = {sauer}, number = 1, pages = {93–110}, title = {Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling}, volume = 132, year = 2016 }

  %0 Journal Article %1 yadav2016neurofilament %A Yadav, Preeti %A Selvaraj, Bhuvaneish T. %A Bender, Florian L. P. %A Behringer, Marcus %A Moradi, Mehri %A Sivadasan, Rajeeve %A Dombert, Benjamin %A Blum, Robert %A Asan, Esther %A Sauer, Markus %A Julien, Jean-Pierre %A Sendtner, Michael %D 2016 %J Acta Neuropathologica %N 1 %P 93--110 %R 10.1007/s00401-016-1564-y %T Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling %U http://dx.doi.org/10.1007/s00401-016-1564-y %V 132 %X In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
• Super-resolution imaging of plasma membrane proteins with click chemistry. Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Soeren; Sauer, Markus. In Frontiers in Cell and Developmental Biology, 4, S. 98-. 2016.
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  Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.

  @article{mateosgil2016superresolution, abstract = {Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.}, author = {Mateos-Gil, Pablo and Letschert, Sebastian and Doose, Soeren and Sauer, Markus}, journal = {Frontiers in Cell and Developmental Biology}, keywords = {sauer}, pages = {98–}, title = {Super-resolution imaging of plasma membrane proteins with click chemistry}, volume = 4, year = 2016 }

  %0 Journal Article %1 mateosgil2016superresolution %A Mateos-Gil, Pablo %A Letschert, Sebastian %A Doose, Soeren %A Sauer, Markus %D 2016 %J Frontiers in Cell and Developmental Biology %P 98-- %T Super-resolution imaging of plasma membrane proteins with click chemistry %U http://journal.frontiersin.org/article/10.3389/fcell.2016.00098 %V 4 %X Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.
• Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule. Djuzenova, Cholpon; Fiedler, Vanessa; Katzer, Astrid; Michel, Konstanze; Deckert, Stefanie; Zimmermann, Heiko; Sukhorukov, Vladimir; Flentje, Michael. In Oncotarget, 5. 2016.
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  Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells

  @article{OT9501, abstract = {Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells}, author = {Djuzenova, Cholpon and Fiedler, Vanessa and Katzer, Astrid and Michel, Konstanze and Deckert, Stefanie and Zimmermann, Heiko and Sukhorukov, Vladimir and Flentje, Michael}, journal = {Oncotarget}, keywords = {vladimir}, title = {Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule}, volume = 5, year = 2016 }

  %0 Journal Article %1 OT9501 %A Djuzenova, Cholpon %A Fiedler, Vanessa %A Katzer, Astrid %A Michel, Konstanze %A Deckert, Stefanie %A Zimmermann, Heiko %A Sukhorukov, Vladimir %A Flentje, Michael %D 2016 %J Oncotarget %T Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule %U http://www.ncbi.nlm.nih.gov/pubmed/27224913 %V 5 %X Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells
• Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition. Werner, Christian; Pauli, Martin; Doose, Sören; Weishaupt, Andreas; Haselmann, Holger; Grünewald, Benedikt; Sauer, Markus; Heckmann, Manfred; Toyka, Klaus V.; Asan, Esther; Sommer, Claudia; Geis, Christian. In Brain, 139(2), S. 365–379. 2016.
  • [ Abstract ]
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  See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

  @article{werner2016human, abstract = {See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.}, author = {Werner, Christian and Pauli, Martin and Doose, Sören and Weishaupt, Andreas and Haselmann, Holger and Grünewald, Benedikt and Sauer, Markus and Heckmann, Manfred and Toyka, Klaus V. and Asan, Esther and Sommer, Claudia and Geis, Christian}, journal = {Brain}, keywords = {werner}, month = {02}, number = 2, pages = {365–379}, title = {Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition}, volume = 139, year = 2016 }

  %0 Journal Article %1 werner2016human %A Werner, Christian %A Pauli, Martin %A Doose, Sören %A Weishaupt, Andreas %A Haselmann, Holger %A Grünewald, Benedikt %A Sauer, Markus %A Heckmann, Manfred %A Toyka, Klaus V. %A Asan, Esther %A Sommer, Claudia %A Geis, Christian %D 2016 %J Brain %N 2 %P 365--379 %T Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition %U http://dx.doi.org/10.1093/brain/awv324 %V 139 %X See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.
• Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission. Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska; Dalby, Nils Ole; Doose, Sören; Sauer, Markus; Kneussel, Matthias; Strømgaard, Kristian. In Nature Chemical Biology, 13, S. 153-. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
  • [ Abstract ]
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  γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

  @article{maric2016gephyrinbinding, abstract = {γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.}, author = {Maric, Hans Michael and Hausrat, Torben Johann and Neubert, Franziska and Dalby, Nils Ole and Doose, Sören and Sauer, Markus and Kneussel, Matthias and Strømgaard, Kristian}, journal = {Nature Chemical Biology}, keywords = {maric}, month = 11, pages = {153–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission}, volume = 13, year = 2016 }

  %0 Journal Article %1 maric2016gephyrinbinding %A Maric, Hans Michael %A Hausrat, Torben Johann %A Neubert, Franziska %A Dalby, Nils Ole %A Doose, Sören %A Sauer, Markus %A Kneussel, Matthias %A Strømgaard, Kristian %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Chemical Biology %P 153-- %T Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission %U https://doi.org/10.1038/nchembio.2246 %V 13 %X γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.
• A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells. Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle. In The Journal of Immunology, 196(9), S. 3951–3962. 2016.
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  Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.

  @article{collenburg2016functionalized, abstract = {Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.}, author = {Collenburg, Lena and Walter, Tim and Burgert, Anne and Müller, Nora and Seibel, Jürgen and Japtok, Lukasz and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle}, journal = {The Journal of Immunology}, keywords = {sauer}, month = {05}, number = 9, pages = {3951–3962}, title = {A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells}, volume = 196, year = 2016 }

  %0 Journal Article %1 collenburg2016functionalized %A Collenburg, Lena %A Walter, Tim %A Burgert, Anne %A Müller, Nora %A Seibel, Jürgen %A Japtok, Lukasz %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %D 2016 %J The Journal of Immunology %N 9 %P 3951--3962 %T A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells %U http://www.jimmunol.org/content/196/9/3951.abstract %V 196 %X Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
• The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Dugar, Gaurav; Svensson, Sarah L.; Bischler, Thorsten; Wäldchen, Sina; Reinhardt, Richard; Sauer, Markus; Sharma, Cynthia M. In Nature Communications, 7, S. 11667-. The Author(s), 2016.
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  The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.

  @article{dugar2016csrafliw, abstract = {The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.}, author = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Wäldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.}, journal = {Nature Communications}, keywords = {sauer}, month = {05}, pages = {11667–}, publisher = {The Author(s)}, title = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni}, volume = 7, year = 2016 }

  %0 Journal Article %1 dugar2016csrafliw %A Dugar, Gaurav %A Svensson, Sarah L. %A Bischler, Thorsten %A Wäldchen, Sina %A Reinhardt, Richard %A Sauer, Markus %A Sharma, Cynthia M. %D 2016 %I The Author(s) %J Nature Communications %P 11667-- %T The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni %U http://dx.doi.org/10.1038/ncomms11667 %V 7 %X The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.
• Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism. Schulze, Andrea; Beliu, Gerti; Helmerich, Dominic A; Schubert, Jonathan; Pearl, Laurence H; Prodromou, Chrisostomos; Neuweiler, Hannes. In Nat Chem Biol, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
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  The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

  @article{schulze2016cooperation, abstract = {The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.}, author = {Schulze, Andrea and Beliu, Gerti and Helmerich, Dominic A and Schubert, Jonathan and Pearl, Laurence H and Prodromou, Chrisostomos and Neuweiler, Hannes}, journal = {Nat Chem Biol}, keywords = {hannes}, month = {06}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 schulze2016cooperation %A Schulze, Andrea %A Beliu, Gerti %A Helmerich, Dominic A %A Schubert, Jonathan %A Pearl, Laurence H %A Prodromou, Chrisostomos %A Neuweiler, Hannes %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Chem Biol %T Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism %U http://dx.doi.org/10.1038/nchembio.2111 %V advance online publication %X The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.
• Optochemokine Tandem for Light-Control of Intracellular Ca²⁺.. Feldbauer, Katrin; Schlegel, Jan; Weissbecker, Juliane; Sauer, Frank; Wood, Phillip G.; Bamberg, Ernst; Terpitz, Ulrich. In PLoS ONE, 11(10), S. e0165344-. Public Library of Science, 2016.
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  <p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>

  @article{feldbauer2016optochemokine, abstract = {<p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>}, author = {Feldbauer, Katrin and Schlegel, Jan and Weissbecker, Juliane and Sauer, Frank and Wood, Phillip G. and Bamberg, Ernst and Terpitz, Ulrich}, journal = {PLoS ONE}, keywords = {terpitz}, month = 10, number = 10, pages = {e0165344–}, publisher = {Public Library of Science}, title = {Optochemokine Tandem for Light-Control of Intracellular Ca²⁺}, volume = 11, year = 2016 }

  %0 Journal Article %1 feldbauer2016optochemokine %A Feldbauer, Katrin %A Schlegel, Jan %A Weissbecker, Juliane %A Sauer, Frank %A Wood, Phillip G. %A Bamberg, Ernst %A Terpitz, Ulrich %D 2016 %I Public Library of Science %J PLoS ONE %N 10 %P e0165344-- %T Optochemokine Tandem for Light-Control of Intracellular Ca²⁺ %U http://dx.doi.org/10.1371/journal.pone.0165344 %V 11 %X <p>An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca²⁺-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca²⁺ by tandem endosomes into the cytosol via CatCh was visualized using the Ca²⁺-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca²⁺ in response to light.</p>
• OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors. Jung, L A; Gebhardt, A; Koelmel, W; Ade, C P; Walz, S; Kuper, J; von Eyss, B; Letschert, S; Redel, C; d’Artista, L; Biankin, A; Zender, L; Sauer, M; Wolf, E; Evan, G; Kisker, C; Eilers, M. In Oncogene, 36, S. 1911-. Macmillan Publishers Limited, part of Springer Nature., 2016.
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  MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

  @article{jung2016omomyc, abstract = {MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.}, author = {Jung, L A and Gebhardt, A and Koelmel, W and Ade, C P and Walz, S and Kuper, J and von Eyss, B and Letschert, S and Redel, C and d'Artista, L and Biankin, A and Zender, L and Sauer, M and Wolf, E and Evan, G and Kisker, C and Eilers, M}, journal = {Oncogene}, keywords = {sauer}, month = 10, pages = {1911–}, publisher = {Macmillan Publishers Limited, part of Springer Nature.}, title = {OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors}, volume = 36, year = 2016 }

  %0 Journal Article %1 jung2016omomyc %A Jung, L A %A Gebhardt, A %A Koelmel, W %A Ade, C P %A Walz, S %A Kuper, J %A von Eyss, B %A Letschert, S %A Redel, C %A d'Artista, L %A Biankin, A %A Zender, L %A Sauer, M %A Wolf, E %A Evan, G %A Kisker, C %A Eilers, M %D 2016 %I Macmillan Publishers Limited, part of Springer Nature. %J Oncogene %P 1911-- %T OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors %U http://dx.doi.org/10.1038/onc.2016.354 %V 36 %X MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.
• Photometry unlocks 3D information from 2D localization microscopy data. Franke, Christian; Sauer, Markus; van de Linde, Sebastian. In Nat Meth, advance online publication. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2016.
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  We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

  @article{franke2016photometry, abstract = {We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.}, author = {Franke, Christian and Sauer, Markus and van de Linde, Sebastian}, journal = {Nat Meth}, keywords = {linde}, month = 11, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Photometry unlocks 3D information from 2D localization microscopy data}, volume = {advance online publication}, year = 2016 }

  %0 Journal Article %1 franke2016photometry %A Franke, Christian %A Sauer, Markus %A van de Linde, Sebastian %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Photometry unlocks 3D information from 2D localization microscopy data %U http://dx.doi.org/10.1038/nmeth.4073 %V advance online publication %X We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

• Interactions of Human Autoantibodies with Hippocampal GABAergic Synaptic Transmission – Analyzing Antibody-Induced Effects ex vivo. Haselmann, Holger; Röpke, Luise; Werner, Christian; Kunze, Albrecht; Geis, Christian. In Frontiers in Neurology, 6. 2015.
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  Autoantibodies (aAB) to the presynaptic located enzyme glutamate decarboxylase 65 (GAD65) are a characteristic attribute for a variety of autoimmune diseases of the central nervous system including subtypes of limbic encephalitis, stiff person-syndrome, cerebellar ataxia, and Batten’s disease. Clinical signs of hyperexcitability and improvement of disease symptoms upon immunotherapy in some of these disorders suggest a possible pathogenic role of associated aAB. Recent experimental studies report inconsistent results regarding a direct pathogenic influence of anti-GAD65 aAB affecting inhibitory synaptic transmission in central GABAergic pathways. We here provide a method for direct evaluation of aAB-induced pathomechanisms in the intact hippocampal network. Purified patient IgG fractions containing aAB to GAD65 together with fixable lipophilic styryl dyes (FMdyes) are stereotactically injected into the hilus and the dentate gyrus in anesthetized mice. Twenty-four hours after intrahippocampal injection, acute hippocampal slices are prepared and transferred to a patch-clamp recording setup equipped with a fluorescence light source. Intraneural incorporated FMdyes show correct injection site for patch-clamp recording. Whole-cell patch-clamp recordings are performed from granule cells in the dentate gyrus and extracellular stimulation is applied in the border area of the dentate gyrus-hilus region to stimulate GABAergic afferents arising from parvalbumin positive basket cells. GABA-A receptor mediated inhibitory postsynaptic currents (IPSC) and miniature IPSC are recorded after blocking glutamatergic transmission. This approach allows investigation of potential aAB-induced effects on GABA-A receptor signaling ex vivo in an intact neuronal network. This offers several advantages compared to experimental procedures used in previous studies by in vitro AB preincubation of primary neurons or slice preparations. Furthermore, this method requires only small amounts of patient material that are often limited in rare diseases.

  @article{haselmann2015interactions, abstract = {Autoantibodies (aAB) to the presynaptic located enzyme glutamate decarboxylase 65 (GAD65) are a characteristic attribute for a variety of autoimmune diseases of the central nervous system including subtypes of limbic encephalitis, stiff person-syndrome, cerebellar ataxia, and Batten’s disease. Clinical signs of hyperexcitability and improvement of disease symptoms upon immunotherapy in some of these disorders suggest a possible pathogenic role of associated aAB. Recent experimental studies report inconsistent results regarding a direct pathogenic influence of anti-GAD65 aAB affecting inhibitory synaptic transmission in central GABAergic pathways. We here provide a method for direct evaluation of aAB-induced pathomechanisms in the intact hippocampal network. Purified patient IgG fractions containing aAB to GAD65 together with fixable lipophilic styryl dyes (FMdyes) are stereotactically injected into the hilus and the dentate gyrus in anesthetized mice. Twenty-four hours after intrahippocampal injection, acute hippocampal slices are prepared and transferred to a patch-clamp recording setup equipped with a fluorescence light source. Intraneural incorporated FMdyes show correct injection site for patch-clamp recording. Whole-cell patch-clamp recordings are performed from granule cells in the dentate gyrus and extracellular stimulation is applied in the border area of the dentate gyrus-hilus region to stimulate GABAergic afferents arising from parvalbumin positive basket cells. GABA-A receptor mediated inhibitory postsynaptic currents (IPSC) and miniature IPSC are recorded after blocking glutamatergic transmission. This approach allows investigation of potential aAB-induced effects on GABA-A receptor signaling ex vivo in an intact neuronal network. This offers several advantages compared to experimental procedures used in previous studies by in vitro AB preincubation of primary neurons or slice preparations. Furthermore, this method requires only small amounts of patient material that are often limited in rare diseases.}, author = {Haselmann, Holger and Röpke, Luise and Werner, Christian and Kunze, Albrecht and Geis, Christian}, journal = {Frontiers in Neurology}, keywords = {werner}, title = {Interactions of Human Autoantibodies with Hippocampal GABAergic Synaptic Transmission – Analyzing Antibody-Induced Effects ex vivo}, volume = 6, year = 2015 }

  %0 Journal Article %1 haselmann2015interactions %A Haselmann, Holger %A Röpke, Luise %A Werner, Christian %A Kunze, Albrecht %A Geis, Christian %D 2015 %J Frontiers in Neurology %T Interactions of Human Autoantibodies with Hippocampal GABAergic Synaptic Transmission – Analyzing Antibody-Induced Effects ex vivo %U https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2015.00136 %V 6 %X Autoantibodies (aAB) to the presynaptic located enzyme glutamate decarboxylase 65 (GAD65) are a characteristic attribute for a variety of autoimmune diseases of the central nervous system including subtypes of limbic encephalitis, stiff person-syndrome, cerebellar ataxia, and Batten’s disease. Clinical signs of hyperexcitability and improvement of disease symptoms upon immunotherapy in some of these disorders suggest a possible pathogenic role of associated aAB. Recent experimental studies report inconsistent results regarding a direct pathogenic influence of anti-GAD65 aAB affecting inhibitory synaptic transmission in central GABAergic pathways. We here provide a method for direct evaluation of aAB-induced pathomechanisms in the intact hippocampal network. Purified patient IgG fractions containing aAB to GAD65 together with fixable lipophilic styryl dyes (FMdyes) are stereotactically injected into the hilus and the dentate gyrus in anesthetized mice. Twenty-four hours after intrahippocampal injection, acute hippocampal slices are prepared and transferred to a patch-clamp recording setup equipped with a fluorescence light source. Intraneural incorporated FMdyes show correct injection site for patch-clamp recording. Whole-cell patch-clamp recordings are performed from granule cells in the dentate gyrus and extracellular stimulation is applied in the border area of the dentate gyrus-hilus region to stimulate GABAergic afferents arising from parvalbumin positive basket cells. GABA-A receptor mediated inhibitory postsynaptic currents (IPSC) and miniature IPSC are recorded after blocking glutamatergic transmission. This approach allows investigation of potential aAB-induced effects on GABA-A receptor signaling ex vivo in an intact neuronal network. This offers several advantages compared to experimental procedures used in previous studies by in vitro AB preincubation of primary neurons or slice preparations. Furthermore, this method requires only small amounts of patient material that are often limited in rare diseases.
• β-Structure within the Denatured State of the Helical Protein Domain BBL. Thukral, Lipi; Schwarze, Simone; Daidone, Isabella; Neuweiler, Hannes. In Journal of Molecular Biology. 2015.
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  Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.

  @article{thukralstructure, abstract = {Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.}, author = {Thukral, Lipi and Schwarze, Simone and Daidone, Isabella and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, title = {β-Structure within the Denatured State of the Helical Protein Domain BBL}, year = 2015 }

  %0 Journal Article %1 thukralstructure %A Thukral, Lipi %A Schwarze, Simone %A Daidone, Isabella %A Neuweiler, Hannes %D 2015 %J Journal of Molecular Biology %R http://dx.doi.org/10.1016/j.jmb.2015.08.007 %T β-Structure within the Denatured State of the Helical Protein Domain BBL %U http://www.sciencedirect.com/science/article/pii/S0022283615004520 %X Abstract Protein denatured states are the origin of both healthy and toxic conformational species. Denatured states of ultrafast folding proteins are of interest in mechanistic studies because they are energetically close to the kinetic bottleneck of folding. However, their transient nature makes them elusive to experiment. Here, we generated the denatured state of the helical domain BBL that is poised to fold in microseconds by a single-point mutation and combined circular dichroism spectroscopy, single-molecule fluorescence fluctuation analysis, and computer simulation to characterize its structure and dynamics. Circular dichroism showed a largely unfolded ensemble with marginal helix but significant β-sheet content. Main-chain structure and dynamics were unaffected by side-chain interactions that stabilize the native state, as revealed by site-directed mutagenesis and nanosecond loop closure kinetics probed by fluorescence correlation spectroscopy. Replica-exchange and constant-temperature molecular dynamics simulations showed a highly collapsed, hydrogen-bonded denatured state containing turn and β-sheet structure and few nucleating helices in an otherwise unfolded ensemble. An irregular β-hairpin element that connects helices in the native fold was poised to be formed. The surprising observation of β-structure in regions that form helices in the native state is reconciled by a generic low-energy pathway from the northwest quadrant of Ramachandran space to the helical basin present under folding conditions, proposed recently. Our results show that, indeed, rapid nucleation of helix emanates from β-structure formed early within a collapsed ensemble of unfolded conformers.
• Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution. Schücker, Katharina; Holm, Thorge; Franke, Christian; Sauer, Markus; Benavente, Ricardo. In Proceedings of the National Academy of Sciences, 112(7), S. 2029–2033. 2015.
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  Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.

  @article{Schücker17022015, abstract = {Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.}, author = {Schücker, Katharina and Holm, Thorge and Franke, Christian and Sauer, Markus and Benavente, Ricardo}, journal = {Proceedings of the National Academy of Sciences}, keywords = {sauer}, number = 7, pages = {2029-2033}, title = {Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution}, volume = 112, year = 2015 }

  %0 Journal Article %1 Schücker17022015 %A Schücker, Katharina %A Holm, Thorge %A Franke, Christian %A Sauer, Markus %A Benavente, Ricardo %D 2015 %J Proceedings of the National Academy of Sciences %N 7 %P 2029-2033 %R 10.1073/pnas.1414814112 %T Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution %U http://www.pnas.org/content/112/7/2029.abstract %V 112 %X Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.
• Tailoring recombinant protein quality by rational media design. Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé. In Biotechnology Progress, 31(3), S. 615–629. 2015.
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  Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

  @article{bruhlmann2015tailoring, abstract = {Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015}, author = {Brühlmann, David and Jordan, Martin and Hemberger, Jürgen and Sauer, Markus and Stettler, Matthieu and Broly, Hervé}, journal = {Biotechnology Progress}, keywords = {sauer}, number = 3, pages = {615–629}, title = {Tailoring recombinant protein quality by rational media design}, volume = 31, year = 2015 }

  %0 Journal Article %1 bruhlmann2015tailoring %A Brühlmann, David %A Jordan, Martin %A Hemberger, Jürgen %A Sauer, Markus %A Stettler, Matthieu %A Broly, Hervé %D 2015 %J Biotechnology Progress %N 3 %P 615--629 %R 10.1002/btpr.2089 %T Tailoring recombinant protein quality by rational media design %U http://dx.doi.org/10.1002/btpr.2089 %V 31 %X Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015
• Quantifying molecular colocalization in live cell fluorescence microscopy. Humpert, Fabian; Yahiatène, Idir; Lummer, Martina; Sauer, Markus; Huser, Thomas. In Journal of Biophotonics, 8(1-2), S. 124–132. WILEY-VCH Verlag, 2015.
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  One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

  @article{humpert2015quantifying, abstract = {One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)}, author = {Humpert, Fabian and Yahiatène, Idir and Lummer, Martina and Sauer, Markus and Huser, Thomas}, journal = {Journal of Biophotonics}, keywords = {sauer}, number = {1-2}, pages = {124–132}, publisher = {WILEY-VCH Verlag}, title = {Quantifying molecular colocalization in live cell fluorescence microscopy}, volume = 8, year = 2015 }

  %0 Journal Article %1 humpert2015quantifying %A Humpert, Fabian %A Yahiatène, Idir %A Lummer, Martina %A Sauer, Markus %A Huser, Thomas %D 2015 %I WILEY-VCH Verlag %J Journal of Biophotonics %N 1-2 %P 124--132 %R 10.1002/jbio.201300146 %T Quantifying molecular colocalization in live cell fluorescence microscopy %U http://dx.doi.org/10.1002/jbio.201300146 %V 8 %X One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)
• Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy. Grimm, Jonathan B.; Klein, Teresa; Kopek, Benjamin G.; Shtengel, Gleb; Hess, Harald F.; Sauer, Markus; Lavis, Luke D. In Angewandte Chemie, S. n/a-n/a. WILEY-VCH Verlag, 2015.
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  The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.

  @article{grimm2015synthesis, abstract = {The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.}, author = {Grimm, Jonathan B. and Klein, Teresa and Kopek, Benjamin G. and Shtengel, Gleb and Hess, Harald F. and Sauer, Markus and Lavis, Luke D.}, journal = {Angewandte Chemie}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy}, year = 2015 }

  %0 Journal Article %1 grimm2015synthesis %A Grimm, Jonathan B. %A Klein, Teresa %A Kopek, Benjamin G. %A Shtengel, Gleb %A Hess, Harald F. %A Sauer, Markus %A Lavis, Luke D. %D 2015 %I WILEY-VCH Verlag %J Angewandte Chemie %P n/a--n/a %R 10.1002/ange.201509649 %T Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201509649 %X The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.
• Super-resolution microscopy of the synaptic active zone. Ehmann, Nadine; Sauer, Markus; Kittel, Robert J. In Frontiers in Cellular Neuroscience, 9(7). 2015.
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  Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.

  @article{10.3389/fncel.2015.00007, abstract = {Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.}, author = {Ehmann, Nadine and Sauer, Markus and Kittel, Robert J}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 7, title = {Super-resolution microscopy of the synaptic active zone}, volume = 9, year = 2015 }

  %0 Journal Article %1 10.3389/fncel.2015.00007 %A Ehmann, Nadine %A Sauer, Markus %A Kittel, Robert J %D 2015 %J Frontiers in Cellular Neuroscience %N 7 %R 10.3389/fncel.2015.00007 %T Super-resolution microscopy of the synaptic active zone %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00007/abstract %V 9 %X Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.
• Artifacts in single-molecule localization microscopy. Burgert, Anne; Letschert, Sebastian; Doose, Sören; Sauer, Markus. In Histochemistry and Cell Biology, 144, S. 123–131. 2015.
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  Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

  @article{burgert2015artifacts, abstract = {Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.}, author = {Burgert, Anne and Letschert, Sebastian and Doose, Sören and Sauer, Markus}, journal = {Histochemistry and Cell Biology}, keywords = {sauer}, pages = {123-131}, series = 2, title = {Artifacts in single-molecule localization microscopy}, volume = 144, year = 2015 }

  %0 Journal Article %1 burgert2015artifacts %A Burgert, Anne %A Letschert, Sebastian %A Doose, Sören %A Sauer, Markus %B 2 %D 2015 %J Histochemistry and Cell Biology %P 123-131 %T Artifacts in single-molecule localization microscopy %U http://dx.doi.org/10.1007/s00418-015-1340-4 %V 144 %X Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.
• Stiff person-syndrome IgG affects presynaptic GABAergic release mechanisms. Werner, Christian; Haselmann, Holger; Weishaupt, Andreas; Toyka, Klaus V.; Sommer, Claudia; Geis, Christian. In Journal of Neural Transmission, 122(3), S. 357–362. 2015.
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  The majority of patients with stiff person-syndrome (SPS) are characterized by autoantibodies to glutamate decarboxylase 65 (GAD65). In previous passive-transfer studies, SPS immunoglobulin G (IgG) induced SPS core symptoms. We here provide evidence that SPS-IgG causes a higher frequency of spontaneous vesicle fusions. Sustained GABAergic transmission and presynaptic GABAergic vesicle pool size remained unchanged. Since these findings cannot be attributed to anti-GAD65 autoantibodies alone, we propose that additional autoantibodies with so far undefined antigen specificity might affect presynaptic release mechanisms.

  @article{werner2015stiff, abstract = {The majority of patients with stiff person-syndrome (SPS) are characterized by autoantibodies to glutamate decarboxylase 65 (GAD65). In previous passive-transfer studies, SPS immunoglobulin G (IgG) induced SPS core symptoms. We here provide evidence that SPS-IgG causes a higher frequency of spontaneous vesicle fusions. Sustained GABAergic transmission and presynaptic GABAergic vesicle pool size remained unchanged. Since these findings cannot be attributed to anti-GAD65 autoantibodies alone, we propose that additional autoantibodies with so far undefined antigen specificity might affect presynaptic release mechanisms.}, author = {Werner, Christian and Haselmann, Holger and Weishaupt, Andreas and Toyka, Klaus V. and Sommer, Claudia and Geis, Christian}, journal = {Journal of Neural Transmission}, keywords = {werner}, number = 3, pages = {357–362}, title = {Stiff person-syndrome IgG affects presynaptic GABAergic release mechanisms}, volume = 122, year = 2015 }

  %0 Journal Article %1 werner2015stiff %A Werner, Christian %A Haselmann, Holger %A Weishaupt, Andreas %A Toyka, Klaus V. %A Sommer, Claudia %A Geis, Christian %D 2015 %J Journal of Neural Transmission %N 3 %P 357--362 %R 10.1007/s00702-014-1268-1 %T Stiff person-syndrome IgG affects presynaptic GABAergic release mechanisms %U https://doi.org/10.1007/s00702-014-1268-1 %V 122 %X The majority of patients with stiff person-syndrome (SPS) are characterized by autoantibodies to glutamate decarboxylase 65 (GAD65). In previous passive-transfer studies, SPS immunoglobulin G (IgG) induced SPS core symptoms. We here provide evidence that SPS-IgG causes a higher frequency of spontaneous vesicle fusions. Sustained GABAergic transmission and presynaptic GABAergic vesicle pool size remained unchanged. Since these findings cannot be attributed to anti-GAD65 autoantibodies alone, we propose that additional autoantibodies with so far undefined antigen specificity might affect presynaptic release mechanisms.
• Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation. Paul, Mila M; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J; Heckmann, Manfred. In Frontiers in Cellular Neuroscience, 9(29). 2015.
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  The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.

  @article{10.3389/fncel.2015.00029, abstract = {The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.}, author = {Paul, Mila M and Pauli, Martin and Ehmann, Nadine and Hallermann, Stefan and Sauer, Markus and Kittel, Robert J and Heckmann, Manfred}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 29, title = {Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation}, volume = 9, year = 2015 }

  %0 Journal Article %1 10.3389/fncel.2015.00029 %A Paul, Mila M %A Pauli, Martin %A Ehmann, Nadine %A Hallermann, Stefan %A Sauer, Markus %A Kittel, Robert J %A Heckmann, Manfred %D 2015 %J Frontiers in Cellular Neuroscience %N 29 %R 10.3389/fncel.2015.00029 %T Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00029/abstract %V 9 %X The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
• Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy. Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U.; Westerling, Katherine M.; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L. In PLoS ONE, 10(3), S. e0119990. Public Library of Science, 2015.
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  Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

  @article{10.1371/journal.pone.0119990, abstract = {Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of  3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.}, author = {Andronic, Joseph and Shirakashi, Ryo and Pickel, Simone U. and Westerling, Katherine M. and Klein, Teresa and Holm, Thorge and Sauer, Markus and Sukhorukov, Vladimir L.}, journal = {PLoS ONE}, keywords = {sauer}, month = {03}, number = 3, pages = {e0119990}, publisher = {Public Library of Science}, title = {Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy}, volume = 10, year = 2015 }

  %0 Journal Article %1 10.1371/journal.pone.0119990 %A Andronic, Joseph %A Shirakashi, Ryo %A Pickel, Simone U. %A Westerling, Katherine M. %A Klein, Teresa %A Holm, Thorge %A Sauer, Markus %A Sukhorukov, Vladimir L. %D 2015 %I Public Library of Science %J PLoS ONE %N 3 %P e0119990 %R 10.1371/journal.pone.0119990 %T Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy %U http://dx.doi.org/10.1371%2Fjournal.pone.0119990 %V 10 %X Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
• Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung. Terpitz, U. In Naturwissenschaftliche Rundschau, 68(9), S. 465–466. 2015.
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  @article{terpitz2015rhodopsin, author = {Terpitz, U.}, journal = {Naturwissenschaftliche Rundschau}, keywords = {terpitz}, number = 9, pages = {465-466}, title = {Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung}, volume = 68, year = 2015 }

  %0 Journal Article %1 terpitz2015rhodopsin %A Terpitz, U. %D 2015 %J Naturwissenschaftliche Rundschau %N 9 %P 465-466 %T Ein Rhodopsin des Pilzes Fusarium fujikuroi reguliert die Sporenkeimung %V 68
• Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium. Schneider, Johannes; Klein, Teresa; Mielich-Süss, Benjamin; Koch, Gudrun; Franke, Christian; Kuipers, Oscar P.; Kovács, Ákos T.; Sauer, Markus; Lopez, Daniel. In PLoS Genet, 11(4), S. e1005140-. Public Library of Science, 2015.
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  <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>

  @article{schneider2015spatiotemporal, abstract = {<title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>}, author = {Schneider, Johannes and Klein, Teresa and Mielich-Süss, Benjamin and Koch, Gudrun and Franke, Christian and Kuipers, Oscar P. and Kovács, Ákos T. and Sauer, Markus and Lopez, Daniel}, journal = {PLoS Genet}, keywords = {sauer}, month = {04}, number = 4, pages = {e1005140–}, publisher = {Public Library of Science}, title = {Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium}, volume = 11, year = 2015 }

  %0 Journal Article %1 schneider2015spatiotemporal %A Schneider, Johannes %A Klein, Teresa %A Mielich-Süss, Benjamin %A Koch, Gudrun %A Franke, Christian %A Kuipers, Oscar P. %A Kovács, Ákos T. %A Sauer, Markus %A Lopez, Daniel %D 2015 %I Public Library of Science %J PLoS Genet %N 4 %P e1005140-- %T Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium %U http://dx.doi.org/10.1371%2Fjournal.pgen.1005140 %V 11 %X <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>
• Electro-microinjection of fish eggs with an immobile capillary electrode. Shirakashi, Ryo; Yasui, Tatsuo; Memmel, Simon; Sukhorukov, Vladimir L. In Biomicrofluidics, 9, S. 064109. 2015.
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  Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.

  @article{shirakashi2015electromicroinjection, abstract = {Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.}, author = {Shirakashi, Ryo and Yasui, Tatsuo and Memmel, Simon and Sukhorukov, Vladimir L.}, journal = {Biomicrofluidics}, keywords = {vladimir}, pages = {064109}, series = 6, title = {Electro-microinjection of fish eggs with an immobile capillary electrode}, volume = 9, year = 2015 }

  %0 Journal Article %1 shirakashi2015electromicroinjection %A Shirakashi, Ryo %A Yasui, Tatsuo %A Memmel, Simon %A Sukhorukov, Vladimir L. %B 6 %D 2015 %J Biomicrofluidics %P 064109 %T Electro-microinjection of fish eggs with an immobile capillary electrode %U http://scitation.aip.org/content/aip/journal/bmf/9/6/10.1063/1.4936573 %V 9 %X Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.
• A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer. von Eyss, Björn; Jaenicke, Laura A; Kortlever, Roderik M; Royla, Nadine; Wiese, Katrin E; Letschert, Sebastian; McDuffus, Leigh-Anne; Sauer, Markus; Rosenwald, Andreas; Evan, Gerard I; Kempa, Stefan; Eilers, Martin. In Cancer Cell, 28(6), S. 743–757. 2015.
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  Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

  @article{voneyss2015mycdriven, abstract = {Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.}, author = {von Eyss, Björn and Jaenicke, Laura A and Kortlever, Roderik M and Royla, Nadine and Wiese, Katrin E and Letschert, Sebastian and McDuffus, Leigh-Anne and Sauer, Markus and Rosenwald, Andreas and Evan, Gerard I and Kempa, Stefan and Eilers, Martin}, journal = {Cancer Cell}, keywords = {sauer}, month = 12, number = 6, pages = {743–757}, title = {A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer}, volume = 28, year = 2015 }

  %0 Journal Article %1 voneyss2015mycdriven %A von Eyss, Björn %A Jaenicke, Laura A %A Kortlever, Roderik M %A Royla, Nadine %A Wiese, Katrin E %A Letschert, Sebastian %A McDuffus, Leigh-Anne %A Sauer, Markus %A Rosenwald, Andreas %A Evan, Gerard I %A Kempa, Stefan %A Eilers, Martin %D 2015 %J Cancer Cell %N 6 %P 743--757 %R http://dx.doi.org/10.1016/j.ccell.2015.10.013 %T A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer %U http://www.sciencedirect.com/science/article/pii/S1535610815003906 %V 28 %X Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.
• Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns. Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus. In ACS Nano, 9(8), S. 8122–8130. American Chemical Society, 2015.
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  We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

  @article{hennig2015quantitative, abstract = {We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.}, author = {Hennig, Simon and van de Linde, Sebastian and Bergmann, Stephan and Huser, Thomas and Sauer, Markus}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {linde}, month = {08}, number = 8, pages = {8122–8130}, publisher = {American Chemical Society}, title = {Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns}, volume = 9, year = 2015 }

  %0 Journal Article %1 hennig2015quantitative %A Hennig, Simon %A van de Linde, Sebastian %A Bergmann, Stephan %A Huser, Thomas %A Sauer, Markus %B ACS Nano %D 2015 %I American Chemical Society %J ACS Nano %N 8 %P 8122--8130 %R 10.1021/acsnano.5b02220 %T Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns %U http://dx.doi.org/10.1021/acsnano.5b02220 %V 9 %X We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.
• Light-induced cell damage in live-cell super-resolution microscopy. Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In Scientific Reports, 5, S. 15348-. Nature Publishing Group, 2015.
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  Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

  @article{waldchen2015lightinduced, abstract = {Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of  1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only  50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.}, author = {Wäldchen, Sina and Lehmann, Julian and Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {Scientific Reports}, keywords = {linde}, month = {09}, pages = {15348–}, publisher = {Nature Publishing Group}, title = {Light-induced cell damage in live-cell super-resolution microscopy}, volume = 5, year = 2015 }

  %0 Journal Article %1 waldchen2015lightinduced %A Wäldchen, Sina %A Lehmann, Julian %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2015 %I Nature Publishing Group %J Scientific Reports %P 15348-- %R 10.1038/srep15348 %T Light-induced cell damage in live-cell super-resolution microscopy %U http://dx.doi.org/10.1038/srep15348 %V 5 %X Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
• Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes. Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus. In Nano Lett., 15(2), S. 1374–1381. American Chemical Society, 2015.
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  Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  @article{hennig2015instant, abstract = {Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.}, author = {Hennig, Simon and van de Linde, Sebastian and Lummer, Martina and Simonis, Matthias and Huser, Thomas and Sauer, Markus}, booktitle = {Nano Letters}, journal = {Nano Lett.}, keywords = {linde}, month = {02}, number = 2, pages = {1374–1381}, publisher = {American Chemical Society}, title = {Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes}, volume = 15, year = 2015 }

  %0 Journal Article %1 hennig2015instant %A Hennig, Simon %A van de Linde, Sebastian %A Lummer, Martina %A Simonis, Matthias %A Huser, Thomas %A Sauer, Markus %B Nano Letters %D 2015 %I American Chemical Society %J Nano Lett. %N 2 %P 1374--1381 %R 10.1021/nl504660t %T Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes %U http://dx.doi.org/10.1021/nl504660t %V 15 %X Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.
• The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination. García-Martínez, Jorge; Brunk, Michael; Avalos, Javier; Terpitz, Ulrich. In Sci. Rep., 5. Macmillan Publishers Limited. All rights reserved, 2015.
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  Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.

  @article{garciamartinez2015rhodopsin, abstract = {Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.}, author = {García-Martínez, Jorge and Brunk, Michael and Avalos, Javier and Terpitz, Ulrich}, journal = {Sci. Rep.}, keywords = {terpitz}, month = {01}, publisher = {Macmillan Publishers Limited. All rights reserved}, title = {The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination}, volume = 5, year = 2015 }

  %0 Journal Article %1 garciamartinez2015rhodopsin %A García-Martínez, Jorge %A Brunk, Michael %A Avalos, Javier %A Terpitz, Ulrich %D 2015 %I Macmillan Publishers Limited. All rights reserved %J Sci. Rep. %T The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination %U http://dx.doi.org/10.1038/srep07798 %V 5 %X Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO− mutant and carO+ control strains showed a faster development of light-exposed carO− germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.
• Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors. Maric, Hans Michael; Kasaragod, Vikram Babu; Haugaard-Kedström, Linda; Hausrat, Torben Johann; Kneussel, Matthias; Schindelin, Hermann; Strømgaard, Kristian. In Angewandte Chemie International Edition, 54(2), S. 490–494. John Wiley & Sons, Ltd, 2015.
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  Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.

  @article{maric2015design, abstract = {Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Haugaard-Kedström, Linda and Hausrat, Torben Johann and Kneussel, Matthias and Schindelin, Hermann and Strømgaard, Kristian}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {maric}, month = {01}, number = 2, pages = {490–494}, publisher = {John Wiley & Sons, Ltd}, title = {Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors}, volume = 54, year = 2015 }

  %0 Journal Article %1 maric2015design %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Haugaard-Kedström, Linda %A Hausrat, Torben Johann %A Kneussel, Matthias %A Schindelin, Hermann %A Strømgaard, Kristian %B Angewandte Chemie International Edition %D 2015 %I John Wiley & Sons, Ltd %J Angewandte Chemie International Edition %N 2 %P 490--494 %R 10.1002/anie.201409043 %T Design and Synthesis of High-Affinity Dimeric Inhibitors Targeting the Interactions between Gephyrin and Inhibitory Neurotransmitter Receptors %U https://doi.org/10.1002/anie.201409043 %V 54 %X Abstract Gephyrin is the central scaffolding protein for inhibitory neurotransmitter receptors in the brain. Here we describe the development of dimeric peptides that inhibit the interaction between gephyrin and these receptors, a process which is fundamental to numerous synaptic functions and diseases of the brain. We first identified receptor-derived minimal gephyrin-binding peptides that displayed exclusive binding towards native gephyrin from brain lysates. We then designed and synthesized a series of dimeric ligands, which led to a remarkable 1220-fold enhancement of the gephyrin affinity (KD=6.8?nM). In X-ray crystal structures we visualized the simultaneous dimer-to-dimer binding in atomic detail, revealing compound-specific binding modes. Thus, we defined the molecular basis of the affinity-enhancing effect of multivalent gephyrin inhibitors and provide conceptually novel compounds with therapeutic potential, which will allow further elucidation of the gephyrin?receptor interplay.
• Structural analysis of herpes simplex virus by optical super-resolution imaging. Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F. In Nat Commun, 6. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2015.
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  Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

  @article{laine2015structural, abstract = {Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.}, author = {Laine, Romain F. and Albecka, Anna and van de Linde, Sebastian and Rees, Eric J. and Crump, Colin M. and Kaminski, Clemens F.}, journal = {Nat Commun}, keywords = {linde}, month = {01}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Structural analysis of herpes simplex virus by optical super-resolution imaging}, volume = 6, year = 2015 }

  %0 Journal Article %1 laine2015structural %A Laine, Romain F. %A Albecka, Anna %A van de Linde, Sebastian %A Rees, Eric J. %A Crump, Colin M. %A Kaminski, Clemens F. %D 2015 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T Structural analysis of herpes simplex virus by optical super-resolution imaging %U http://dx.doi.org/10.1038/ncomms6980 %V 6 %X Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.
• Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in \{PTEN} and p53 status. Djuzenova, Cholpon S.; Fiedler, Vanessa; Memmel, Simon; Katzer, Astrid; Hartmann, Susanne; Krohne, Georg; Zimmermann, Heiko; Scholz, Claus-Jürgen; Polat, Bülent; Flentje, Michael; Sukhorukov, Vladimir L. In Experimental Cell Research, 330(2), S. 346–357. 2015.
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  Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.

  @article{Djuzenova2015346, abstract = {Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.}, author = {Djuzenova, Cholpon S. and Fiedler, Vanessa and Memmel, Simon and Katzer, Astrid and Hartmann, Susanne and Krohne, Georg and Zimmermann, Heiko and Scholz, Claus-Jürgen and Polat, Bülent and Flentje, Michael and Sukhorukov, Vladimir L.}, journal = {Experimental Cell Research}, keywords = {vladimir}, number = 2, pages = {346 - 357}, title = {Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in \{PTEN\} and p53 status}, volume = 330, year = 2015 }

  %0 Journal Article %1 Djuzenova2015346 %A Djuzenova, Cholpon S. %A Fiedler, Vanessa %A Memmel, Simon %A Katzer, Astrid %A Hartmann, Susanne %A Krohne, Georg %A Zimmermann, Heiko %A Scholz, Claus-Jürgen %A Polat, Bülent %A Flentje, Michael %A Sukhorukov, Vladimir L. %D 2015 %J Experimental Cell Research %N 2 %P 346 - 357 %R http://dx.doi.org/10.1016/j.yexcr.2014.08.013 %T Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in \{PTEN} and p53 status %U http://www.sciencedirect.com/science/article/pii/S0014482714003413 %V 330 %X Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.

• Eight years of single-molecule localization microscopy. Klein, Teresa; Proppert, Sven; Sauer, Markus. In Histochemistry and Cell Biology, 141(6), S. 561–575. 2014.
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  Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

  @article{klein2014eight, abstract = {Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.}, author = {Klein, Teresa and Proppert, Sven and Sauer, Markus}, journal = {Histochemistry and Cell Biology}, keywords = {sauer}, number = 6, pages = {561–575}, title = {Eight years of single-molecule localization microscopy}, volume = 141, year = 2014 }

  %0 Journal Article %1 klein2014eight %A Klein, Teresa %A Proppert, Sven %A Sauer, Markus %D 2014 %J Histochemistry and Cell Biology %N 6 %P 561--575 %R 10.1007/s00418-014-1184-3 %T Eight years of single-molecule localization microscopy %U https://doi.org/10.1007/s00418-014-1184-3 %V 141 %X Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.
• Microsecond folding and domain motions of a spider silk protein structural switch. Ries, Julia; Schwarze, Simone; Johnson, Christopher M.; Neuweiler, Hannes. In Journal of the American Chemical Society, (ja), S. null. 2014.
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  Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ~100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.

  @article{doi:10.1021/ja508760a, abstract = {Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in  100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.}, author = {Ries, Julia and Schwarze, Simone and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Journal of the American Chemical Society}, keywords = {hannes}, note = {PMID: 25382060}, number = {ja}, pages = {null}, title = {Microsecond folding and domain motions of a spider silk protein structural switch}, year = 2014 }

  %0 Journal Article %1 doi:10.1021/ja508760a %A Ries, Julia %A Schwarze, Simone %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2014 %J Journal of the American Chemical Society %N ja %P null %R 10.1021/ja508760a %T Microsecond folding and domain motions of a spider silk protein structural switch %U http://dx.doi.org/10.1021/ja508760a %X Web spiders rapidly assemble protein monomers, so-called spidroins, into extraordinarily tough silk fibers. The process involves the pH-triggered self-association of the spidroin N-terminal domain (NTD), which contains a structural switch connecting spidroins to super-molecules. Single-molecule spectroscopy can detect conformational heterogeneity that is hidden to conventional methods, but motions of the NTD are beyond the resolution limit. Here, we engineered probes for 1-nm conformational changes based on the phenomenon of fluorescence quenching by photoinduced electron transfer into the isolated NTD of a spidroin from the nursery web spider Euprosthenops australis. Correlation analysis of single-molecule fluorescence fluctuations uncovered site-dependent nano-to-microsecond movement of secondary and tertiary structure. Kinetic amplitudes were most pronounced for helices that are part of the association interface and where structural studies show large displacements between monomeric and dimeric conformations. A single tryptophan at the center of the five-helix bundle toggled conformations in ~100 µs and in a pH-dependent manner. Equilibrium denaturation and temperature-jump relaxation experiments revealed cooperative and ultrafast folding in only 60 µs. We deduced a free-energy surface that exhibits native-state ruggedness with apparently similar barrier heights to folding and native motions. Observed equilibrium dynamics within the domain suggest a conformational selection mechanism in the rapid association of spidroins through their NTDs during silk synthesis by web spiders.
• Super-Resolution Imaging of Plasma Membrane Glycans. Letschert, Sebastian; Göhler, Antonia; Franke, Christian; Bertleff-Zieschang, Nadja; Memmel, Elisabeth; Doose, Sören; Seibel, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, S. n/a-n/a. WILEY-VCH Verlag, 2014.
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  Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.

  @article{ANIE:ANIE201406045, abstract = {Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.}, author = {Letschert, Sebastian and Göhler, Antonia and Franke, Christian and Bertleff-Zieschang, Nadja and Memmel, Elisabeth and Doose, Sören and Seibel, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Super-Resolution Imaging of Plasma Membrane Glycans}, year = 2014 }

  %0 Journal Article %1 ANIE:ANIE201406045 %A Letschert, Sebastian %A Göhler, Antonia %A Franke, Christian %A Bertleff-Zieschang, Nadja %A Memmel, Elisabeth %A Doose, Sören %A Seibel, Jürgen %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %P n/a--n/a %R 10.1002/anie.201406045 %T Super-Resolution Imaging of Plasma Membrane Glycans %U http://dx.doi.org/10.1002/anie.201406045 %X Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.
• A Blueprint for Cost-Efficient Localization Microscopy. Holm, Thorge; Klein, Teresa; Löschberger, Anna; Klamp, Tobias; Wiebusch, Gerd; van de Linde, Sebastian; Sauer, Markus. In ChemPhysChem, 15(4), S. 651–654. WILEY-VCH Verlag, 2014.
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  Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.

  @article{CPHC:CPHC201300739, abstract = {Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.}, author = {Holm, Thorge and Klein, Teresa and Löschberger, Anna and Klamp, Tobias and Wiebusch, Gerd and van de Linde, Sebastian and Sauer, Markus}, journal = {ChemPhysChem}, keywords = {linde}, number = 4, pages = {651–654}, publisher = {WILEY-VCH Verlag}, title = {A Blueprint for Cost-Efficient Localization Microscopy}, volume = 15, year = 2014 }

  %0 Journal Article %1 CPHC:CPHC201300739 %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Klamp, Tobias %A Wiebusch, Gerd %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J ChemPhysChem %N 4 %P 651--654 %R 10.1002/cphc.201300739 %T A Blueprint for Cost-Efficient Localization Microscopy %U http://dx.doi.org/10.1002/cphc.201300739 %V 15 %X Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.
• Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution. Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus. In Journal of Cell Science, 127(20), S. 4351–4355. 2014.
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  Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

  @article{Löschberger15102014, abstract = {Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.}, author = {Löschberger, Anna and Franke, Christian and Krohne, Georg and van de Linde, Sebastian and Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {linde}, number = 20, pages = {4351-4355}, title = {Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution}, volume = 127, year = 2014 }

  %0 Journal Article %1 Löschberger15102014 %A Löschberger, Anna %A Franke, Christian %A Krohne, Georg %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %J Journal of Cell Science %N 20 %P 4351-4355 %R 10.1242/jcs.156620 %T Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution %U http://jcs.biologists.org/content/127/20/4351.abstract %V 127 %X Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
• The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress. Mehlitz, Adrian; Karunakaran, Karthika; Herweg, Jo-Ana; Krohne, Georg; van de Linde, Sebastian; Rieck, Elke; Sauer, Markus; Rudel, Thomas. In Cellular Microbiology, S. n/a-n/a. 2014.
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  Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.

  @article{CMI:CMI12278, abstract = {Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.}, author = {Mehlitz, Adrian and Karunakaran, Karthika and Herweg, Jo-Ana and Krohne, Georg and van de Linde, Sebastian and Rieck, Elke and Sauer, Markus and Rudel, Thomas}, journal = {Cellular Microbiology}, keywords = {linde}, pages = {n/a–n/a}, title = {The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress}, year = 2014 }

  %0 Journal Article %1 CMI:CMI12278 %A Mehlitz, Adrian %A Karunakaran, Karthika %A Herweg, Jo-Ana %A Krohne, Georg %A van de Linde, Sebastian %A Rieck, Elke %A Sauer, Markus %A Rudel, Thomas %D 2014 %J Cellular Microbiology %P n/a--n/a %R 10.1111/cmi.12278 %T The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress %U http://dx.doi.org/10.1111/cmi.12278 %X Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.
• How to switch a fluorophore: from undesired blinking to controlled photoswitching. van de Linde, Sebastian; Sauer, Markus. In Chem. Soc. Rev. 2014, 43(4), S. 1076–1087. The Royal Society of Chemistry, 2014.
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  Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.

  @article{C3CS60195A, abstract = {Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.}, author = {van de Linde, Sebastian and Sauer, Markus}, journal = {Chem. Soc. Rev. 2014}, keywords = {linde}, pages = {1076-1087}, publisher = {The Royal Society of Chemistry}, title = {How to switch a fluorophore: from undesired blinking to controlled photoswitching}, volume = {43(4)}, year = 2014 }

  %0 Journal Article %1 C3CS60195A %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I The Royal Society of Chemistry %J Chem. Soc. Rev. 2014 %P 1076-1087 %R 10.1039/C3CS60195A %T How to switch a fluorophore: from undesired blinking to controlled photoswitching %U http://dx.doi.org/10.1039/C3CS60195A %V 43(4) %X Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.
• Timing Protein Assembly in Neurons. Sauer, Markus. In Chemistry & Biology, 21(6), S. 703–704. 2014.
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  Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.

  @article{sauer2014timing, abstract = {Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.}, author = {Sauer, Markus}, journal = {Chemistry & Biology}, keywords = {sauer}, number = 6, pages = {703–704}, title = {Timing Protein Assembly in Neurons}, volume = 21, year = 2014 }

  %0 Journal Article %1 sauer2014timing %A Sauer, Markus %D 2014 %J Chemistry & Biology %N 6 %P 703--704 %R https://doi.org/10.1016/j.chembiol.2014.06.001 %T Timing Protein Assembly in Neurons %U http://www.sciencedirect.com/science/article/pii/S1074552114001847 %V 21 %X Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.
• High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons. Andreska, Thomas; Aufmkolk, Sarah; Sauer, Markus; Blum, Robert. In Frontiers in Cellular Neuroscience, 8(107). 2014.
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  In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.

  @article{10.3389/fncel.2014.00107, abstract = {In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of  20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of  60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.}, author = {Andreska, Thomas and Aufmkolk, Sarah and Sauer, Markus and Blum, Robert}, journal = {Frontiers in Cellular Neuroscience}, keywords = {sauer}, number = 107, title = {High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons}, volume = 8, year = 2014 }

  %0 Journal Article %1 10.3389/fncel.2014.00107 %A Andreska, Thomas %A Aufmkolk, Sarah %A Sauer, Markus %A Blum, Robert %D 2014 %J Frontiers in Cellular Neuroscience %N 107 %R 10.3389/fncel.2014.00107 %T High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2014.00107/abstract %V 8 %X In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.
• Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Löschberger, Anna; Niehörster, Thomas; Sauer, Markus. In Biotechnology Journal, S. n/a-n/a. WILEY-VCH Verlag, 2014.
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  Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

  @article{BIOT:BIOT201400026, abstract = {Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.}, author = {Löschberger, Anna and Niehörster, Thomas and Sauer, Markus}, journal = {Biotechnology Journal}, keywords = {sauer}, pages = {n/a–n/a}, publisher = {WILEY-VCH Verlag}, title = {Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx}, year = 2014 }

  %0 Journal Article %1 BIOT:BIOT201400026 %A Löschberger, Anna %A Niehörster, Thomas %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Biotechnology Journal %P n/a--n/a %R 10.1002/biot.201400026 %T Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx %U http://dx.doi.org/10.1002/biot.201400026 %X Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.
• PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching. Sauer, Markus; Neuweiler, Hannes. In Methods Mol Biol - Fluorescence Spectroscopy and Microscopy, Bd. 1076, Y. Engelborghs, A. J. Visser (Hrsg.), S. 597–615. Humana Press, 2014.
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  Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.

  @incollection{year={2014}, abstract = {Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.}, author = {Sauer, Markus and Neuweiler, Hannes}, booktitle = {Methods Mol Biol - Fluorescence Spectroscopy and Microscopy}, editor = {Engelborghs, Yves and Visser, Antonie J.W.G.}, keywords = {hannes}, pages = {597-615}, publisher = {Humana Press}, series = {Methods in Molecular Biology}, title = {PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching}, volume = 1076, year = 2014 }

  %0 Book Section %1 year={2014} %A Sauer, Markus %A Neuweiler, Hannes %B Methods Mol Biol - Fluorescence Spectroscopy and Microscopy %D 2014 %E Engelborghs, Yves %E Visser, Antonie J.W.G. %I Humana Press %P 597-615 %R 10.1007/978-1-62703-649-8_27 %T PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching %U http://dx.doi.org/10.1007/978-1-62703-649-8_27 %V 1076 %X Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples. %@ 978-1-62703-648-1
• Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity. Bielopolski, Noa; Lam, Alice D.; Bar-On, Dana; Sauer, Markus; Stuenkel, Edward L.; Ashery, Uri. In Journal of Biological Chemistry, 289, S. 17087–17099. 2014.
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  Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

  @article{bielopolski2014differential, abstract = {Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.}, author = {Bielopolski, Noa and Lam, Alice D. and Bar-On, Dana and Sauer, Markus and Stuenkel, Edward L. and Ashery, Uri}, journal = {Journal of Biological Chemistry}, keywords = {sauer}, pages = {17087-17099}, series = 24, title = {Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity}, volume = 289, year = 2014 }

  %0 Journal Article %1 bielopolski2014differential %A Bielopolski, Noa %A Lam, Alice D. %A Bar-On, Dana %A Sauer, Markus %A Stuenkel, Edward L. %A Ashery, Uri %B 24 %D 2014 %J Journal of Biological Chemistry %P 17087-17099 %T Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity %U http://www.jbc.org/content/289/24/17087.abstract %V 289 %X Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.
• Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM). Whelan, Donna R.; Holm, Thorge; Sauer, Markus; Bell, Toby D. M. In Australian Journal of Chemistry, 67, S. 179–183. 2014.
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  The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

  @article{whelan2014focus, abstract = {The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.}, author = {Whelan, Donna R. and Holm, Thorge and Sauer, Markus and Bell, Toby D. M.}, journal = {Australian Journal of Chemistry}, keywords = {sauer}, pages = {179-183}, series = 2, title = {Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)}, volume = 67, year = 2014 }

  %0 Journal Article %1 whelan2014focus %A Whelan, Donna R. %A Holm, Thorge %A Sauer, Markus %A Bell, Toby D. M. %B 2 %D 2014 %J Australian Journal of Chemistry %P 179-183 %T Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM) %U http://www.publish.csiro.au/paper/CH13499 %V 67 %X The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.
• Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status. Memmel, Simon; Sukhorukov, Vladimir L.; Höring, Marcus; Westerling, Katherine; Fiedler, Vanessa; Katzer, Astrid; Krohne, Georg; Flentje, Michael; Djuzenova, Cholpon S. In PLoS ONE, 9(1), S. e87052. Public Library of Science, 2014.
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  Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.

  @article{10.1371/journal.pone.0087052, abstract = {Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.}, author = {Memmel, Simon and Sukhorukov, Vladimir L. and Höring, Marcus and Westerling, Katherine and Fiedler, Vanessa and Katzer, Astrid and Krohne, Georg and Flentje, Michael and Djuzenova, Cholpon S.}, journal = {PLoS ONE}, keywords = {vladimir}, month = {01}, number = 1, pages = {e87052}, publisher = {Public Library of Science}, title = {Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status}, volume = 9, year = 2014 }

  %0 Journal Article %1 10.1371/journal.pone.0087052 %A Memmel, Simon %A Sukhorukov, Vladimir L. %A Höring, Marcus %A Westerling, Katherine %A Fiedler, Vanessa %A Katzer, Astrid %A Krohne, Georg %A Flentje, Michael %A Djuzenova, Cholpon S. %D 2014 %I Public Library of Science %J PLoS ONE %N 1 %P e87052 %R 10.1371/journal.pone.0087052 %T Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status %U http://dx.doi.org/10.1371%2Fjournal.pone.0087052 %V 9 %X Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
• Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging. Proppert, Sven; Wolter, Steve; Holm, Thorge; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In Opt. Express, 22(9), S. 10304–10316. OSA, 2014.
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  In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

  @article{Proppert:14, abstract = {In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.}, author = {Proppert, Sven and Wolter, Steve and Holm, Thorge and Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {Opt. Express}, keywords = {linde}, month = {05}, number = 9, pages = {10304–10316}, publisher = {OSA}, title = {Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging}, volume = 22, year = 2014 }

  %0 Journal Article %1 Proppert:14 %A Proppert, Sven %A Wolter, Steve %A Holm, Thorge %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I OSA %J Opt. Express %N 9 %P 10304--10316 %R 10.1364/OE.22.010304 %T Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging %U http://www.opticsexpress.org/abstract.cfm?URI=oe-22-9-10304 %V 22 %X In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
• Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Ehmann, Nadine; van de Linde, Sebastian; Alon, Amit; Ljaschenko, Dmitrij; Keung, Xi Zhen; Holm, Thorge; Rings, Annika; DiAntonio, Aaron; Hallermann, Stefan; Ashery, Uri; Heckmann, Manfred; Sauer, Markus; Kittel, Robert J. In Nat Commun, 5. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2014.
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  The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

  @article{ehmann2014quantitative, abstract = {The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing  137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.}, author = {Ehmann, Nadine and van de Linde, Sebastian and Alon, Amit and Ljaschenko, Dmitrij and Keung, Xi Zhen and Holm, Thorge and Rings, Annika and DiAntonio, Aaron and Hallermann, Stefan and Ashery, Uri and Heckmann, Manfred and Sauer, Markus and Kittel, Robert J.}, journal = {Nat Commun}, keywords = {linde}, month = {08}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states}, volume = 5, year = 2014 }

  %0 Journal Article %1 ehmann2014quantitative %A Ehmann, Nadine %A van de Linde, Sebastian %A Alon, Amit %A Ljaschenko, Dmitrij %A Keung, Xi Zhen %A Holm, Thorge %A Rings, Annika %A DiAntonio, Aaron %A Hallermann, Stefan %A Ashery, Uri %A Heckmann, Manfred %A Sauer, Markus %A Kittel, Robert J. %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states %U http://dx.doi.org/10.1038/ncomms5650 %V 5 %X The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
• Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency. Maric, Hans Michael; Kasaragod, Vikram Babu; Schindelin, Hermann. In ACS Chem. Biol., 9(11), S. 2554–2562. American Chemical Society, 2014.
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  Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.

  @article{maric2014modulation, abstract = {Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Schindelin, Hermann}, booktitle = {ACS Chemical Biology}, journal = {ACS Chem. Biol.}, keywords = {maric}, month = 11, number = 11, pages = {2554–2562}, publisher = {American Chemical Society}, title = {Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency}, volume = 9, year = 2014 }

  %0 Journal Article %1 maric2014modulation %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Schindelin, Hermann %B ACS Chemical Biology %D 2014 %I American Chemical Society %J ACS Chem. Biol. %N 11 %P 2554--2562 %R 10.1021/cb500303a %T Modulation of Gephyrin-Glycine Receptor Affinity by Multivalency %U https://doi.org/10.1021/cb500303a %V 9 %X Gephyrin is a major determinant for the accumulation and anchoring of glycine receptors (GlyRs) and the majority of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic sites. Here we explored the interaction of gephyrin with a dimeric form of a GlyR β-subunit receptor-derived peptide. A 2 Å crystal structure of the C-terminal domain of gephyrin (GephE) in complex with a 15-residue peptide derived from the GlyR β-subunit defined the core binding site, which we targeted with the dimeric peptide. Biophysical analyses via differential scanning calorimetry (DSC), thermofluor, and isothermal titration calorimetry (ITC) demonstrated that this dimeric ligand is capable of binding simultaneously to two receptor binding sites and that this multivalency results in a 25-fold enhanced affinity. Our study therefore suggests that the oligomeric state of gephyrin and the number of gephyrin-binding subunits in the pentameric GABAARs and GlyRs together control postsynaptic receptor clustering.
• Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin. Maric, Hans Michael; Kasaragod, Vikram Babu; Hausrat, Torben Johann; Kneussel, Matthias; Tretter, Verena; Strømgaard, Kristian; Schindelin, Hermann. In Nature Communications, 5, S. 5767-. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2014.
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  γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.

  @article{maric2014molecular, abstract = {γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.}, author = {Maric, Hans Michael and Kasaragod, Vikram Babu and Hausrat, Torben Johann and Kneussel, Matthias and Tretter, Verena and Strømgaard, Kristian and Schindelin, Hermann}, journal = {Nature Communications}, keywords = {maric}, month = 12, pages = {5767–}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin}, volume = 5, year = 2014 }

  %0 Journal Article %1 maric2014molecular %A Maric, Hans Michael %A Kasaragod, Vikram Babu %A Hausrat, Torben Johann %A Kneussel, Matthias %A Tretter, Verena %A Strømgaard, Kristian %A Schindelin, Hermann %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Communications %P 5767-- %T Molecular basis of the alternative recruitment of GABAA versus glycine receptors through gephyrin %U https://doi.org/10.1038/ncomms6767 %V 5 %X γ-Aminobutyric acid type A and glycine receptors (GABAARs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABAARs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin–GABAAR α3 complex, revealing that the N-terminal region of the α3 peptide occupies the same binding site as the GlyR β subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABAAR α subunits, engages in unique interactions. Thermodynamic dissections of the gephyrin–receptor interactions identify two residues as primary determinants for gephyrin’s subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABAARs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin–GABAAR interaction.

• Localization microscopy coming of age: from concepts to biological impact. Sauer, Markus. In Journal of Cell Science, 126(16), S. 3505–3513. 2013.
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  Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.

  @article{Sauer15082013, abstract = {Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.}, author = {Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {sauer}, number = 16, pages = {3505-3513}, title = {Localization microscopy coming of age: from concepts to biological impact}, volume = 126, year = 2013 }

  %0 Journal Article %1 Sauer15082013 %A Sauer, Markus %D 2013 %J Journal of Cell Science %N 16 %P 3505-3513 %R 10.1242/jcs.123612 %T Localization microscopy coming of age: from concepts to biological impact %U http://jcs.biologists.org/content/126/16/3505.abstract %V 126 %X Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.
• Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives. Kolesinska, Beata; Podwysocka, Dominika J.; Rueping, Magnus A.; Seebach, Dieter; Kamena, Faustin; Walde, Peter; Sauer, Markus; Windschiegl, Barbara; Meyer-Ács, Mira; Vor der Brüggen, Marc; Giehring, Sebastian. In Chemistry & Biodiversity, 10(1), S. 1–38. WILEY-VCH Verlag, 2013.
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  After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.

  @article{CBDV:CBDV201200393, abstract = {After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.}, author = {Kolesinska, Beata and Podwysocka, Dominika J. and Rueping, Magnus A. and Seebach, Dieter and Kamena, Faustin and Walde, Peter and Sauer, Markus and Windschiegl, Barbara and Meyer-Ács, Mira and Vor der Brüggen, Marc and Giehring, Sebastian}, journal = {Chemistry & Biodiversity}, keywords = {sauer}, number = 1, pages = {1–38}, publisher = {WILEY-VCH Verlag}, title = {Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives}, volume = 10, year = 2013 }

  %0 Journal Article %1 CBDV:CBDV201200393 %A Kolesinska, Beata %A Podwysocka, Dominika J. %A Rueping, Magnus A. %A Seebach, Dieter %A Kamena, Faustin %A Walde, Peter %A Sauer, Markus %A Windschiegl, Barbara %A Meyer-Ács, Mira %A Vor der Brüggen, Marc %A Giehring, Sebastian %D 2013 %I WILEY-VCH Verlag %J Chemistry & Biodiversity %N 1 %P 1--38 %R 10.1002/cbdv.201200393 %T Permeation through Phospholipid Bilayers, Skin-Cell Penetration, Plasma Stability, and CD Spectra of α- and β-Oligoproline Derivatives %U http://dx.doi.org/10.1002/cbdv.201200393 %V 10 %X After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1–8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.
• Investigating Cellular Structures at the Nanoscale with Organic Fluorophores. van de Linde, Sebastian; Aufmkolk, Sarah; Franke, Christian; Holm, Thorge; Klein, Teresa; Löschberger, Anna; Proppert, Sven; Wolter, Steve; Sauer, Markus. In Chemistry & Biology, 20(1), S. 8–18. 2013.
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  Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.

  @article{van de Linde20138, abstract = {Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.}, author = {van de Linde, Sebastian and Aufmkolk, Sarah and Franke, Christian and Holm, Thorge and Klein, Teresa and Löschberger, Anna and Proppert, Sven and Wolter, Steve and Sauer, Markus}, journal = {Chemistry & Biology}, keywords = {linde}, number = 1, pages = {8 - 18}, title = {Investigating Cellular Structures at the Nanoscale with Organic Fluorophores}, volume = 20, year = 2013 }

  %0 Journal Article %1 van de Linde20138 %A van de Linde, Sebastian %A Aufmkolk, Sarah %A Franke, Christian %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Proppert, Sven %A Wolter, Steve %A Sauer, Markus %D 2013 %J Chemistry & Biology %N 1 %P 8 - 18 %R 10.1016/j.chembiol.2012.11.004 %T Investigating Cellular Structures at the Nanoscale with Organic Fluorophores %U http://www.sciencedirect.com/science/article/pii/S1074552112004280 %V 20 %X Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.
• Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Andronic, Joseph; Bobak, Nicole; Bittner, Stefan; Ehling, Petra; Kleinschnitz, Christoph; Herrmann, Alexander M.; Zimmermann, Heiko; Sauer, Markus; Wiendl, Heinz; Budde, Thomas; Meuth, Sven G.; Sukhorukov, Vladimir L. In Biochimica et Biophysica Acta (BBA) - Biomembranes, 1828(2), S. 699–707. 2013.
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  Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.

  @article{Andronic2013699, abstract = {Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.}, author = {Andronic, Joseph and Bobak, Nicole and Bittner, Stefan and Ehling, Petra and Kleinschnitz, Christoph and Herrmann, Alexander M. and Zimmermann, Heiko and Sauer, Markus and Wiendl, Heinz and Budde, Thomas and Meuth, Sven G. and Sukhorukov, Vladimir L.}, journal = {Biochimica et Biophysica Acta (BBA) - Biomembranes}, keywords = {sauer}, number = 2, pages = {699 - 707}, title = {Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes}, volume = 1828, year = 2013 }

  %0 Journal Article %1 Andronic2013699 %A Andronic, Joseph %A Bobak, Nicole %A Bittner, Stefan %A Ehling, Petra %A Kleinschnitz, Christoph %A Herrmann, Alexander M. %A Zimmermann, Heiko %A Sauer, Markus %A Wiendl, Heinz %A Budde, Thomas %A Meuth, Sven G. %A Sukhorukov, Vladimir L. %D 2013 %J Biochimica et Biophysica Acta (BBA) - Biomembranes %N 2 %P 699 - 707 %R 10.1016/j.bbamem.2012.09.028 %T Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes %U http://www.sciencedirect.com/science/article/pii/S0005273612003495 %V 1828 %X Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.
• Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines. Hartmann, Susanne; Günther, Nadine; Biehl, Marlene; Katzer, Astrid; Kuger, Sebastian; Worschech, Eike; Sukhorukov, Vladimir L.; Krohne, Georg; Zimmermann, Heiko; Flentje, Michael; Djuzenova, Cholpon S. In Cancer Letters, 331(2), S. 200–210. 2013.
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  This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.

  @article{Hartmann2013200, abstract = {This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.}, author = {Hartmann, Susanne and Günther, Nadine and Biehl, Marlene and Katzer, Astrid and Kuger, Sebastian and Worschech, Eike and Sukhorukov, Vladimir L. and Krohne, Georg and Zimmermann, Heiko and Flentje, Michael and Djuzenova, Cholpon S.}, journal = {Cancer Letters}, keywords = {vladimir}, number = 2, pages = {200 - 210}, title = {Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines}, volume = 331, year = 2013 }

  %0 Journal Article %1 Hartmann2013200 %A Hartmann, Susanne %A Günther, Nadine %A Biehl, Marlene %A Katzer, Astrid %A Kuger, Sebastian %A Worschech, Eike %A Sukhorukov, Vladimir L. %A Krohne, Georg %A Zimmermann, Heiko %A Flentje, Michael %A Djuzenova, Cholpon S. %D 2013 %J Cancer Letters %N 2 %P 200 - 210 %R 10.1016/j.canlet.2012.12.027 %T Hsp90 inhibition by NVP-AUY922 and NVP-BEP800 decreases migration and invasion of irradiated normoxic and hypoxic tumor cell lines %U http://www.sciencedirect.com/science/article/pii/S0304383513000335 %V 331 %X This study explores the impact of Hsp90 inhibitors NVP-AUY922 and NVP-BEP800 in combination with ionizing radiation (IR) on the migration and invasion of lung carcinoma A549 and glioblastoma SNB19 cells, under normoxia or hypoxia. Independent of oxygen concentration, both drugs decreased the migration and invasion rates of non-irradiated tumor cells. Combined drug-IR treatment under hypoxia inhibited cell invasion to a greater extent than did each treatment alone. Decreased migration of cells correlated with altered expression of several matrix-associated proteins (FAK/p-FAK, Erk2, RhoA) and impaired F-actin modulation. The anti-metastatic efficacy of the Hsp90 inhibitors could be useful in combinational therapies of cancer.
• Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale. Steger, Katrin; Bollmann, Stefan; Noe, Frank; Doose, Soren. In Phys. Chem. Chem. Phys., S. -. The Royal Society of Chemistry, 2013.
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  Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.

  @article{C3CP50644D, abstract = {Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.}, author = {Steger, Katrin and Bollmann, Stefan and Noe, Frank and Doose, Soren}, journal = {Phys. Chem. Chem. Phys.}, keywords = {doose}, pages = {-}, publisher = {The Royal Society of Chemistry}, title = {Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale}, year = 2013 }

  %0 Journal Article %1 C3CP50644D %A Steger, Katrin %A Bollmann, Stefan %A Noe, Frank %A Doose, Soren %D 2013 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %P - %R 10.1039/C3CP50644D %T Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale %U http://dx.doi.org/10.1039/C3CP50644D %X Signal fluctuations in a fluorescence time trace on nanosecond time scales can be induced by specific quenching interactions that report on the dynamics of biomolecules. Fluorescence correlation spectroscopy is an analysis tool to investigate dynamic processes on time scales from pico- to milliseconds or longer. Under certain conditions{,} e.g. in a solvent of high viscosity{,} a fluorescence labeled dynamic biomolecule yields multiple independent correlation decays due to rotational and translational diffusion{,} fluorescence quenching interactions{,} and fluorophore photophysics. We compared parameter estimation for FCS data with multiple correlation decays by dynamical fingerprint analysis and by the non-linear Levenberg-Marquardt fitting procedure and identified conditions for which dynamical fingerprint analysis can be of advantage. In this context we identified a previously unrecognized photophysical process in ATTO655 that introduces fluorescence intermittency on nanosecond time scales that is absent in MR121. The optimized fitting procedure is used to resolve the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes.
• A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants. Lummer, Martina; Humpert, Fabian; Wiedenlübbert, Matthias; Sauer, Markus; Schüttpelz, Mark; Staiger, Dorothee. In Molecular Plant, 6(5), S. 1518–1530. 2013.
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  Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.

  @article{lummer2013reversibly, abstract = {Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.}, author = {Lummer, Martina and Humpert, Fabian and Wiedenlübbert, Matthias and Sauer, Markus and Schüttpelz, Mark and Staiger, Dorothee}, journal = {Molecular Plant}, keywords = {sauer}, number = 5, pages = {1518–1530}, title = {A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants}, volume = 6, year = 2013 }

  %0 Journal Article %1 lummer2013reversibly %A Lummer, Martina %A Humpert, Fabian %A Wiedenlübbert, Matthias %A Sauer, Markus %A Schüttpelz, Mark %A Staiger, Dorothee %D 2013 %J Molecular Plant %N 5 %P 1518--1530 %R https://doi.org/10.1093/mp/sst040 %T A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants %U http://www.sciencedirect.com/science/article/pii/S1674205214602240 %V 6 %X Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.
• A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology. Zimmermann, Ulrich; Bitter, Rebecca; Marchiori, Paulo Eduardo Ribeiro; Rüger, Simon; Ehrenberger, Wilhelm; Sukhorukov, Vladimir L.; Schüttler, Annika; Ribeiro, Rafael Vasconcelos. In Theoretical and Experimental Plant Physiology, 25, S. 2–11. scielo, 2013.
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  The non-invasive, magnetic leaf patch clamp pressure probe (also termed ZIM-probe) allows for the first time to measure continuously turgor pressure changes of plant leaves over long periods of time with high precision and in real time. The probe has become an important tool in plant physiology, molecular biology and ecology, but also in agriculture because the probe is very robust and user-friendly. Growers receive the information about the water status of their plants by wireless telemetry, mobile network and internet on an as-needed basis and can thus adjust very precisely both the timing of irrigation and the quantity of water to apply. Effects of air and leaf temperature, relative humidity, illumination and wind on turgor pressure can be monitored very sensitively both under indoor and outdoor conditions. Even the effects of blue and red light as well as of oscillations of stomata aperture on turgor pressure can be monitored by the probe with high sensitivity. Similarly, water deficit due to increase of the osmotic pressure in the nutrition solutions resulted in significant changes of the probe signals. Multiple probe readings open up new possibilities to resolve (together with other techniques) the mechanisms of short- and long-distance water transport, particularly how plants can cope with water shortage. The applications of the magnetic probe are numerous and one can expect highly interesting developments in plant water relations in the nearest future.

  @article{zimmermann2013noninvasive, abstract = {The non-invasive, magnetic leaf patch clamp pressure probe (also termed ZIM-probe) allows for the first time to measure continuously turgor pressure changes of plant leaves over long periods of time with high precision and in real time. The probe has become an important tool in plant physiology, molecular biology and ecology, but also in agriculture because the probe is very robust and user-friendly. Growers receive the information about the water status of their plants by wireless telemetry, mobile network and internet on an as-needed basis and can thus adjust very precisely both the timing of irrigation and the quantity of water to apply. Effects of air and leaf temperature, relative humidity, illumination and wind on turgor pressure can be monitored very sensitively both under indoor and outdoor conditions. Even the effects of blue and red light as well as of oscillations of stomata aperture on turgor pressure can be monitored by the probe with high sensitivity. Similarly, water deficit due to increase of the osmotic pressure in the nutrition solutions resulted in significant changes of the probe signals. Multiple probe readings open up new possibilities to resolve (together with other techniques) the mechanisms of short- and long-distance water transport, particularly how plants can cope with water shortage. The applications of the magnetic probe are numerous and one can expect highly interesting developments in plant water relations in the nearest future.}, author = {Zimmermann, Ulrich and Bitter, Rebecca and Marchiori, Paulo Eduardo Ribeiro and Rüger, Simon and Ehrenberger, Wilhelm and Sukhorukov, Vladimir L. and Schüttler, Annika and Ribeiro, Rafael Vasconcelos}, journal = {Theoretical and Experimental Plant Physiology}, keywords = {vladimir}, month = {00}, pages = {2 - 11}, publisher = {scielo}, title = {A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology}, volume = 25, year = 2013 }

  %0 Journal Article %1 zimmermann2013noninvasive %A Zimmermann, Ulrich %A Bitter, Rebecca %A Marchiori, Paulo Eduardo Ribeiro %A Rüger, Simon %A Ehrenberger, Wilhelm %A Sukhorukov, Vladimir L. %A Schüttler, Annika %A Ribeiro, Rafael Vasconcelos %D 2013 %I scielo %J Theoretical and Experimental Plant Physiology %P 2 - 11 %T A non-invasive plant-based probe for continuous monitoring of water stress in real time: a new tool for irrigation scheduling and deeper insight into drought and salinity stress physiology %U http://dx.doi.org/10.1590/S2197-00252013000100002 %V 25 %X The non-invasive, magnetic leaf patch clamp pressure probe (also termed ZIM-probe) allows for the first time to measure continuously turgor pressure changes of plant leaves over long periods of time with high precision and in real time. The probe has become an important tool in plant physiology, molecular biology and ecology, but also in agriculture because the probe is very robust and user-friendly. Growers receive the information about the water status of their plants by wireless telemetry, mobile network and internet on an as-needed basis and can thus adjust very precisely both the timing of irrigation and the quantity of water to apply. Effects of air and leaf temperature, relative humidity, illumination and wind on turgor pressure can be monitored very sensitively both under indoor and outdoor conditions. Even the effects of blue and red light as well as of oscillations of stomata aperture on turgor pressure can be monitored by the probe with high sensitivity. Similarly, water deficit due to increase of the osmotic pressure in the nutrition solutions resulted in significant changes of the probe signals. Multiple probe readings open up new possibilities to resolve (together with other techniques) the mechanisms of short- and long-distance water transport, particularly how plants can cope with water shortage. The applications of the magnetic probe are numerous and one can expect highly interesting developments in plant water relations in the nearest future.
• Elements of Transcriptional Machinery Are Compatible among Plants and Mammals. Wolf, Annette; Akrap, Nina; Marg, Berenice; Galliardt, Helena; Heiligentag, Martyna; Humpert, Fabian; Sauer, Markus; Kaltschmidt, Barbara; Kaltschmidt, Christian; Seidel, Thorsten. In PLOS ONE, 8(1), S. e53737-. Public Library of Science, 2013.
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  In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.

  @article{wolf2013elements, abstract = {In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.}, author = {Wolf, Annette and Akrap, Nina and Marg, Berenice and Galliardt, Helena and Heiligentag, Martyna and Humpert, Fabian and Sauer, Markus and Kaltschmidt, Barbara and Kaltschmidt, Christian and Seidel, Thorsten}, journal = {PLOS ONE}, keywords = {sauer}, month = {01}, number = 1, pages = {e53737–}, publisher = {Public Library of Science}, title = {Elements of Transcriptional Machinery Are Compatible among Plants and Mammals}, volume = 8, year = 2013 }

  %0 Journal Article %1 wolf2013elements %A Wolf, Annette %A Akrap, Nina %A Marg, Berenice %A Galliardt, Helena %A Heiligentag, Martyna %A Humpert, Fabian %A Sauer, Markus %A Kaltschmidt, Barbara %A Kaltschmidt, Christian %A Seidel, Thorsten %D 2013 %I Public Library of Science %J PLOS ONE %N 1 %P e53737-- %T Elements of Transcriptional Machinery Are Compatible among Plants and Mammals %U https://doi.org/10.1371/journal.pone.0053737 %V 8 %X In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
• Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes. Terpitz, Ulrich; Sukhorukov, Vladimir L.; Zimmermann, Dirk. In ASSAY and Drug Development Technologies., 11(1), S. 9–16. 2013.
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  Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.

  @article{terpitz2012prototype, abstract = {Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.}, author = {Terpitz, Ulrich and Sukhorukov, Vladimir L. and Zimmermann, Dirk}, journal = {ASSAY and Drug Development Technologies.}, keywords = {terpitz}, month = {02}, number = 1, pages = {9-16}, title = {Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes}, volume = 11, year = 2013 }

  %0 Journal Article %1 terpitz2012prototype %A Terpitz, Ulrich %A Sukhorukov, Vladimir L. %A Zimmermann, Dirk %D 2013 %J ASSAY and Drug Development Technologies. %N 1 %P 9-16 %T Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane-Potential Measurements by Metal Electrodes %U http://dx.doi.org/10.1089/adt.2012.455 %V 11 %X Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.
• Methylene blue- and thiol-based oxygen depletion for super-resolution imaging. Schäfer, Philip; van de Linde, Sebastian; Lehmann, Julian; Sauer, Markus; Doose, Sören. In Analytical Chemistry, 85(6), S. 3393–3400. 2013.
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  Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

  @article{doi:10.1021/ac400035k, abstract = {Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.}, author = {Schäfer, Philip and van de Linde, Sebastian and Lehmann, Julian and Sauer, Markus and Doose, Sören}, journal = {Analytical Chemistry}, keywords = {linde}, month = {02}, number = 6, pages = {3393 - 3400}, title = {Methylene blue- and thiol-based oxygen depletion for super-resolution imaging}, volume = 85, year = 2013 }

  %0 Journal Article %1 doi:10.1021/ac400035k %A Schäfer, Philip %A van de Linde, Sebastian %A Lehmann, Julian %A Sauer, Markus %A Doose, Sören %D 2013 %J Analytical Chemistry %N 6 %P 3393 - 3400 %R 10.1021/ac400035k %T Methylene blue- and thiol-based oxygen depletion for super-resolution imaging %U http://pubs.acs.org/doi/abs/10.1021/ac400035k %V 85 %X Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.
• Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics. Seibel, Jürgen; König, Simone; Göhler, Antonia; Doose, Sören; Memmel, Elisabeth; Bertleff, Nadja; Sauer, Markus. In Expert Rev Proteomics, 10(1), S. 25–31. Expert Reviews, 2013.
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  Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.

  @article{seibel2013investigating, abstract = {Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.}, author = {Seibel, Jürgen and König, Simone and Göhler, Antonia and Doose, Sören and Memmel, Elisabeth and Bertleff, Nadja and Sauer, Markus}, booktitle = {Expert Review of Proteomics}, journal = {Expert Rev Proteomics}, keywords = {sauer}, month = {02}, number = 1, pages = {25–31}, publisher = {Expert Reviews}, title = {Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics}, volume = 10, year = 2013 }

  %0 Journal Article %1 seibel2013investigating %A Seibel, Jürgen %A König, Simone %A Göhler, Antonia %A Doose, Sören %A Memmel, Elisabeth %A Bertleff, Nadja %A Sauer, Markus %B Expert Review of Proteomics %D 2013 %I Expert Reviews %J Expert Rev Proteomics %N 1 %P 25--31 %R 10.1586/epr.12.72 %T Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics %U http://dx.doi.org/10.1586/epr.12.72 %V 10 %X Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.
• Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule. Kuger, Sebastian; Graus, Dorothea; Brendtke, Rico; Gunther, Nadine; Katzer, Astrid; Lutyj, Paul; Polat, Bulent; Chatterjee, Manik; Sukhorukov, Vladimir L; Flentje, Michael; Djuzenova, Cholpon S. In Transl Oncol., 6(2), S. 169–179. 2013.
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  Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.

  @article{s2013radiosensitization, abstract = {Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.}, author = {Kuger, Sebastian and Graus, Dorothea and Brendtke, Rico and Gunther, Nadine and Katzer, Astrid and Lutyj, Paul and Polat, Bulent and Chatterjee, Manik and Sukhorukov, Vladimir L and Flentje, Michael and Djuzenova, Cholpon S}, journal = {Transl Oncol.}, keywords = {vladimir}, month = {04}, number = 2, pages = {169-179}, title = {Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule.}, volume = 6, year = 2013 }

  %0 Journal Article %1 s2013radiosensitization %A Kuger, Sebastian %A Graus, Dorothea %A Brendtke, Rico %A Gunther, Nadine %A Katzer, Astrid %A Lutyj, Paul %A Polat, Bulent %A Chatterjee, Manik %A Sukhorukov, Vladimir L %A Flentje, Michael %A Djuzenova, Cholpon S %D 2013 %J Transl Oncol. %N 2 %P 169-179 %T Radiosensitization of Glioblastoma Cell Lines by the Dual PI3K and mTOR Inhibitor NVP-BEZ235 Depends on Drug-Irradiation Schedule. %U http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3610553/ %V 6 %X Previous studies have shown that the dual phosphatidylinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor NVP-BEZ235 radiosensitizes tumor cells if added shortly before ionizing radiation (IR) and kept in culture medium thereafter. The present study explores the impact of inhibitor and IR schedule on the radiosensitizing ability of NVP-BEZ235 in four human glioblastoma cell lines. Two different drug-IR treatment schedules were compared. In schedule I, cells were treated with NVP-BEZ235 for 24 hours before IR and the drug was removed before IR. In schedule II, the cells were exposed to NVP-BEZ235 1 hour before, during, and up to 48 hours after IR. The cellular response was analyzed by colony counts, expression of marker proteins of the PI3K/AKT/mTOR pathway, cell cycle, and DNA damage. We found that under schedule I, NVP-BEZ235 did not radiosensitize cells, which were mostly arrested in G1 phase during IR exposure. In addition, the drug-pretreated and irradiated cells exhibited less DNA damage but increased expressions of phospho-AKT and phospho-mTOR, compared to controls. In contrast, NVP-BEZ235 strongly enhanced the radiosensitivity of cells treated according to schedule II. Possible reasons of radiosensitization by NVP-BEZ235 under schedule II might be the protracted DNA repair, prolonged G2/M arrest, and, to some extent, apoptosis. In addition, the PI3K pathway was downregulated by the NVP-BEZ235 at the time of irradiation under schedule II, as contrasted with its activation in schedule I. We found that, depending on the drug-IR schedule, the NVP-BEZ235 can act either as a strong radiosensitizer or as a cytostatic agent in glioblastoma cells.
• Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay. Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus. In Sci. Rep., 3. Macmillan Publishers Limited. All rights reserved, 2013.
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  There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

  @article{klamp2013highly, abstract = {There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of  5 pM and a linear detection range spanning three orders of magnitude.}, author = {Klamp, Tobias and Camps, Marta and Nieto, Benjamin and Guasch, Francesc and Ranasinghe, Rohan T. and Wiedemann, Jens and Petrášek, Zdeněk and Schwille, Petra and Klenerman, David and Sauer, Markus}, journal = {Sci. Rep.}, keywords = {sauer}, month = {05}, publisher = {Macmillan Publishers Limited. All rights reserved}, title = {Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay}, volume = 3, year = 2013 }

  %0 Journal Article %1 klamp2013highly %A Klamp, Tobias %A Camps, Marta %A Nieto, Benjamin %A Guasch, Francesc %A Ranasinghe, Rohan T. %A Wiedemann, Jens %A Petrášek, Zdeněk %A Schwille, Petra %A Klenerman, David %A Sauer, Markus %D 2013 %I Macmillan Publishers Limited. All rights reserved %J Sci. Rep. %T Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay %U http://dx.doi.org/10.1038/srep01852 %V 3 %X There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.
• The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening. Schwarze, Simone; Zwettler, Fabian U.; Johnson, Christopher M.; Neuweiler, Hannes. In Nat Commun, 4. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2013.
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  Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.

  @article{schwarze2013nterminal, abstract = {Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.}, author = {Schwarze, Simone and Zwettler, Fabian U. and Johnson, Christopher M. and Neuweiler, Hannes}, journal = {Nat Commun}, keywords = {hannes}, month = 11, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening}, volume = 4, year = 2013 }

  %0 Journal Article %1 schwarze2013nterminal %A Schwarze, Simone %A Zwettler, Fabian U. %A Johnson, Christopher M. %A Neuweiler, Hannes %D 2013 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T The N-terminal domains of spider silk proteins assemble ultrafast and protected from charge screening %U http://dx.doi.org/10.1038/ncomms3815 %V 4 %X Web spiders assemble spidroin monomers into silk fibres of unrivalled tensile strength at remarkably high spinning speeds of up to 1 m s−1. The spidroin N-terminal domain contains a charge-driven, pH-sensitive relay that controls self-association by an elusive mechanism. The underlying kinetics have not yet been reported. Here we engineer a fluorescence switch into the isolated N-terminal domain from spidroin 1 of the major ampullate gland of the nursery web spider E. australis that monitors dimerization. We observe ultrafast association that is surprisingly insensitive to salt, contrasting the classical screening effects in accelerated, charged protein interfaces. To gain deeper mechanistic insight, we mutate each of the protonatable residue side chains and probe their contributions. Two vicinal aspartic acids are critically involved in an unusual process of accelerated protein association that is protected from screening by electrolytes, potentially facilitating the rapid synthesis of silk fibres by web spiders.

• Concomitant measurements of stem sap flow and leaf turgor pressure in olive trees using the leaf patch clamp pressure probe. Rodriguez-Dominguez, C.M.; Ehrenberger, W.; Sann, C.; Rüger, S.; Sukhorukov, V.; Martín-Palomo, M.J.; Diaz-Espejo, A.; Cuevas, M.V.; Torres-Ruiz, J.M.; Perez-Martin, A.; Zimmermann, U.; Fernández, J.E. In Agricultural Water Management, 114, S. 50–58. 2012.
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  Stem sap flow (Q) and leaf turgor pressure (Pc) were measured simultaneously on 4-year-old, 2.4 m tall ‘Arbequina’ olive trees in a hedgerow orchard. Measurements were performed on well-watered control trees as well as on 60RDI and 30RDI trees (RDI = regulated deficit irrigation). The 60RDI trees received 59.2% of the crop water needs (ETc), and the 30RDI trees received 29.4% of ETc. Pc was determined non-invasively using the magnetic leaf patch clamp pressure probe (ZIM probe). The patch pressure Pp measured by the probe is inversely correlated with turgor pressure at Pc > ca. 50 kPa. Pc is coupled with xylem pressure; thus Pp yields information about the development of tension in xylem. In the case of the control trees a positive correlation between Q and Pp was generally found, i.e. Q increased usually with increasing Pp and decreased with decreasing Pp, as expected. However, Q peaking did not always coincided with Pp peaking at noon. Occasionally, Q peaking preceded or followed Pp peaking with a time difference of up to 3 h in both cases. Under some circumstances, the onset of Q after sunrise was greatly delayed, even though a pronounced increase of Pp was observed. A delayed onset of Q after sunrise resulted in hysteresis phenomena, i.e. the linear increase of Q and Pp in the morning hours did not coincide with the corresponding decrease of Q and Pp in the afternoon. The development of severe water stress (Pc < ca. 50 kPa) associated with the increase in the intercellular spaces of the spongy tissue in the leaves resulted in inverted diurnal Pp curves, i.e. minimum Pp values were recorded at noon and maximum Pp values during the night on the 30RDI trees. The effects were reversible as shown by re-watering. By contrast, the magnitude of Q decreased continuously from the turgescent state to the state of severe water stress; maximum Q values were still recorded around noon. The data suggests that short-range tension forces are responsible for water lifting in olive trees and that water uptake from water storage reservoirs must play an important role in the supply of the leaves with water. Furthermore, for setting of irrigation thresholds the finding of shape changes of the Pp curves upon severe water stress seems to be a useful indicator. Such shape changes are detected and monitored more sensitively than changes in the magnitude of sap flow rates or of turgor pressure.

  @article{RodriguezDominguez201250, abstract = {Stem sap flow (Q) and leaf turgor pressure (Pc) were measured simultaneously on 4-year-old, 2.4 m tall ‘Arbequina’ olive trees in a hedgerow orchard. Measurements were performed on well-watered control trees as well as on 60RDI and 30RDI trees (RDI = regulated deficit irrigation). The 60RDI trees received 59.2% of the crop water needs (ETc), and the 30RDI trees received 29.4% of ETc. Pc was determined non-invasively using the magnetic leaf patch clamp pressure probe (ZIM probe). The patch pressure Pp measured by the probe is inversely correlated with turgor pressure at Pc > ca. 50 kPa. Pc is coupled with xylem pressure; thus Pp yields information about the development of tension in xylem. In the case of the control trees a positive correlation between Q and Pp was generally found, i.e. Q increased usually with increasing Pp and decreased with decreasing Pp, as expected. However, Q peaking did not always coincided with Pp peaking at noon. Occasionally, Q peaking preceded or followed Pp peaking with a time difference of up to 3 h in both cases. Under some circumstances, the onset of Q after sunrise was greatly delayed, even though a pronounced increase of Pp was observed. A delayed onset of Q after sunrise resulted in hysteresis phenomena, i.e. the linear increase of Q and Pp in the morning hours did not coincide with the corresponding decrease of Q and Pp in the afternoon. The development of severe water stress (Pc < ca. 50 kPa) associated with the increase in the intercellular spaces of the spongy tissue in the leaves resulted in inverted diurnal Pp curves, i.e. minimum Pp values were recorded at noon and maximum Pp values during the night on the 30RDI trees. The effects were reversible as shown by re-watering. By contrast, the magnitude of Q decreased continuously from the turgescent state to the state of severe water stress; maximum Q values were still recorded around noon. The data suggests that short-range tension forces are responsible for water lifting in olive trees and that water uptake from water storage reservoirs must play an important role in the supply of the leaves with water. Furthermore, for setting of irrigation thresholds the finding of shape changes of the Pp curves upon severe water stress seems to be a useful indicator. Such shape changes are detected and monitored more sensitively than changes in the magnitude of sap flow rates or of turgor pressure.}, author = {Rodriguez-Dominguez, C.M. and Ehrenberger, W. and Sann, C. and Rüger, S. and Sukhorukov, V. and Martín-Palomo, M.J. and Diaz-Espejo, A. and Cuevas, M.V. and Torres-Ruiz, J.M. and Perez-Martin, A. and Zimmermann, U. and Fernández, J.E.}, journal = {Agricultural Water Management}, keywords = {vladimir}, note = {<ce:title>For a better use and distribution of water</ce:title>}, pages = {50 - 58}, title = {Concomitant measurements of stem sap flow and leaf turgor pressure in olive trees using the leaf patch clamp pressure probe}, volume = 114, year = 2012 }

  %0 Journal Article %1 RodriguezDominguez201250 %A Rodriguez-Dominguez, C.M. %A Ehrenberger, W. %A Sann, C. %A Rüger, S. %A Sukhorukov, V. %A Martín-Palomo, M.J. %A Diaz-Espejo, A. %A Cuevas, M.V. %A Torres-Ruiz, J.M. %A Perez-Martin, A. %A Zimmermann, U. %A Fernández, J.E. %D 2012 %J Agricultural Water Management %P 50 - 58 %R http://dx.doi.org/10.1016/j.agwat.2012.07.007 %T Concomitant measurements of stem sap flow and leaf turgor pressure in olive trees using the leaf patch clamp pressure probe %U http://www.sciencedirect.com/science/article/pii/S0378377412001916 %V 114 %X Stem sap flow (Q) and leaf turgor pressure (Pc) were measured simultaneously on 4-year-old, 2.4 m tall ‘Arbequina’ olive trees in a hedgerow orchard. Measurements were performed on well-watered control trees as well as on 60RDI and 30RDI trees (RDI = regulated deficit irrigation). The 60RDI trees received 59.2% of the crop water needs (ETc), and the 30RDI trees received 29.4% of ETc. Pc was determined non-invasively using the magnetic leaf patch clamp pressure probe (ZIM probe). The patch pressure Pp measured by the probe is inversely correlated with turgor pressure at Pc > ca. 50 kPa. Pc is coupled with xylem pressure; thus Pp yields information about the development of tension in xylem. In the case of the control trees a positive correlation between Q and Pp was generally found, i.e. Q increased usually with increasing Pp and decreased with decreasing Pp, as expected. However, Q peaking did not always coincided with Pp peaking at noon. Occasionally, Q peaking preceded or followed Pp peaking with a time difference of up to 3 h in both cases. Under some circumstances, the onset of Q after sunrise was greatly delayed, even though a pronounced increase of Pp was observed. A delayed onset of Q after sunrise resulted in hysteresis phenomena, i.e. the linear increase of Q and Pp in the morning hours did not coincide with the corresponding decrease of Q and Pp in the afternoon. The development of severe water stress (Pc < ca. 50 kPa) associated with the increase in the intercellular spaces of the spongy tissue in the leaves resulted in inverted diurnal Pp curves, i.e. minimum Pp values were recorded at noon and maximum Pp values during the night on the 30RDI trees. The effects were reversible as shown by re-watering. By contrast, the magnitude of Q decreased continuously from the turgescent state to the state of severe water stress; maximum Q values were still recorded around noon. The data suggests that short-range tension forces are responsible for water lifting in olive trees and that water uptake from water storage reservoirs must play an important role in the supply of the leaves with water. Furthermore, for setting of irrigation thresholds the finding of shape changes of the Pp curves upon severe water stress seems to be a useful indicator. Such shape changes are detected and monitored more sensitively than changes in the magnitude of sap flow rates or of turgor pressure.
• Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters. Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian; Heilemann, Mike; Nudelman, German; Nachliel, Esther; Gutman, Menachem; Sauer, Markus; Ashery, Uri. In Journal of Biological Chemistry. 2012.
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  Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

  @article{Bar-On14062012, abstract = {Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.}, author = {Bar-On, Dana and Wolter, Steve and van de Linde, Sebastian and Heilemann, Mike and Nudelman, German and Nachliel, Esther and Gutman, Menachem and Sauer, Markus and Ashery, Uri}, journal = {Journal of Biological Chemistry}, keywords = {mike}, title = {Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters}, year = 2012 }

  %0 Journal Article %1 Bar-On14062012 %A Bar-On, Dana %A Wolter, Steve %A van de Linde, Sebastian %A Heilemann, Mike %A Nudelman, German %A Nachliel, Esther %A Gutman, Menachem %A Sauer, Markus %A Ashery, Uri %D 2012 %J Journal of Biological Chemistry %R 10.1074/jbc.M112.353250 %T Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters %U http://www.jbc.org/content/early/2012/06/14/jbc.M112.353250.abstract %X Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.
• Gephyrin, the enigmatic organizer at GABAergic synapses. Tretter, Verena; Mukherjee, Jayanta; Maric, Hans; Schindelin, Hermann; Sieghart, Werner; Moss, Stephen. In Frontiers in Cellular Neuroscience, 6. 2012.
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  GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.

  @article{tretter2012gephyrin, abstract = {GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.}, author = {Tretter, Verena and Mukherjee, Jayanta and Maric, Hans and Schindelin, Hermann and Sieghart, Werner and Moss, Stephen}, journal = {Frontiers in Cellular Neuroscience}, keywords = {maric}, series = 23, title = {Gephyrin, the enigmatic organizer at GABAergic synapses}, volume = 6, year = 2012 }

  %0 Journal Article %1 tretter2012gephyrin %A Tretter, Verena %A Mukherjee, Jayanta %A Maric, Hans %A Schindelin, Hermann %A Sieghart, Werner %A Moss, Stephen %B 23 %D 2012 %J Frontiers in Cellular Neuroscience %T Gephyrin, the enigmatic organizer at GABAergic synapses %U https://www.frontiersin.org/article/10.3389/fncel.2012.00023 %V 6 %X GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin. Gephyrin-interacting domains have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but is also a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signalling proteins into close spatial proximity.
• Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast. Lando, David; Endesfelder, Ulrike; Berger, Harald; Subramanian, Lakxmi; Dunne, Paul D.; McColl, James; Klenerman, David; Carr, Antony M.; Sauer, Markus; Allshire, Robin C.; Heilemann, Mike; Laue, Ernest D. In Open Biology, 2(7). 2012.
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  The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

  @article{LandoJuly2012, abstract = {The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.}, author = {Lando, David and Endesfelder, Ulrike and Berger, Harald and Subramanian, Lakxmi and Dunne, Paul D. and McColl, James and Klenerman, David and Carr, Antony M. and Sauer, Markus and Allshire, Robin C. and Heilemann, Mike and Laue, Ernest D.}, journal = {Open Biology}, keywords = {mike}, number = 7, title = {Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast}, volume = 2, year = 2012 }

  %0 Journal Article %1 LandoJuly2012 %A Lando, David %A Endesfelder, Ulrike %A Berger, Harald %A Subramanian, Lakxmi %A Dunne, Paul D. %A McColl, James %A Klenerman, David %A Carr, Antony M. %A Sauer, Markus %A Allshire, Robin C. %A Heilemann, Mike %A Laue, Ernest D. %D 2012 %J Open Biology %N 7 %R 10.1098/rsob.120078 %T Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast %U http://rsob.royalsocietypublishing.org/content/2/7/120078.abstract %V 2 %X The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
• Live-Cell Super-Resolution Imaging with Synthetic Fluorophores. van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus. In Annual Review of Physical Chemistry, 63(1), S. null. 2012.
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  Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

  @article{doi:10.1146/annurev-physchem-032811-112012, abstract = {Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.}, author = {van de Linde, Sebastian and Heilemann, Mike and Sauer, Markus}, journal = {Annual Review of Physical Chemistry}, keywords = {mike}, number = 1, pages = {null}, title = {Live-Cell Super-Resolution Imaging with Synthetic Fluorophores}, volume = 63, year = 2012 }

  %0 Journal Article %1 doi:10.1146/annurev-physchem-032811-112012 %A van de Linde, Sebastian %A Heilemann, Mike %A Sauer, Markus %D 2012 %J Annual Review of Physical Chemistry %N 1 %P null %R 10.1146/annurev-physchem-032811-112012 %T Live-Cell Super-Resolution Imaging with Synthetic Fluorophores %U http://www.annualreviews.org/doi/abs/10.1146/annurev-physchem-032811-112012 %V 63 %X Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
• Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding. Göhler, Antonia; Büchner, Claudia; André, Sabine; Doose, Sören; Kaltner, Herbert; Gabius, Hans-Joachim. In Biochimie, 94(12), S. 2649–2655. 2012.
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  Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.

  @article{Göhler20122649, abstract = {Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.}, author = {Göhler, Antonia and Büchner, Claudia and André, Sabine and Doose, Sören and Kaltner, Herbert and Gabius, Hans-Joachim}, journal = {Biochimie}, keywords = {doose}, number = 12, pages = {2649 - 2655}, title = {Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding}, volume = 94, year = 2012 }

  %0 Journal Article %1 Göhler20122649 %A Göhler, Antonia %A Büchner, Claudia %A André, Sabine %A Doose, Sören %A Kaltner, Herbert %A Gabius, Hans-Joachim %D 2012 %J Biochimie %N 12 %P 2649 - 2655 %R 10.1016/j.biochi.2012.08.001 %T Analysis of homodimeric avian and human galectins by two methods based on fluorescence spectroscopy: Different structural alterations upon oxidation and ligand binding %U http://www.sciencedirect.com/science/article/pii/S0300908412003100 %V 94 %X Spectroscopic monitoring is applied to detect structural alterations for homodimeric adhesion/growth-regulatory galectins. Mammalian galectin-1 and the avian ortholog CG-1B, due to their distinct patterns of cysteine positioning, can undergo oxidation. When monitoring tryptophan fluorescence anisotropy comparatively, an indicator of structural changes affecting rotational diffusion, segmental motion and/or fluorescence life time, reductions are seen in both cases upon oxidation. The decrease was especially marked for the human protein, more than 2-fold compared to the avian lectin. Using this approach to analyze binding of lactose, equilibrium and kinetic binding constants of both proteins were similar. This result is corroborated by fluorescence correlation spectroscopy with labeled proteins. Of note, the diffusion constant of CG-1B increased by 5.6% in the presence of lactose, as has been seen for the human protein. When processing the other two homodimeric avian galectins (CG-1A, CG-2) accordingly it was revealed that sequence homology does not translate into identical behavior. The diffusion constant of CG-1A was not affected, a slight decrease (−3.8%) was observed for CG-2. Obviously, alterations induced by oxidation and responses to ligand binding are different between these closely related proteins. Methodologically, the two spectroscopic techniques are proven to be sensitive and robust sensors for detecting intergalectin differences.
• Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. Löschberger, Anna; van de Linde, Sebastian; Dabauvalle, Marie-Christine; Rieger, Bernd; Heilemann, Mike; Krohne, Georg; Sauer, Markus. In Journal of Cell Science, 125(3), S. 570–575. 2012.
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  One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

  @article{Löschberger01022012, abstract = {One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of  15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.}, author = {Löschberger, Anna and van de Linde, Sebastian and Dabauvalle, Marie-Christine and Rieger, Bernd and Heilemann, Mike and Krohne, Georg and Sauer, Markus}, journal = {Journal of Cell Science}, keywords = {mike}, number = 3, pages = {570-575}, title = {Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution}, volume = 125, year = 2012 }

  %0 Journal Article %1 Löschberger01022012 %A Löschberger, Anna %A van de Linde, Sebastian %A Dabauvalle, Marie-Christine %A Rieger, Bernd %A Heilemann, Mike %A Krohne, Georg %A Sauer, Markus %D 2012 %J Journal of Cell Science %N 3 %P 570-575 %R 10.1242/jcs.098822 %T Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution %U http://jcs.biologists.org/content/125/3/570.abstract %V 125 %X One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.
• Live-Cell Super-Resolution Imaging Goes Multicolor. Klein, Teresa; van de Linde, Sebastian; Sauer, Markus. In ChemBioChem, 13(13), S. 1861–1863. WILEY-VCH Verlag, 2012.
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  New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.

  @article{CBIC:CBIC201200347, abstract = {New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.}, author = {Klein, Teresa and van de Linde, Sebastian and Sauer, Markus}, journal = {ChemBioChem}, keywords = {linde}, number = 13, pages = {1861–1863}, publisher = {WILEY-VCH Verlag}, title = {Live-Cell Super-Resolution Imaging Goes Multicolor}, volume = 13, year = 2012 }

  %0 Journal Article %1 CBIC:CBIC201200347 %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I WILEY-VCH Verlag %J ChemBioChem %N 13 %P 1861--1863 %R 10.1002/cbic.201200347 %T Live-Cell Super-Resolution Imaging Goes Multicolor %U http://dx.doi.org/10.1002/cbic.201200347 %V 13 %X New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.
• Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV)-Jurkat Cell. SHIRAKASHI, Ryo; SUKHORUKOV, Vladimir L.; REUSS, Randolph; SCHULZ, Alexander; ZIMMERMANN, Ulrich. In Journal of Thermal Science and Technology, 7(4), S. 589–602. 2012.
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  Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.

  @article{RyoSHIRAKASHI2012, abstract = {Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.}, author = {SHIRAKASHI, Ryo and SUKHORUKOV, Vladimir L. and REUSS, Randolph and SCHULZ, Alexander and ZIMMERMANN, Ulrich}, journal = {Journal of Thermal Science and Technology}, keywords = {vladimir}, number = 4, pages = {589-602}, title = {Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV)-Jurkat Cell}, volume = 7, year = 2012 }

  %0 Journal Article %1 RyoSHIRAKASHI2012 %A SHIRAKASHI, Ryo %A SUKHORUKOV, Vladimir L. %A REUSS, Randolph %A SCHULZ, Alexander %A ZIMMERMANN, Ulrich %D 2012 %J Journal of Thermal Science and Technology %N 4 %P 589-602 %T Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV)-Jurkat Cell %U http://japanlinkcenter.org/DN/JST.JSTAGE/jtst/7.589 %V 7 %X Here we describe a new method to deliver membrane impermeable cryo-/lyo-protective agents (CPAs) into the cytosol of living cells via their electrofusion with Giant Unilamellar Vesicles (GUVs) containing large amounts of CPAs. Membrane electrofusion is commonly believed to be triggered by the irreversible electrical breakdown of the membrane at contact region induced by a DC field pulse. Therefore, analysis of the temporal changes of the membrane potential distribution in a cell-GUV pair is is necessary to achieve optimum electrofusion conditions with respect to the field pulse strength and duration. In this study, the GUV-Jurkat cell electrofusion rates under various pulse strength and length were measured. In addition, we calculated the transient membrane potentials in a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The relevant electric properties of both fusion partners reported previously were used for the analysis to validate the quantitative calculation results. Both experimental results and theoretical calculation suggest that; 1) GUV-Jurkat cell pairs should be stretched by AC field prior to electrofusion, 2) the whole membrane contact zone should undergo electrical breakdown at the same time to accomplish electrofusion, 3) after applying an electric field for around 1µsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10µsec, membrane potential is saturated, 5) the irreversible breakdown occurs at a membrane voltage of about 3V.
• Superresolution Optical Fluctuation Imaging (SOFI). Dertinger, Thomas; Colyer, Ryan; Vogel, Robert; Heilemann, Mike; Sauer, Markus; Enderlein, Jörg; Weiss, Shimon. In Advances in Experimental Medicine and Biology, 733, E. Zahavy; A. Ordentlich; S. Yitzhaki; A. Shafferman (Hrsg.), S. 17–21. Springer Netherlands, 2012.
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  Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.

  @article{springerlink:10.1007/978-94-007-2555-3_2, abstract = {Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.}, author = {Dertinger, Thomas and Colyer, Ryan and Vogel, Robert and Heilemann, Mike and Sauer, Markus and Enderlein, Jörg and Weiss, Shimon}, booktitle = {Nano-Biotechnology for Biomedical and Diagnostic Research}, editor = {Zahavy, Eran and Ordentlich, Arie and Yitzhaki, Shmuel and Shafferman, Avigdor}, journal = {Advances in Experimental Medicine and Biology}, keywords = {mike}, note = {10.1007/978-94-007-2555-3_2}, pages = {17-21}, publisher = {Springer Netherlands}, series = {Advances in Experimental Medicine and Biology}, title = {Superresolution Optical Fluctuation Imaging (SOFI)}, volume = 733, year = 2012 }

  %0 Journal Article %1 springerlink:10.1007/978-94-007-2555-3_2 %A Dertinger, Thomas %A Colyer, Ryan %A Vogel, Robert %A Heilemann, Mike %A Sauer, Markus %A Enderlein, Jörg %A Weiss, Shimon %B Nano-Biotechnology for Biomedical and Diagnostic Research %D 2012 %E Zahavy, Eran %E Ordentlich, Arie %E Yitzhaki, Shmuel %E Shafferman, Avigdor %I Springer Netherlands %J Advances in Experimental Medicine and Biology %P 17-21 %T Superresolution Optical Fluctuation Imaging (SOFI) %U http://dx.doi.org/10.1007/978-94-007-2555-3_2 %V 733 %X Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances. %@ 978-94-007-2555-3
• Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies. Terpitz, Ulrich; Letschert, Sebastian; Bonda, Ulrich; Spahn, Christoph; Guan, Chonglin; Sauer, Markus; Zimmermann, Ulrich; Bamberg, Ernst; Zimmermann, Dirk; Sukhorukov, Vladimir. In Journal of Membrane Biology, S. 1–12. Springer New York, 2012.
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  The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

  @article{springerlink:10.1007/s00232-012-9484-9, abstract = {The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.}, author = {Terpitz, Ulrich and Letschert, Sebastian and Bonda, Ulrich and Spahn, Christoph and Guan, Chonglin and Sauer, Markus and Zimmermann, Ulrich and Bamberg, Ernst and Zimmermann, Dirk and Sukhorukov, Vladimir}, journal = {Journal of Membrane Biology}, keywords = {terpitz}, note = {10.1007/s00232-012-9484-9}, pages = {1-12}, publisher = {Springer New York}, title = {Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies}, year = 2012 }

  %0 Journal Article %1 springerlink:10.1007/s00232-012-9484-9 %A Terpitz, Ulrich %A Letschert, Sebastian %A Bonda, Ulrich %A Spahn, Christoph %A Guan, Chonglin %A Sauer, Markus %A Zimmermann, Ulrich %A Bamberg, Ernst %A Zimmermann, Dirk %A Sukhorukov, Vladimir %D 2012 %I Springer New York %J Journal of Membrane Biology %P 1-12 %T Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies %U http://dx.doi.org/10.1007/s00232-012-9484-9 %X The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
• Fluorescence localization microscopy: The transition from concept to biological research tool. Owen, Dylan M.; Sauer, Markus; Gaus, Katharina. In cib, 5(1942-0889), S. 0–1. Landes Bioscience Inc., 2012.
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  Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.

  @article{owen2012fluorescence, abstract = {Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.}, author = {Owen, Dylan M. and Sauer, Markus and Gaus, Katharina}, journal = {cib}, keywords = {sauer}, month = {07}, number = {1942-0889}, pages = {0—1}, publisher = {Landes Bioscience Inc.}, title = {Fluorescence localization microscopy: The transition from concept to biological research tool}, volume = 5, year = 2012 }

  %0 Journal Article %1 owen2012fluorescence %A Owen, Dylan M. %A Sauer, Markus %A Gaus, Katharina %D 2012 %I Landes Bioscience Inc. %J cib %N 1942-0889 %P 0---1 %T Fluorescence localization microscopy: The transition from concept to biological research tool %U http://www.landesbioscience.com/journals/cib/article/20348/ %V 5 %X Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.
• rapidSTORM: accurate, fast open-source software for localization microscopy. Wolter, Steve; Loschberger, Anna; Holm, Thorge; Aufmkolk, Sarah; Dabauvalle, Marie-Christine; van de Linde, Sebastian; Sauer, Markus. In Nat Meth, 9(11), S. 1040–1041. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2012.
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  To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.

  @article{wolter2012rapidstorm, abstract = {To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.}, author = {Wolter, Steve and Loschberger, Anna and Holm, Thorge and Aufmkolk, Sarah and Dabauvalle, Marie-Christine and van de Linde, Sebastian and Sauer, Markus}, journal = {Nat Meth}, keywords = {linde}, month = 11, number = 11, pages = {1040–1041}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {rapidSTORM: accurate, fast open-source software for localization microscopy}, volume = 9, year = 2012 }

  %0 Journal Article %1 wolter2012rapidstorm %A Wolter, Steve %A Loschberger, Anna %A Holm, Thorge %A Aufmkolk, Sarah %A Dabauvalle, Marie-Christine %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %N 11 %P 1040--1041 %T rapidSTORM: accurate, fast open-source software for localization microscopy %U http://dx.doi.org/10.1038/nmeth.2224 %V 9 %X To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.
• Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation. Shirakashi, Ryo; Mischke, Miriam; Fischer, Peter; Memmel, Simon; Krohne, Georg; Fuhr, Günter R; Zimmermann, Heiko; Sukhorukov, Vladimir L. In Biochemical and biophysical research communications. 2012.
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  The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during prehatching development by means of the electrorotation {(ROT)} technique. Due to their layered structure, medaka eggs exhibited up to three {ROT} peaks in the {kHz-MHz} frequency range. During development from blastula to early somite stage, {ROT} spectra varied only slightly. But as the embryo progressed to the late-somite stage, the {ROT} peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the {ROT} spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field {ROT} peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.

  @article{shirakashi_changes_2012, abstract = {The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during prehatching development by means of the electrorotation {(ROT)} technique. Due to their layered structure, medaka eggs exhibited up to three {ROT} peaks in the {kHz-MHz} frequency range. During development from blastula to early somite stage, {ROT} spectra varied only slightly. But as the embryo progressed to the late-somite stage, the {ROT} peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the {ROT} spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field {ROT} peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.}, author = {Shirakashi, Ryo and Mischke, Miriam and Fischer, Peter and Memmel, Simon and Krohne, Georg and Fuhr, Günter R and Zimmermann, Heiko and Sukhorukov, Vladimir L}, journal = {Biochemical and biophysical research communications}, keywords = {vladimir}, month = 10, note = {{PMID:} 23063978}, title = {Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation}, year = 2012 }

  %0 Journal Article %1 shirakashi_changes_2012 %A Shirakashi, Ryo %A Mischke, Miriam %A Fischer, Peter %A Memmel, Simon %A Krohne, Georg %A Fuhr, Günter R %A Zimmermann, Heiko %A Sukhorukov, Vladimir L %D 2012 %J Biochemical and biophysical research communications %R 10.1016/j.bbrc.2012.10.019 %T Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation %U http://dx.doi.org/10.1016/j.bbrc.2012.10.019 %X The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during prehatching development by means of the electrorotation {(ROT)} technique. Due to their layered structure, medaka eggs exhibited up to three {ROT} peaks in the {kHz-MHz} frequency range. During development from blastula to early somite stage, {ROT} spectra varied only slightly. But as the embryo progressed to the late-somite stage, the {ROT} peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the {ROT} spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field {ROT} peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.
• Hsp90 inhibitors, NVP-AUY922 and NVP-BEP800 may exert a significant radiosensitization on tumor cells along with a cell type-specific cytotoxicity. Niewidok, Natalia; Wack, Linda-Jacqueline; Schiessl, Sarah; Stingl, Lavinia; Katzer, Astrid; Polat, Bülent; Sukhorukov, Vladimir L; Flentje, Michael; Djuzenova, Cholpon S. In Transl. Oncology, 5(5), S. 356–369. 2012.
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  Targeting Hsp90 provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration is analyzed. Cellular response was evaluated at different time intervals (0.5, 24 and 48 h) after IR-alone, drug-alone, or combined drug-IR treatments by colony counts, expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR/drug-induced cell cycle arrest, DNA damage and repair. A short 30-min exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone, and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the anti-apoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells. The data show that, simultaneous Hsp90 inhibition and irradiation may induce cell-type specific radiosensitization as well as cytotoxicity against tumor cells.

  @article{noauthororeditor2012hsp90, abstract = {Targeting Hsp90 provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration is analyzed. Cellular response was evaluated at different time intervals (0.5, 24 and 48 h) after IR-alone, drug-alone, or combined drug-IR treatments by colony counts, expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR/drug-induced cell cycle arrest, DNA damage and repair. A short 30-min exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone, and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the anti-apoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells. The data show that, simultaneous Hsp90 inhibition and irradiation may induce cell-type specific radiosensitization as well as cytotoxicity against tumor cells.}, author = {Niewidok, Natalia and Wack, Linda-Jacqueline and Schiessl, Sarah and Stingl, Lavinia and Katzer, Astrid and Polat, Bülent and Sukhorukov, Vladimir L and Flentje, Michael and Djuzenova, Cholpon S}, journal = {Transl. Oncology}, keywords = {vladimir}, month = 10, number = 5, pages = {356-369}, title = {Hsp90 inhibitors, NVP-AUY922 and NVP-BEP800 may exert a significant radiosensitization on tumor cells along with a cell type-specific cytotoxicity}, volume = 5, year = 2012 }

  %0 Journal Article %1 noauthororeditor2012hsp90 %A Niewidok, Natalia %A Wack, Linda-Jacqueline %A Schiessl, Sarah %A Stingl, Lavinia %A Katzer, Astrid %A Polat, Bülent %A Sukhorukov, Vladimir L %A Flentje, Michael %A Djuzenova, Cholpon S %D 2012 %J Transl. Oncology %N 5 %P 356-369 %T Hsp90 inhibitors, NVP-AUY922 and NVP-BEP800 may exert a significant radiosensitization on tumor cells along with a cell type-specific cytotoxicity %U http://www.transonc.com/abstract.php?msid=328 %V 5 %X Targeting Hsp90 provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration is analyzed. Cellular response was evaluated at different time intervals (0.5, 24 and 48 h) after IR-alone, drug-alone, or combined drug-IR treatments by colony counts, expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR/drug-induced cell cycle arrest, DNA damage and repair. A short 30-min exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone, and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the anti-apoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells. The data show that, simultaneous Hsp90 inhibition and irradiation may induce cell-type specific radiosensitization as well as cytotoxicity against tumor cells.
• Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments. Yahiatène, Idir; Doose, Sören; Huser, Thomas; Sauer, Markus. In Analytical Chemistry, 84(6), S. 2729–2736. 2012.
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  We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.

  @article{doi:10.1021/ac2030283, abstract = {We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.}, author = {Yahiatène, Idir and Doose, Sören and Huser, Thomas and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 6, pages = {2729-2736}, title = {Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments}, volume = 84, year = 2012 }

  %0 Journal Article %1 doi:10.1021/ac2030283 %A Yahiatène, Idir %A Doose, Sören %A Huser, Thomas %A Sauer, Markus %D 2012 %J Analytical Chemistry %N 6 %P 2729-2736 %R 10.1021/ac2030283 %T Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments %U http://pubs.acs.org/doi/abs/10.1021/ac2030283 %V 84 %X We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.
• Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia. CS, Djuzenova; C, Blassl; K, Roloff; S, Kuger; A, Katzer; N, Niewidok; N, Gunther; B, Polat; VL, Sukhorukov; M, Flentje. In Cancer Biol Ther., 13(6), S. 425–434. 2012.
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  NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone gammaH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone gammaH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.

  @article{cs2012hsp90, abstract = {NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone gammaH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone gammaH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.}, author = {CS, Djuzenova and C, Blassl and K, Roloff and S, Kuger and A, Katzer and N, Niewidok and N, Gunther and B, Polat and VL, Sukhorukov and M, Flentje}, journal = {Cancer Biol Ther.}, keywords = {vladimir}, month = {04}, number = 6, pages = {425-434}, title = {Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia}, volume = 13, year = 2012 }

  %0 Journal Article %1 cs2012hsp90 %A CS, Djuzenova %A C, Blassl %A K, Roloff %A S, Kuger %A A, Katzer %A N, Niewidok %A N, Gunther %A B, Polat %A VL, Sukhorukov %A M, Flentje %D 2012 %J Cancer Biol Ther. %N 6 %P 425-434 %T Hsp90 inhibitor NVP-AUY922 enhances radiation sensitivity of tumor cell lines under hypoxia %U http://dx.doi.org/10.4161/cbt.19294 %V 13 %X NVP-AUY922, a novel inhibitor of Hsp90, was shown to enhance the effect of ionizing radiation (IR) on tumor cells under normoxic conditions. Since low oxygen tension is a common feature of solid tumors, we explore in the present study the impact of hypoxia on the combined treatment of lung carcinoma A549 and glioblastoma SNB19 cell lines with NVP-AUY922 and IR. Cellular analysis included the colony-forming ability, expression of CAIX, Hsp90, Hsp70, Raf-1, Akt, cell cycle progression and associated proteins, as well as DNA damage measured by histone gammaH2AX. The clonogenic assay revealed that in both cell lines NVP-AUY922 enhanced the radiotoxicity under hypoxic exposure to a level similar to that observed under oxic conditions. Irrespective of oxygen supply during drug treatment, NVP-AUY922 also reduced the expression of anti-apoptotic proteins Raf-1 and Akt. As judged by the levels of histone gammaH2AX, drug-treated hypoxic cells exhibited a lower repair rate of DNA double-strand breaks than normoxic cells. The drug-IR mediated changes in the cell cycle, i.e., S-phase depletion and G 2/M arrest, developed not directly during hypoxic exposure but first upon 24 h reoxygenation. Under both oxygen tensions, Hsp90 inhibition downregulated the cell cycle-associated proteins, Cdk1, Cdk4 and pRb. The finding that NVP-AUY922 can enhance the in vitro radiosensitivity of hypoxic tumor cells may have implications for the combined modality treatment of solid tumors.
• Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images. WOLTER, S.; SAUER, M. In Journal of Microscopy, 245(1), S. 109–109. Blackwell Publishing Ltd, 2012.
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  @article{JMI:JMI3562, author = {WOLTER, S. and SAUER, M.}, journal = {Journal of Microscopy}, keywords = {mike}, number = 1, pages = {109–109}, publisher = {Blackwell Publishing Ltd}, title = {Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images}, volume = 245, year = 2012 }

  %0 Journal Article %1 JMI:JMI3562 %A WOLTER, S. %A SAUER, M. %D 2012 %I Blackwell Publishing Ltd %J Journal of Microscopy %N 1 %P 109--109 %R 10.1111/j.1365-2818.2011.03562.x %T Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images %U http://dx.doi.org/10.1111/j.1365-2818.2011.03562.x %V 245
• Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state. Ehrenberger, W.; Rüger, S.; Rodríguez-Domínguez, C. M.; Díaz-Espejo, A.; Fernández, J.E.; Moreno, J.; Zimmermann, D.; Sukhorukov, V. L.; Zimmermann, U. In Plant Biology, 14(4), S. 666–674. Blackwell Publishing Ltd, 2012.
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  The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, Pp, in response to an externally applied magnetic force. Pp is inversely coupled with leaf turgor pressure, Pc, i.e. at high Pc values the Pp values are small and at low Pc values the Pp values are high. This relationship between Pc and Pp could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (Pc dropped below ca. 50 kPa), Pp curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum Pp value, and during darkness a peak Pp value is recorded. This reversal of the Pp curves was completely reversible. Upon watering, the original diurnal Pp changes were re-established within 2–3 days. Olive trees in the field showed a similar turnover of the shape of the Pp curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the Pp curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal Pp curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental Pp curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero Pc.

  @article{PLB:PLB545, abstract = {The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, Pp, in response to an externally applied magnetic force. Pp is inversely coupled with leaf turgor pressure, Pc, i.e. at high Pc values the Pp values are small and at low Pc values the Pp values are high. This relationship between Pc and Pp could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (Pc dropped below ca. 50 kPa), Pp curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum Pp value, and during darkness a peak Pp value is recorded. This reversal of the Pp curves was completely reversible. Upon watering, the original diurnal Pp changes were re-established within 2–3 days. Olive trees in the field showed a similar turnover of the shape of the Pp curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the Pp curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal Pp curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental Pp curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero Pc.}, author = {Ehrenberger, W. and Rüger, S. and Rodríguez-Domínguez, C. M. and Díaz-Espejo, A. and Fernández, J.E. and Moreno, J. and Zimmermann, D. and Sukhorukov, V. L. and Zimmermann, U.}, journal = {Plant Biology}, keywords = {vladimir}, number = 4, pages = {666–674}, publisher = {Blackwell Publishing Ltd}, title = {Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state}, volume = 14, year = 2012 }

  %0 Journal Article %1 PLB:PLB545 %A Ehrenberger, W. %A Rüger, S. %A Rodríguez-Domínguez, C. M. %A Díaz-Espejo, A. %A Fernández, J.E. %A Moreno, J. %A Zimmermann, D. %A Sukhorukov, V. L. %A Zimmermann, U. %D 2012 %I Blackwell Publishing Ltd %J Plant Biology %N 4 %P 666--674 %R 10.1111/j.1438-8677.2011.00545.x %T Leaf patch clamp pressure probe measurements on olive leaves in a nearly turgorless state %U http://dx.doi.org/10.1111/j.1438-8677.2011.00545.x %V 14 %X The non-invasive leaf patch clamp pressure (LPCP) probe measures the attenuated pressure of a leaf patch, Pp, in response to an externally applied magnetic force. Pp is inversely coupled with leaf turgor pressure, Pc, i.e. at high Pc values the Pp values are small and at low Pc values the Pp values are high. This relationship between Pc and Pp could also be verified for 2-m tall olive trees under laboratory conditions using the cell turgor pressure probe. When the laboratory plants were subjected to severe water stress (Pc dropped below ca. 50 kPa), Pp curves show reverse diurnal changes, i.e. during the light regime (high transpiration) a minimum Pp value, and during darkness a peak Pp value is recorded. This reversal of the Pp curves was completely reversible. Upon watering, the original diurnal Pp changes were re-established within 2–3 days. Olive trees in the field showed a similar turnover of the shape of the Pp curves upon drought, despite pronounced fluctuations in microclimate. The reversal of the Pp curves is most likely due to accumulation of air in the leaves. This assumption was supported with cross-sections through leaves subjected to prolonged drought. In contrast to well-watered leaves, microscopic inspection of leaves exhibiting inverse diurnal Pp curves revealed large air-filled areas in parenchyma tissue. Significantly larger amounts of air could also be extracted from water-stressed leaves than from well-watered leaves using the cell turgor pressure probe. Furthermore, theoretical analysis of the experimental Pp curves shows that the propagation of pressure through the nearly turgorless leaf must be exclusively dictated by air. Equations are derived that provide valuable information about the water status of olive leaves close to zero Pc.
• Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53. Lum, Jenifer K.; Neuweiler, Hannes; Fersht, Alan R. In Journal of the American Chemical Society, 134(3), S. 1617–1622. 2012.
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  The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.

  @article{doi:10.1021/ja2078619, abstract = {The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.}, author = {Lum, Jenifer K. and Neuweiler, Hannes and Fersht, Alan R.}, journal = {Journal of the American Chemical Society}, keywords = {hannes}, number = 3, pages = {1617-1622}, title = {Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53}, volume = 134, year = 2012 }

  %0 Journal Article %1 doi:10.1021/ja2078619 %A Lum, Jenifer K. %A Neuweiler, Hannes %A Fersht, Alan R. %D 2012 %J Journal of the American Chemical Society %N 3 %P 1617-1622 %R 10.1021/ja2078619 %T Long-Range Modulation of Chain Motions within the Intrinsically Disordered Transactivation Domain of Tumor Suppressor p53 %U http://pubs.acs.org/doi/abs/10.1021/ja2078619 %V 134 %X The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.

• Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy. Jensen, Mette H.; Sukumaran, Madhav; Johnson, Christopher M.; Greger, Ingo H.; Neuweiler, Hannes. In Journal of Molecular Biology, 414(1), S. 96–105. 2011.
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  Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.

  @article{Jensen201196, abstract = {Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.}, author = {Jensen, Mette H. and Sukumaran, Madhav and Johnson, Christopher M. and Greger, Ingo H. and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, number = 1, pages = {96 - 105}, title = {Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy}, volume = 414, year = 2011 }

  %0 Journal Article %1 Jensen201196 %A Jensen, Mette H. %A Sukumaran, Madhav %A Johnson, Christopher M. %A Greger, Ingo H. %A Neuweiler, Hannes %D 2011 %J Journal of Molecular Biology %N 1 %P 96 - 105 %R 10.1016/j.jmb.2011.09.037 %T Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy %U http://www.sciencedirect.com/science/article/pii/S0022283611010771 %V 414 %X Ionotropic glutamate receptors (iGluRs) mediate excitatory neurotransmission in the central nervous system and play key roles in brain development and disease. iGluRs have two distinct extracellular domains, but the functional role of the distal N-terminal domain (NTD) is poorly understood. Crystal structures of the NTD from some non-N-methyl-d-aspartate (NMDA) iGluRs are consistent with a rigid body that facilitates receptor assembly but suggest an additional dynamic role that could modulate signaling. Here, we moved beyond spatial and temporal limitations of conventional protein single-molecule spectroscopy by employing correlation analysis of extrinsic oxazine fluorescence fluctuations. We observed nanosecond (ns)-to-microsecond (μs) motions of loop segments and helices within a region of an AMPA-type iGluR NTD, which has been identified previously to be structurally variable. Our data reveal that the AMPA receptor NTD undergoes rapid conformational fluctuations, suggesting an inherent allosteric capacity for this domain in addition to its established assembly function.
• Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Lummer, Martina; Humpert, Fabian; Steuwe, Christian; Caesar, Katharina; Schüttpelz, Mark; Sauer, Markus; Staiger, Dorothee. In Traffic, 12(6), S. 693–702. Blackwell Publishing Ltd, 2011.
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  Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.

  @article{lummer2011reversible, abstract = {Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.}, author = {Lummer, Martina and Humpert, Fabian and Steuwe, Christian and Caesar, Katharina and Schüttpelz, Mark and Sauer, Markus and Staiger, Dorothee}, journal = {Traffic}, keywords = {sauer}, number = 6, pages = {693–702}, publisher = {Blackwell Publishing Ltd}, title = {Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants}, volume = 12, year = 2011 }

  %0 Journal Article %1 lummer2011reversible %A Lummer, Martina %A Humpert, Fabian %A Steuwe, Christian %A Caesar, Katharina %A Schüttpelz, Mark %A Sauer, Markus %A Staiger, Dorothee %D 2011 %I Blackwell Publishing Ltd %J Traffic %N 6 %P 693--702 %R 10.1111/j.1600-0854.2011.01180.x %T Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants %U http://dx.doi.org/10.1111/j.1600-0854.2011.01180.x %V 12 %X Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.
• Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy. Gohler, Antonia; Buchner, Claudia; Andre, Sabine; Doose, Soren; Kaltner, Herbert; Gabius, H.-J. In Analyst, 136(24), S. 5270–5276. The Royal Society of Chemistry, 2011.
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  Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.

  @article{C1AN15692F, abstract = {Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.}, author = {Gohler, Antonia and Buchner, Claudia and Andre, Sabine and Doose, Soren and Kaltner, Herbert and Gabius, H.-J.}, journal = {Analyst}, keywords = {doose}, number = 24, pages = {5270-5276}, publisher = {The Royal Society of Chemistry}, title = {Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy}, volume = 136, year = 2011 }

  %0 Journal Article %1 C1AN15692F %A Gohler, Antonia %A Buchner, Claudia %A Andre, Sabine %A Doose, Soren %A Kaltner, Herbert %A Gabius, H.-J. %D 2011 %I The Royal Society of Chemistry %J Analyst %N 24 %P 5270-5276 %R 10.1039/C1AN15692F %T Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy %U http://dx.doi.org/10.1039/C1AN15692F %V 136 %X Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence{,} robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r0 was about 0.2{,} altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited{,} no labeling is needed. Owing to tryptophan{'}s presence in carbohydrate-binding sites{,} also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases{,} glycosidases){,} this assay procedure can have relevance beyond galectins.
• Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments. Noe, Frank; Doose, Sören; Daidone, Isabella; Löllmann, Marc; Sauer, Markus; Chodera, John D.; Smith, Jeremy C. In Proceedings of the National Academy of Sciences. 2011.
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  There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprints� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.

  @article{Noe, abstract = {There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprintsâ€� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.}, author = {Noe, Frank and Doose, Sören and Daidone, Isabella and Löllmann, Marc and Sauer, Markus and Chodera, John D. and Smith, Jeremy C.}, journal = {Proceedings of the National Academy of Sciences}, keywords = {sauer}, title = {Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments}, year = 2011 }

  %0 Journal Article %1 Noe %A Noe, Frank %A Doose, Sören %A Daidone, Isabella %A Löllmann, Marc %A Sauer, Markus %A Chodera, John D. %A Smith, Jeremy C. %D 2011 %J Proceedings of the National Academy of Sciences %T Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments %U http://dx.doi.org/10.1073/pnas.1004646108 %X There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprintsâ€� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.
• Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV) -Jurkat Cell (Measurement of Fusion Ratio and Electric Field Analysis of Pulsed GUV-Jurkat Cell). SHIRAKASHI, Ryo; REUSS, Randolph; SCHULZ, Alexander; SUKHORUKOV, Vladimir L.; ZIMMERMANN, Ulrich. In TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B, 77(777), S. 1269–1278. 2011.
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  Electrofusion of Giant Unilamellar Vesicle (GUV) and living cell is a gentle method to deliver membrane impermeable cryo-/lyo-protective molecules into living cell for cryo-/lyopreservation. In this method, cellular fusion is believed to be triggered by the irreversible electical breakdown of the membrane at contact region. Nevertheless, as the time change of the membrane potential distribution during an electric pulse is still not clear, the length and strength of electric pulse are hardly designed. In this study, the GUV-Jurkat electrofusion ratios under various pulse strength and length were measured. In addition, we calculated the time change of membrane potential distribution of a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The measured cellular and GUV's electric properties and a GUV-Jurkat profile were used for the analysis to validate the quantitative calculation results. Both results of experiment and calculation suggested that; 1) GUV-Jurkat should be stretched for the electrofusion, 2) whole contact region of membrane should breakdown at the same time to process electrofusion, 3) after applying an electric field for around 1μsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10μsec, membrane potential is relaxed, 5) the irreversible electric breakdown is around 3V.

  @article{2011, abstract = {Electrofusion of Giant Unilamellar Vesicle (GUV) and living cell is a gentle method to deliver membrane impermeable cryo-/lyo-protective molecules into living cell for cryo-/lyopreservation. In this method, cellular fusion is believed to be triggered by the irreversible electical breakdown of the membrane at contact region. Nevertheless, as the time change of the membrane potential distribution during an electric pulse is still not clear, the length and strength of electric pulse are hardly designed. In this study, the GUV-Jurkat electrofusion ratios under various pulse strength and length were measured. In addition, we calculated the time change of membrane potential distribution of a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The measured cellular and GUV's electric properties and a GUV-Jurkat profile were used for the analysis to validate the quantitative calculation results. Both results of experiment and calculation suggested that; 1) GUV-Jurkat should be stretched for the electrofusion, 2) whole contact region of membrane should breakdown at the same time to process electrofusion, 3) after applying an electric field for around 1μsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10μsec, membrane potential is relaxed, 5) the irreversible electric breakdown is around 3V.}, author = {SHIRAKASHI, Ryo and REUSS, Randolph and SCHULZ, Alexander and SUKHORUKOV, Vladimir L. and ZIMMERMANN, Ulrich}, journal = {TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B}, keywords = {vladimir}, number = 777, pages = {1269-1278}, title = {Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV) -Jurkat Cell (Measurement of Fusion Ratio and Electric Field Analysis of Pulsed GUV-Jurkat Cell)}, volume = 77, year = 2011 }

  %0 Journal Article %1 2011 %A SHIRAKASHI, Ryo %A REUSS, Randolph %A SCHULZ, Alexander %A SUKHORUKOV, Vladimir L. %A ZIMMERMANN, Ulrich %D 2011 %J TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B %N 777 %P 1269-1278 %T Effects of a Pulse Electric Field on Electrofusion of Giant Unilamellar Vesicle (GUV) -Jurkat Cell (Measurement of Fusion Ratio and Electric Field Analysis of Pulsed GUV-Jurkat Cell) %U http://dx.doi.org/10.1299/kikaib.77.1269 %V 77 %X Electrofusion of Giant Unilamellar Vesicle (GUV) and living cell is a gentle method to deliver membrane impermeable cryo-/lyo-protective molecules into living cell for cryo-/lyopreservation. In this method, cellular fusion is believed to be triggered by the irreversible electical breakdown of the membrane at contact region. Nevertheless, as the time change of the membrane potential distribution during an electric pulse is still not clear, the length and strength of electric pulse are hardly designed. In this study, the GUV-Jurkat electrofusion ratios under various pulse strength and length were measured. In addition, we calculated the time change of membrane potential distribution of a deformed GUV-Jurkat pair during the electric field application by a finite element method (FEM)-electric field analysis. The measured cellular and GUV's electric properties and a GUV-Jurkat profile were used for the analysis to validate the quantitative calculation results. Both results of experiment and calculation suggested that; 1) GUV-Jurkat should be stretched for the electrofusion, 2) whole contact region of membrane should breakdown at the same time to process electrofusion, 3) after applying an electric field for around 1μsec, membrane potential in contact region becomes homogeneous, 4) after applying an electric field for around 10μsec, membrane potential is relaxed, 5) the irreversible electric breakdown is around 3V.
• Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels. Bobak, Nicole; Bittner, Stefan; Andronic, Joseph; Hartmann, Susanne; Mühlpfordt, Friederike; Schneider-Hohendorf, Tilman; Wolf, Karen; Schmelter, Carsten; Göbel, Kerstin; Meuth, Patrick; Zimmermann, Heiko; Döring, Frank; Wischmeyer, Erhard; Budde, Thomas; Wiendl, Heinz; Meuth, Sven G.; Sukhorukov, Vladimir L. In Biochimica et Biophysica Acta (BBA) - Biomembranes, 1808(8), S. 2036–2044. 2011.
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  A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel KV1.3 and the calcium-activated potassium channel KCa3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of KV1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K2P channels.

  @article{Bobak20112036, abstract = {A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel KV1.3 and the calcium-activated potassium channel KCa3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of KV1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K2P channels.}, author = {Bobak, Nicole and Bittner, Stefan and Andronic, Joseph and Hartmann, Susanne and Mühlpfordt, Friederike and Schneider-Hohendorf, Tilman and Wolf, Karen and Schmelter, Carsten and Göbel, Kerstin and Meuth, Patrick and Zimmermann, Heiko and Döring, Frank and Wischmeyer, Erhard and Budde, Thomas and Wiendl, Heinz and Meuth, Sven G. and Sukhorukov, Vladimir L.}, journal = {Biochimica et Biophysica Acta (BBA) - Biomembranes}, keywords = {vladimir}, number = 8, pages = {2036 - 2044}, title = {Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels}, volume = 1808, year = 2011 }

  %0 Journal Article %1 Bobak20112036 %A Bobak, Nicole %A Bittner, Stefan %A Andronic, Joseph %A Hartmann, Susanne %A Mühlpfordt, Friederike %A Schneider-Hohendorf, Tilman %A Wolf, Karen %A Schmelter, Carsten %A Göbel, Kerstin %A Meuth, Patrick %A Zimmermann, Heiko %A Döring, Frank %A Wischmeyer, Erhard %A Budde, Thomas %A Wiendl, Heinz %A Meuth, Sven G. %A Sukhorukov, Vladimir L. %D 2011 %J Biochimica et Biophysica Acta (BBA) - Biomembranes %N 8 %P 2036 - 2044 %R 10.1016/j.bbamem.2011.04.013 %T Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels %U http://www.sciencedirect.com/science/article/pii/S0005273611001180 %V 1808 %X A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel KV1.3 and the calcium-activated potassium channel KCa3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of KV1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K2P channels.
• Measuring localization performance of super-resolution algorithms on very active samples. Wolter, Steve; Endesfelder, Ulrike; van de Linde, S.; Heilemann, Mike; Sauer, Markus. In Optics Express, 19(8), S. 7020–33. 2011.
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  Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.

  @article{Sauer_2011, abstract = {Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.}, author = {Wolter, Steve and Endesfelder, Ulrike and van de Linde, S. and Heilemann, Mike and Sauer, Markus}, journal = {Optics Express}, keywords = {mike}, number = 8, pages = {7020–33}, title = {Measuring localization performance of super-resolution algorithms on very active samples.}, volume = 19, year = 2011 }

  %0 Journal Article %1 Sauer_2011 %A Wolter, Steve %A Endesfelder, Ulrike %A van de Linde, S. %A Heilemann, Mike %A Sauer, Markus %D 2011 %J Optics Express %N 8 %P 7020--33 %T Measuring localization performance of super-resolution algorithms on very active samples. %U http://www.ncbi.nlm.nih.gov/pubmed/21503016 %V 19 %X Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.
• Backbone-Driven Collapse in Unfolded Protein Chains. Teufel, Daniel P.; Johnson, Christopher M.; Lum, Jenifer K.; Neuweiler, Hannes. In Journal of Molecular Biology, 409(2), S. 250–262. 2011.
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  Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.

  @article{Teufel2011250, abstract = {Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.}, author = {Teufel, Daniel P. and Johnson, Christopher M. and Lum, Jenifer K. and Neuweiler, Hannes}, journal = {Journal of Molecular Biology}, keywords = {hannes}, number = 2, pages = {250 - 262}, title = {Backbone-Driven Collapse in Unfolded Protein Chains}, volume = 409, year = 2011 }

  %0 Journal Article %1 Teufel2011250 %A Teufel, Daniel P. %A Johnson, Christopher M. %A Lum, Jenifer K. %A Neuweiler, Hannes %D 2011 %J Journal of Molecular Biology %N 2 %P 250 - 262 %R DOI: 10.1016/j.jmb.2011.03.066 %T Backbone-Driven Collapse in Unfolded Protein Chains %U http://www.sciencedirect.com/science/article/pii/S002228361100355X %V 409 %X Collapse of unfolded protein chains is an early event in folding. It affects structural properties of intrinsically disordered proteins, which take a considerable fraction of the human proteome. Collapse is generally believed to be driven by hydrophobic forces imposed by the presence of nonpolar amino acid side chains. Contributions from backbone hydrogen bonds to protein folding and stability, however, are controversial. To date, the experimental dissection of side-chain and backbone contributions has not yet been achieved because both types of interactions are integral parts of protein structure. Here, we realized this goal by applying mutagenesis and chemical modification on a set of disordered peptides and proteins. We measured the protein dimensions and kinetics of intra-chain diffusion of modified polypeptides at the level of individual molecules using fluorescence correlation spectroscopy, thereby avoiding artifacts commonly caused by aggregation of unfolded protein material in bulk. We found no contributions from side chains to collapse but, instead, identified backbone interactions as a source sufficient to form globules of native-like dimensions. The presence of backbone hydrogen bonds decreased polypeptide water solubility dramatically and accelerated the nanosecond kinetics of loop closure, in agreement with recent predictions from computer simulation. The presence of side chains, instead, slowed loop closure and modulated the dimensions of intrinsically disordered domains. It appeared that the transient formation of backbone interactions facilitates the diffusive search for productive conformations at the early stage of folding and within intrinsically disordered proteins.
• Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes. Seefeldt, Britta; Altenhoner, Kai; Tosic, Oliver; Geisler, Thomas; Sauer, Markus; Mattay, Jochen. In Photochem. Photobiol. Sci., 10(9), S. 1488–1495. The Royal Society of Chemistry, 2011.
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  We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.

  @article{seefeldt2011kinetic, abstract = {We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.}, author = {Seefeldt, Britta and Altenhoner, Kai and Tosic, Oliver and Geisler, Thomas and Sauer, Markus and Mattay, Jochen}, journal = {Photochem. Photobiol. Sci.}, keywords = {sauer}, number = 9, pages = {1488–1495}, publisher = {The Royal Society of Chemistry}, title = {Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes}, volume = 10, year = 2011 }

  %0 Journal Article %1 seefeldt2011kinetic %A Seefeldt, Britta %A Altenhoner, Kai %A Tosic, Oliver %A Geisler, Thomas %A Sauer, Markus %A Mattay, Jochen %D 2011 %I The Royal Society of Chemistry %J Photochem. Photobiol. Sci. %N 9 %P 1488--1495 %R 10.1039/C1PP05051F %T Kinetic studies on visible-light-switchable photochromic fluorophores based on diarylethenes %U http://dx.doi.org/10.1039/C1PP05051F %V 10 %X We present three recently developed photochromic fluorophores that are based on diarylethenes with elongated conjugated [small pi]-systems. The diarylethenes 1 and 3 can be switched from their open to their closed form with visible light. The diarylethenes 1 and 2 are covalently coupled to a standard rhodamine B-based fluorophore and act as photoswitchable resonance energy acceptors. By controlling their switching state, the fluorescence intensity of the dye can be modulated. The third compound 3 is a diarylethene that shows photoswitchable inherent fluorescence due to its stilbazolium-like structure. Ensemble experiments demonstrate that diarylethene-based photoswitches show superior characteristics regarding their switching performance, thermal stability and fatigue resistance. These attributes make them promising candidates for super-resolution imaging methods that are based on the determinate fluorescence switching of fluorophores between an off- and an on-state.
• Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. van de Linde, Sebastian; Krstic, Ivan; Prisner, Thomas; Doose, Soren; Heilemann, Mike; Sauer, Markus. In Photochem. Photobiol. Sci., 10(4), S. 499–506. The Royal Society of Chemistry, 2011.
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  Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.

  @article{C0PP00317D, abstract = {Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.}, author = {van de Linde, Sebastian and Krstic, Ivan and Prisner, Thomas and Doose, Soren and Heilemann, Mike and Sauer, Markus}, journal = {Photochem. Photobiol. Sci.}, keywords = {mike}, pages = {499-506}, publisher = {The Royal Society of Chemistry}, title = {Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging}, volume = 10, year = 2011 }

  %0 Journal Article %1 C0PP00317D %A van de Linde, Sebastian %A Krstic, Ivan %A Prisner, Thomas %A Doose, Soren %A Heilemann, Mike %A Sauer, Markus %D 2011 %I The Royal Society of Chemistry %J Photochem. Photobiol. Sci. %P 499-506 %R 10.1039/C0PP00317D %T Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging %U http://dx.doi.org/10.1039/C0PP00317D %V 10 %X Radical ions of organic dyes are highly reactive species and have been studied for decades by transient absorption spectroscopy and pulse radiolysis experiments in oxygen-depleted solution. Here we show by continuous wave EPR, absorption, and fluorescence experiments that the triplet state of rhodamine dyes can be photoreduced by thiols to form stable radical anions in aqueous solution with a lifetime of up to several hours. Our data demonstrate that reduction of the triplet state and photoinduced oxidation of reactive intermediates by oxygen represents a general mechanism for reversible photoswitching of dyes in aqueous thiol-containing solutions highlighting the key role of molecular oxygen for super-resolution fluorescence imaging. Since cells contain the thiol glutathione at millimolar concentrations and reactive oxygen species are formed as side products our findings are of consequence for live cell fluorescence microscopy.
• Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions. Bollmann, Stefan; Lollmann, Marc; Sauer, Markus; Doose, Soren. In Phys. Chem. Chem. Phys., 13(28), S. 12874–12882. The Royal Society of Chemistry, 2011.
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  Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.

  @article{C1CP21111K, abstract = {Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.}, author = {Bollmann, Stefan and Lollmann, Marc and Sauer, Markus and Doose, Soren}, journal = {Phys. Chem. Chem. Phys.}, keywords = {sauer}, pages = {12874-12882}, publisher = {The Royal Society of Chemistry}, title = {Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions}, volume = 13, year = 2011 }

  %0 Journal Article %1 C1CP21111K %A Bollmann, Stefan %A Lollmann, Marc %A Sauer, Markus %A Doose, Soren %D 2011 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %P 12874-12882 %R 10.1039/C1CP21111K %T Dimer formation of organic fluorophores reports on biomolecular dynamics under denaturing conditions %U http://dx.doi.org/10.1039/C1CP21111K %V 13 %X Stacking interactions between organic fluorophores can cause formation of non-fluorescent H-dimers. Dimer formation and dissociation of two fluorophores site-specifically incorporated in a biomolecule result in fluorescence intermittency that can report on conformational dynamics. We characterize intramolecular dimerization of two oxazine fluorophores MR121 attached to an unstructured polypeptide. Formation of stable non-fluorescent complexes with nano- to microsecond lifetimes is a prerequisite for analysing the intermittent fluorescence emission by fluorescence correlation spectroscopy and extracting relaxation time constants on nano- to millisecond time scales. Destabilization of the generally very stable homodimers by chemical denaturation reduces the lifetime of H-dimers. We demonstrate that H-dimer formation of an oxazine fluorophore reports on end-to-end contact rates in unstructured glycine-serine polypeptides under denaturing conditions.
• Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin. Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan; Ramsden, Sarah L.; Ramerstorfer, Joachim; Saiepour, Leila; Maric, Hans-Michael; Moss, Stephen J.; Schindelin, Hermann; Harvey, Robert J.; Sieghart, Werner; Harvey, Kirsten. In Journal of Biological Chemistry, 286(43), S. 37702–37711. 2011.
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  The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

  @article{Tretter28102011, abstract = {The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.}, author = {Tretter, Verena and Kerschner, Bernd and Milenkovic, Ivan and Ramsden, Sarah L. and Ramerstorfer, Joachim and Saiepour, Leila and Maric, Hans-Michael and Moss, Stephen J. and Schindelin, Hermann and Harvey, Robert J. and Sieghart, Werner and Harvey, Kirsten}, journal = {Journal of Biological Chemistry}, keywords = {maric}, number = 43, pages = {37702-37711}, title = {Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin}, volume = 286, year = 2011 }

  %0 Journal Article %1 Tretter28102011 %A Tretter, Verena %A Kerschner, Bernd %A Milenkovic, Ivan %A Ramsden, Sarah L. %A Ramerstorfer, Joachim %A Saiepour, Leila %A Maric, Hans-Michael %A Moss, Stephen J. %A Schindelin, Hermann %A Harvey, Robert J. %A Sieghart, Werner %A Harvey, Kirsten %D 2011 %J Journal of Biological Chemistry %N 43 %P 37702-37711 %R 10.1074/jbc.M111.291336 %T Molecular Basis of the γ-Aminobutyric Acid A Receptor α3 Subunit Interaction with the Clustering Protein Gephyrin %U http://www.jbc.org/content/286/43/37702.abstract %V 286 %X The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABAA receptor (GABAAR) subtypes. We report a direct interaction between the GABAAR α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABAAR α3 M3–M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABAAR α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABAARs in recombinant systems. Interestingly, motifs responsible for interactions with GABAAR α2, GABAAR α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABAAR α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABAAR α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABAAR α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.
• Simultaneous radiation and Hsp90 inhibition by NVP-AUY922 or NVP-BEP800 is more efficient in tumor cell killing than drug-first modality followed by irradiation. Niewidok, N.; Wack, L-J; Stingl, L.; Katzer, A.; Sukhorukov, V. L.; Flentje, M.; Djuzenova, C. S. In STRAHLENTHERAPIE UND ONKOLOGIE, 187(1), S. 125–126. URBAN & VOGEL, NEUMARKTER STRASSE 43, D-81673 MUNICH, GERMANY, 2011.
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  @article{ISI:000291236200358, address = {NEUMARKTER STRASSE 43, D-81673 MUNICH, GERMANY}, author = {Niewidok, N. and Wack, L-J and Stingl, L. and Katzer, A. and Sukhorukov, V. L. and Flentje, M. and Djuzenova, C. S.}, journal = {STRAHLENTHERAPIE UND ONKOLOGIE}, keywords = {vladimir}, month = {06}, note = {{17th Annual Meeting of the German-Society-for-Radiation-Oncology, Wiesbaden, GERMANY, JUN 02-05, 2011}}, number = 1, organization = {German Soc Radiat Oncol}, pages = {125-126}, publisher = {URBAN \& VOGEL}, title = {Simultaneous radiation and Hsp90 inhibition by NVP-AUY922 or NVP-BEP800 is more efficient in tumor cell killing than drug-first modality followed by irradiation}, type = {Meeting Abstract}, volume = 187, year = 2011 }

  %0 Journal Article %1 ISI:000291236200358 %A Niewidok, N. %A Wack, L-J %A Stingl, L. %A Katzer, A. %A Sukhorukov, V. L. %A Flentje, M. %A Djuzenova, C. S. %C NEUMARKTER STRASSE 43, D-81673 MUNICH, GERMANY %D 2011 %I URBAN & VOGEL %J STRAHLENTHERAPIE UND ONKOLOGIE %N 1 %P 125-126 %T Simultaneous radiation and Hsp90 inhibition by NVP-AUY922 or NVP-BEP800 is more efficient in tumor cell killing than drug-first modality followed by irradiation %V 187
• Conformational Flexibility of Glycosylated Peptides. Bollmann, Stefan; Burgert, Anne; Plattner, Carolin; Nagel, Lilly; Sewald, Norbert; Löllmann, Marc; Sauer, Markus; Doose, Sören. In ChemPhysChem, 12(16), S. 2907–2911. WILEY-VCH Verlag, 2011.
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  With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides

  @article{CPHC:CPHC201100650, abstract = {With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides}, author = {Bollmann, Stefan and Burgert, Anne and Plattner, Carolin and Nagel, Lilly and Sewald, Norbert and Löllmann, Marc and Sauer, Markus and Doose, Sören}, journal = {ChemPhysChem}, keywords = {sauer}, number = 16, pages = {2907–2911}, publisher = {WILEY-VCH Verlag}, title = {Conformational Flexibility of Glycosylated Peptides}, volume = 12, year = 2011 }

  %0 Journal Article %1 CPHC:CPHC201100650 %A Bollmann, Stefan %A Burgert, Anne %A Plattner, Carolin %A Nagel, Lilly %A Sewald, Norbert %A Löllmann, Marc %A Sauer, Markus %A Doose, Sören %D 2011 %I WILEY-VCH Verlag %J ChemPhysChem %N 16 %P 2907--2911 %R 10.1002/cphc.201100650 %T Conformational Flexibility of Glycosylated Peptides %U http://dx.doi.org/10.1002/cphc.201100650 %V 12 %X With a twist: The conformational dynamics of glycosylated glycine–serine peptides is studied using contact- induced fluorescence quenching analysed by fluorescence correlation spectroscopy. End-to-end contact rates on ns–μs timescales reveal enthalpic and entropic contributions to the reduction of contact formation rates in glycopeptides
• The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin.. Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine; Maric, Hans-Michael; Ramsden, Sarah; Tretter, Verena; Harvey, Kirsten; Davies, Paul A.; Triller, Antoine; Schindelin, Hermann; Moss, Stephen J. In J. Neurosci., 31(41), S. 14677-. 2011.
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  The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.

  @article{mukherjee2011residence, abstract = {The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.}, author = {Mukherjee, Jayanta and Kretschmannova, Karla and Gouzer, Geraldine and Maric, Hans-Michael and Ramsden, Sarah and Tretter, Verena and Harvey, Kirsten and Davies, Paul A. and Triller, Antoine and Schindelin, Hermann and Moss, Stephen J.}, journal = {J. Neurosci.}, keywords = {maric}, month = 10, number = 41, pages = {14677–}, title = {The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin}, volume = 31, year = 2011 }

  %0 Journal Article %1 mukherjee2011residence %A Mukherjee, Jayanta %A Kretschmannova, Karla %A Gouzer, Geraldine %A Maric, Hans-Michael %A Ramsden, Sarah %A Tretter, Verena %A Harvey, Kirsten %A Davies, Paul A. %A Triller, Antoine %A Schindelin, Hermann %A Moss, Stephen J. %D 2011 %J J. Neurosci. %N 41 %P 14677-- %R 10.1523/JNEUROSCI.2001-11.2011 %T The Residence Time of GABA_ARs at Inhibitory Synapses Is Determined by Direct Binding of the Receptor α1 Subunit to Gephyrin %U http://www.jneurosci.org/content/31/41/14677.abstract %V 31 %X The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABAARs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ∼20 μm, mediated by residues 360–375 within the intracellular domain of this receptor subunit. Mutating residues 360–375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360–375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.
• A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins. Kleinlogel, Sonja; Terpitz, Ulrich; Legrum, Barbara; Gokbuget, Deniz; Boyden, Edward S; Bamann, Christian; Wood, Phillip G; Bamberg, Ernst. In Nat Meth, 8(12), S. 1083–1088. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2011.
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  The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

  @article{kleinlogel2011genefusion, abstract = {The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.}, author = {Kleinlogel, Sonja and Terpitz, Ulrich and Legrum, Barbara and Gokbuget, Deniz and Boyden, Edward S and Bamann, Christian and Wood, Phillip G and Bamberg, Ernst}, journal = {Nat Meth}, keywords = {terpitz}, month = 12, number = 12, pages = {1083–1088}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins}, volume = 8, year = 2011 }

  %0 Journal Article %1 kleinlogel2011genefusion %A Kleinlogel, Sonja %A Terpitz, Ulrich %A Legrum, Barbara %A Gokbuget, Deniz %A Boyden, Edward S %A Bamann, Christian %A Wood, Phillip G %A Bamberg, Ernst %D 2011 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %N 12 %P 1083--1088 %T A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins %U http://dx.doi.org/10.1038/nmeth.1766 %V 8 %X The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.
• Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site. Maric, Hans-Michael; Mukherjee, Jayanta; Tretter, Verena; Moss, Stephen J; Schindelin, Hermann. In The Journal of biological chemistry, 286(49), S. 42105–42114. American Society for Biochemistry and Molecular Biology, 2011.
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  Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.

  @article{maric2011gephyrinmediated, abstract = {Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.}, author = {Maric, Hans-Michael and Mukherjee, Jayanta and Tretter, Verena and Moss, Stephen J and Schindelin, Hermann}, journal = {The Journal of biological chemistry}, keywords = {maric}, month = 12, number = 49, pages = {42105–42114}, publisher = {American Society for Biochemistry and Molecular Biology}, title = {Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site}, volume = 286, year = 2011 }

  %0 Journal Article %1 maric2011gephyrinmediated %A Maric, Hans-Michael %A Mukherjee, Jayanta %A Tretter, Verena %A Moss, Stephen J %A Schindelin, Hermann %D 2011 %I American Society for Biochemistry and Molecular Biology %J The Journal of biological chemistry %N 49 %P 42105--42114 %R 10.1074/jbc.M111.303412 %T Gephyrin-mediated γ-aminobutyric acid type A and glycine receptor clustering relies on a common binding site %U https://www.ncbi.nlm.nih.gov/pubmed/22006921 %V 286 %X Gephyrin is the major protein determinant for the clustering of inhibitory neurotransmitter receptors. Earlier analyses revealed that gephyrin tightly binds to residues 398-410 of the glycine receptor β subunit (GlyR β) and, as demonstrated only recently, also interacts with GABA(A) receptors (GABA(A)Rs) containing the α1, α2, and α3 subunits. Here, we dissect the molecular basis underlying the interactions between gephyrin and GABA(A)Rs containing these α-subunits and compare them to the crystal structure of the gephyrin-GlyR β complex. Biophysical and biochemical assays revealed that, in contrast to its tight interaction with GlyR β, gephyrin only loosely interacts with GABA(A)R α2, whereas it has an intermediate affinity for the GABA(A)R α1 and α3 subunits. Despite the wide variation in affinities and the low overall sequence homology among the identified receptor subunits, competition assays confirmed the receptor-gephyrin interaction to be a mutually exclusive process. Selected gephyrin point mutants that critically weaken complex formation with GlyR β also abolished the GABA(A)R α1 and α3 interactions. Additionally, we identified a common binding motif with two conserved aromatic residues that are central for gephyrin binding. Consistent with the biochemical data, mutations of the corresponding residues within the cytoplasmic domain of α2 subunit-containing GABA(A)Rs attenuated clustering of these receptors at postsynaptic sites in hippocampal neurons. Taken together, our experiments provide key insights regarding similarities and differences in the complex formation between gephyrin and GABA(A)Rs compared with GlyRs and, hence, the accumulation of these receptors at postsynaptic sites.
• Direct stochastic optical reconstruction microscopy with standard fluorescent probes. van de Linde, Sebastian; Loschberger, Anna; Klein, Teresa; Heidbreder, Meike; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In Nat. Protocols, 6(7), S. 991–1009. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved., 2011.
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  Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

  @article{sebastian2011direct, abstract = {Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of  20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.}, author = {van de Linde, Sebastian and Loschberger, Anna and Klein, Teresa and Heidbreder, Meike and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {Nat. Protocols}, keywords = {mike}, month = {06}, number = 7, pages = {991–1009}, publisher = {Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.}, title = {Direct stochastic optical reconstruction microscopy with standard fluorescent probes}, volume = 6, year = 2011 }

  %0 Journal Article %1 sebastian2011direct %A van de Linde, Sebastian %A Loschberger, Anna %A Klein, Teresa %A Heidbreder, Meike %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %D 2011 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat. Protocols %N 7 %P 991--1009 %T Direct stochastic optical reconstruction microscopy with standard fluorescent probes %U http://dx.doi.org/10.1038/nprot.2011.336 %V 6 %X Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.
• Single-molecule Photoswitching and Localization. van de Linde, Sebastian; Wolter, Steve; Sauer, Markus. In Aust. J. Chem., 64(5), S. 503–511. 2011.
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  Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

  @article{sebastian2011singlemolecule, abstract = {Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to  20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.}, author = {van de Linde, Sebastian and Wolter, Steve and Sauer, Markus}, journal = {Aust. J. Chem.}, keywords = {linde}, month = {05}, number = 5, pages = {503–511}, title = {Single-molecule Photoswitching and Localization}, volume = 64, year = 2011 }

  %0 Journal Article %1 sebastian2011singlemolecule %A van de Linde, Sebastian %A Wolter, Steve %A Sauer, Markus %D 2011 %J Aust. J. Chem. %N 5 %P 503--511 %T Single-molecule Photoswitching and Localization %U http://dx.doi.org/10.1071/CH10284 %V 64 %X Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ~20Â nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.
• Live-cell dSTORM with SNAP-tag fusion proteins. Klein, Teresa; Loeschberger, Anna; Proppert, Sven; Wolter, Steve; van de Linde, Sebastian; Sauer, Markus. In NATURE METHODS, 8(1), S. 7–9. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2011.
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  In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.

  @article{Klein2011, abstract = {In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Klein, Teresa and Loeschberger, Anna and Proppert, Sven and Wolter, Steve and van de Linde, Sebastian and Sauer, Markus}, journal = {NATURE METHODS}, keywords = {linde}, month = {01}, number = 1, pages = {7-9}, publisher = {NATURE PUBLISHING GROUP}, title = {Live-cell dSTORM with SNAP-tag fusion proteins}, type = {Letter}, volume = 8, year = 2011 }

  %0 Journal Article %1 Klein2011 %A Klein, Teresa %A Loeschberger, Anna %A Proppert, Sven %A Wolter, Steve %A van de Linde, Sebastian %A Sauer, Markus %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2011 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 1 %P 7-9 %T Live-cell dSTORM with SNAP-tag fusion proteins %U http://dx.doi.org/10.1038/nmeth0111-7b %V 8 %X In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.
• In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging. Kaminski Schierle, Gabriele S.; van de Linde, Sebastian; Erdelyi, Miklos; Esbjörner, Elin K.; Klein, Teresa; Rees, Eric; Bertoncini, Carlos W.; Dobson, Christopher M.; Sauer, Markus; Kaminski, Clemens F. In Journal of the American Chemical Society, 133(33), S. 12902–12905. 2011.
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  Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.

  @article{doi:10.1021/ja201651w, abstract = {Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.}, author = {Kaminski Schierle, Gabriele S. and van de Linde, Sebastian and Erdelyi, Miklos and Esbjörner, Elin K. and Klein, Teresa and Rees, Eric and Bertoncini, Carlos W. and Dobson, Christopher M. and Sauer, Markus and Kaminski, Clemens F.}, journal = {Journal of the American Chemical Society}, keywords = {linde}, note = {PMID: 21793568}, number = 33, pages = {12902-12905}, title = {In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging}, volume = 133, year = 2011 }

  %0 Journal Article %1 doi:10.1021/ja201651w %A Kaminski Schierle, Gabriele S. %A van de Linde, Sebastian %A Erdelyi, Miklos %A Esbjörner, Elin K. %A Klein, Teresa %A Rees, Eric %A Bertoncini, Carlos W. %A Dobson, Christopher M. %A Sauer, Markus %A Kaminski, Clemens F. %D 2011 %J Journal of the American Chemical Society %N 33 %P 12902-12905 %R 10.1021/ja201651w %T In Situ Measurements of the Formation and Morphology of Intracellular β-Amyloid Fibrils by Super-Resolution Fluorescence Imaging %U /brokenurl# http://dx.doi.org/10.1021/ja201651w %V 133 %X Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer’s disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.
• Cross-linking of DNA through HMGA1 suggests a DNA scaffold. Vogel, Benjamin; Löschberger, Anna; Sauer, Markus; Hock, Robert. In Nucleic Acids Research. 2011.
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  Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.

  @article{Vogel19052011, abstract = {Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.}, author = {Vogel, Benjamin and Löschberger, Anna and Sauer, Markus and Hock, Robert}, journal = {Nucleic Acids Research}, keywords = {sauer}, title = {Cross-linking of DNA through HMGA1 suggests a DNA scaffold}, year = 2011 }

  %0 Journal Article %1 Vogel19052011 %A Vogel, Benjamin %A Löschberger, Anna %A Sauer, Markus %A Hock, Robert %D 2011 %J Nucleic Acids Research %R 10.1093/nar/gkr396 %T Cross-linking of DNA through HMGA1 suggests a DNA scaffold %U http://nar.oxfordjournals.org/content/early/2011/05/19/nar.gkr396.abstract %X Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.

• DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis. Schlereth, Katharina; Beinoraviciute-Kellner, Rasa; Zeitlinger, Marie K.; Bretz, Anne C.; Sauer, Markus; Charles, Joel P.; Vogiatzi, Fotini; Leich, Ellen; Samans, Birgit; Eilers, Martin; Kisker, Caroline; Rosenwald, Andreas; Stiewe, Thorsten. In MOLECULAR CELL, 38(3), S. 356–368. CELL PRESS, 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA, 2010.
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  p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.

  @article{Schlereth2010, abstract = {p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.}, address = {600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA}, author = {Schlereth, Katharina and Beinoraviciute-Kellner, Rasa and Zeitlinger, Marie K. and Bretz, Anne C. and Sauer, Markus and Charles, Joel P. and Vogiatzi, Fotini and Leich, Ellen and Samans, Birgit and Eilers, Martin and Kisker, Caroline and Rosenwald, Andreas and Stiewe, Thorsten}, journal = {MOLECULAR CELL}, keywords = {sauer}, month = {05}, number = 3, pages = {356-368}, publisher = {CELL PRESS}, title = {DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis}, type = {Article}, volume = 38, year = 2010 }

  %0 Journal Article %1 Schlereth2010 %A Schlereth, Katharina %A Beinoraviciute-Kellner, Rasa %A Zeitlinger, Marie K. %A Bretz, Anne C. %A Sauer, Markus %A Charles, Joel P. %A Vogiatzi, Fotini %A Leich, Ellen %A Samans, Birgit %A Eilers, Martin %A Kisker, Caroline %A Rosenwald, Andreas %A Stiewe, Thorsten %C 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA %D 2010 %I CELL PRESS %J MOLECULAR CELL %N 3 %P 356-368 %T DNA Binding Cooperativity of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis %U http://dx.doi.org/10.1016/j.molcel.2010.02.037 %V 38 %X p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53's tumor suppressor activity.
• Superresolution Optical Fluctuation Imaging with Organic Dyes. Dertinger, Thomas; Heilemann, Mike; Vogel, Robert; Sauer, Markus; Weiss, Shimon. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 49(49), S. 9441–9443. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.

  @article{Dertinger2010, abstract = {SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Dertinger, Thomas and Heilemann, Mike and Vogel, Robert and Sauer, Markus and Weiss, Shimon}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 49, pages = {9441-9443}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Superresolution Optical Fluctuation Imaging with Organic Dyes}, type = {Article}, volume = 49, year = 2010 }

  %0 Journal Article %1 Dertinger2010 %A Dertinger, Thomas %A Heilemann, Mike %A Vogel, Robert %A Sauer, Markus %A Weiss, Shimon %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 49 %P 9441-9443 %T Superresolution Optical Fluctuation Imaging with Organic Dyes %U http://dx.doi.org/10.1002/anie.201004138 %V 49 %X SOFI′s choice: Amongst the variety of superresolution far-field microscopy techniques, the most recently established method is superresolution optical fluctuation imaging, SOFI. SOFI can be quickly performed on samples labeled with conventional organic dyes.
• Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions. Zheng, Zhikun; Nottbohm, Christoph T.; Turchanin, Andrey; Muzik, Heiko; Beyer, Andre; Heilemann, Mike; Sauer, Markus; Goelzhaeuser, Armin. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 49(45), S. 8493–8497. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.

  @article{Zheng2010, abstract = {Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Zheng, Zhikun and Nottbohm, Christoph T. and Turchanin, Andrey and Muzik, Heiko and Beyer, Andre and Heilemann, Mike and Sauer, Markus and Goelzhaeuser, Armin}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 45, pages = {8493-8497}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions}, type = {Article}, volume = 49, year = 2010 }

  %0 Journal Article %1 Zheng2010 %A Zheng, Zhikun %A Nottbohm, Christoph T. %A Turchanin, Andrey %A Muzik, Heiko %A Beyer, Andre %A Heilemann, Mike %A Sauer, Markus %A Goelzhaeuser, Armin %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 45 %P 8493-8497 %T Janus Nanomembranes: A Generic Platform for Chemistry in Two Dimensions %U http://dx.doi.org/10.1002/anie.201004053 %V 49 %X Buttered side up: A one nanometer thick membrane with different functional groups on each side was fabricated from self-assembled monolayers of aromatic molecules. The amino side of this Janus membrane was modified with tetramethylrhodamine (TMR) fluorescent dye, and the thiol side with ATTO647N (see picture). The functionalization and functionality of the nanomembrane were demonstrated by X-ray photoelectron spectroscopy and fluorescence microscopy.
• Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy. Kottke, Tilman; van de Linde, Sebastian; Sauer, Markus; Kakorin, Sergej; Heilemann, Mike. In The Journal of Physical Chemistry Letters, 1(21), S. 3156–3159. 2010.
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  The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.

  @article{doi:10.1021/jz101300t, abstract = {The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.}, author = {Kottke, Tilman and van de Linde, Sebastian and Sauer, Markus and Kakorin, Sergej and Heilemann, Mike}, journal = {The Journal of Physical Chemistry Letters}, keywords = {rapidstorm}, number = 21, pages = {3156-3159}, title = {Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy}, volume = 1, year = 2010 }

  %0 Journal Article %1 doi:10.1021/jz101300t %A Kottke, Tilman %A van de Linde, Sebastian %A Sauer, Markus %A Kakorin, Sergej %A Heilemann, Mike %D 2010 %J The Journal of Physical Chemistry Letters %N 21 %P 3156-3159 %R 10.1021/jz101300t %T Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy %U http://pubs.acs.org/doi/abs/10.1021/jz101300t %V 1 %X The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here, the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the product of photoswitching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.
• Spiropyrans as molecular optical switches. Seefeldt, Britta; Kasper, Robert; Beining, Mirco; Mattay, Jochen; Arden-Jacob, Jutta; Kemnitzer, Norbert; Drexhage, Karl Heinz; Heilemann, Mike; Sauer, Markus. In PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 9(2), S. 213–220. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2010.
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  Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.

  @article{Seefeldt2010, abstract = {Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {Seefeldt, Britta and Kasper, Robert and Beining, Mirco and Mattay, Jochen and Arden-Jacob, Jutta and Kemnitzer, Norbert and Drexhage, Karl Heinz and Heilemann, Mike and Sauer, Markus}, journal = {PHOTOCHEMICAL \& PHOTOBIOLOGICAL SCIENCES}, keywords = {mike}, number = 2, pages = {213-220}, publisher = {ROYAL SOC CHEMISTRY}, title = {Spiropyrans as molecular optical switches}, type = {Article}, volume = 9, year = 2010 }

  %0 Journal Article %1 Seefeldt2010 %A Seefeldt, Britta %A Kasper, Robert %A Beining, Mirco %A Mattay, Jochen %A Arden-Jacob, Jutta %A Kemnitzer, Norbert %A Drexhage, Karl Heinz %A Heilemann, Mike %A Sauer, Markus %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2010 %I ROYAL SOC CHEMISTRY %J PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES %N 2 %P 213-220 %T Spiropyrans as molecular optical switches %U http://dx.doi.org/10.1039/b9pp00118b %V 9 %X Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.
• Single-Molecule STED Microscopy with Photostable Organic Fluorophores. Kasper, Robert; Harke, Benjamin; Forthmann, Carsten; Tinnefeld, Philip; Hell, Stefan W.; Sauer, Markus. In Small, 6(13), T. combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing; oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic S. imaging; resolution enhancement at the single- molecule level. A. lateral resolution of (Hrsg.), S. 1379–1384. WILEY-VCH Verlag, 2010.
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  The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer

  @article{SMLL:SMLL201000203, abstract = {The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer}, author = {Kasper, Robert and Harke, Benjamin and Forthmann, Carsten and Tinnefeld, Philip and Hell, Stefan W. and Sauer, Markus}, editor = {combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing, The and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer}, journal = {Small}, keywords = {sauer}, number = 13, pages = {1379–1384}, publisher = {WILEY-VCH Verlag}, title = {Single-Molecule STED Microscopy with Photostable Organic Fluorophores}, volume = 6, year = 2010 }

  %0 Journal Article %1 SMLL:SMLL201000203 %A Kasper, Robert %A Harke, Benjamin %A Forthmann, Carsten %A Tinnefeld, Philip %A Hell, Stefan W. %A Sauer, Markus %D 2010 %E combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing, The %E imaging, oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED %E of, resolution enhancement at the single-molecule level. A lateral resolution %I WILEY-VCH Verlag %J Small %N 13 %P 1379--1384 %R 10.1002/smll.201000203 %T Single-Molecule STED Microscopy with Photostable Organic Fluorophores %U http://dx.doi.org/10.1002/smll.201000203 %V 6 %X The combination of stimulated emission depletion (STED) microscopy with fluorophore stabilization through a reducing and oxidizing system (ROXS) enables the repetitive measurements necessary for 3D or dynamic STED imaging and resolution enhancement at the single-molecule level. A lateral resolution of <30 nm is obtained for raw image data recorded from single organic fluorophores immobilized in aqueous buffer
• Real-time computation of subdiffraction-resolution fluorescence images. Wolter, S.; Schuettpelz, M.; Tscherepanow, M.; van de Linde, S.; Heilemann, M.; Sauer, M. In JOURNAL OF MICROSCOPY-OXFORD, 237(1), S. 12–22. WILEY-BLACKWELL PUBLISHING, INC, COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA, 2010.
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  P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.

  @article{Wolter2010, abstract = {P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.}, address = {COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA}, author = {Wolter, S. and Schuettpelz, M. and Tscherepanow, M. and van de Linde, S. and Heilemann, M. and Sauer, M.}, journal = {JOURNAL OF MICROSCOPY-OXFORD}, keywords = {rapidstorm}, month = {01}, number = 1, pages = {12-22}, publisher = {WILEY-BLACKWELL PUBLISHING, INC}, title = {Real-time computation of subdiffraction-resolution fluorescence images}, type = {Article}, volume = 237, year = 2010 }

  %0 Journal Article %1 Wolter2010 %A Wolter, S. %A Schuettpelz, M. %A Tscherepanow, M. %A van de Linde, S. %A Heilemann, M. %A Sauer, M. %C COMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USA %D 2010 %I WILEY-BLACKWELL PUBLISHING, INC %J JOURNAL OF MICROSCOPY-OXFORD %N 1 %P 12-22 %T Real-time computation of subdiffraction-resolution fluorescence images %U http://dx.doi.org/10.1111/j.1365-2818.2009.03287.x %V 237 %X P>In the recent past, single-molecule based localization or photoswitching microscopy methods such as stochastic optical reconstruction microscopy (STORM) or photoactivated localization microscopy (PALM) have been successfully implemented for subdiffraction-resolution fluorescence imaging. However, the computational effort needed to localize numerous fluorophores is tremendous, causing long data processing times and thereby limiting the applicability of the technique. Here we present a new computational scheme for data processing consisting of noise reduction, detection of likely fluorophore positions, high-precision fluorophore localization and subsequent visualization of found fluorophore positions in a super-resolution image. We present and benchmark different algorithms for noise reduction and demonstrate the use of non-maximum suppression to quickly find likely fluorophore positions in high depth and very noisy images. The algorithm is evaluated and compared in terms of speed, accuracy and robustness by means of simulated data. On real biological samples, we find that real-time data processing is possible and that super-resolution imaging with organic fluorophores of cellular structures with similar to 20 nm optical resolution can be completed in less than 10 s.
• Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains. Daidone, Isabella; Neuweiler, Hannes; Doose, Sören; Sauer, Markus; Smith, Jeremy C. In PLOS COMPUTATIONAL BIOLOGY, 6(1). PUBLIC LIBRARY SCIENCE, 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA, 2010.
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  Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.

  @article{Daidone2010, abstract = {Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.}, address = {185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA}, author = {Daidone, Isabella and Neuweiler, Hannes and Doose, Sören and Sauer, Markus and Smith, Jeremy C.}, journal = {PLOS COMPUTATIONAL BIOLOGY}, keywords = {hannes}, month = {01}, number = 1, publisher = {PUBLIC LIBRARY SCIENCE}, title = {Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains}, type = {Article}, volume = 6, year = 2010 }

  %0 Journal Article %1 Daidone2010 %A Daidone, Isabella %A Neuweiler, Hannes %A Doose, Sören %A Sauer, Markus %A Smith, Jeremy C. %C 185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USA %D 2010 %I PUBLIC LIBRARY SCIENCE %J PLOS COMPUTATIONAL BIOLOGY %N 1 %T Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains %U http://dx.doi.org/10.1371/journal.pcbi.1000645 %V 6 %X Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.
• Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles. Terpitz, Ulrich; Zimmermann, Dirk. In BIOTECHNIQUES, 48(1), S. 68–70. INFORMA HEALTHCARE, 52 VANDERBILT AVE, NEW YORK, NY 10017 USA, 2010.
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  The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

  @article{Terpitz2010, abstract = {The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.}, address = {52 VANDERBILT AVE, NEW YORK, NY 10017 USA}, author = {Terpitz, Ulrich and Zimmermann, Dirk}, journal = {BIOTECHNIQUES}, keywords = {terpitz}, month = {01}, number = 1, pages = {68-70}, publisher = {INFORMA HEALTHCARE}, title = {Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles}, type = {Article}, volume = 48, year = 2010 }

  %0 Journal Article %1 Terpitz2010 %A Terpitz, Ulrich %A Zimmermann, Dirk %C 52 VANDERBILT AVE, NEW YORK, NY 10017 USA %D 2010 %I INFORMA HEALTHCARE %J BIOTECHNIQUES %N 1 %P 68-70 %T Isolation of guard cells from fresh epidermis using a piezo-power microdissection system with vibration-attenuated needles %U http://dx.doi.org/10.2144/000113346 %V 48 %X The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.
• Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility. Endesfelder, Ulrike; van de Linde, Sebastian; Wolter, Steve; Sauer, Markus; Heilemann, Mike. In CHEMPHYSCHEM, 11(4), S. 836–840. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2010.
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  Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.

  @article{Endesfelder2010, abstract = {Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Endesfelder, Ulrike and van de Linde, Sebastian and Wolter, Steve and Sauer, Markus and Heilemann, Mike}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {03}, number = 4, pages = {836-840}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility}, type = {Article}, volume = 11, year = 2010 }

  %0 Journal Article %1 Endesfelder2010 %A Endesfelder, Ulrike %A van de Linde, Sebastian %A Wolter, Steve %A Sauer, Markus %A Heilemann, Mike %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2010 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 4 %P 836-840 %T Subdiffraction-Resolution Fluorescence Microscopy of Myosin-Actin Motility %U http://dx.doi.org/10.1002/cphc.200900944 %V 11 %X Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of similar to 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of similar to 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of similar to 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to similar to 0.18 mu m s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.
• The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging. van de Linde, Sebastian; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 260–266. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Linde2010, abstract = {Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {van de Linde, Sebastian and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {mike}, month = {09}, number = {4, Sp. Iss. SI}, pages = {260-266}, publisher = {ELSEVIER SCIENCE BV}, title = {The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Linde2010 %A van de Linde, Sebastian %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 260-266 %T The effect of photoswitching kinetics and labeling densities on super-resolution fluorescence imaging %U http://dx.doi.org/10.1016/j.jbiotec.2010.02.010 %V 149 %X Super-resolution fluorescence imaging methods based on reversible photoswitching of fluorophores with subsequent localization currently develop to promising tools for cellular imaging. Since most of these methods rely on the transfer of the majority of fluorophores to a non-fluorescent dark state and precise localization of separated fluorescent fluorophores, the photophysical properties of photoswitchable fluorophores have to be carefully controlled. The achievable resolution and herewith the ability to resolve a structural feature depends not only on the brightness of the fluorophores, but also on the labeling density and on the stability or lifetime of the non-fluorescent dark state. Here, we discuss how the ratio of off- and on-switching of a fluorophore affects resolution. We compare experimental data with theoretical simulations and present a strategy to customize photoswitching characteristics to achieve optimal optical resolution. (C) 2010 Elsevier B.V. All rights reserved.
• Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy. Goehler, Antonia; Andre, Sabine; Kaltner, Herbert; Sauer, Markus; Gabius, Hans-Joachim; Doose, Sören. In BIOPHYSICAL JOURNAL, 98(12), S. 3044–3053. CELL PRESS, 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA, 2010.
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  Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.

  @article{Goehler2010, abstract = {Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.}, address = {600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA}, author = {Goehler, Antonia and Andre, Sabine and Kaltner, Herbert and Sauer, Markus and Gabius, Hans-Joachim and Doose, Sören}, journal = {BIOPHYSICAL JOURNAL}, keywords = {sauer}, month = {06}, number = 12, pages = {3044-3053}, publisher = {CELL PRESS}, title = {Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy}, type = {Article}, volume = 98, year = 2010 }

  %0 Journal Article %1 Goehler2010 %A Goehler, Antonia %A Andre, Sabine %A Kaltner, Herbert %A Sauer, Markus %A Gabius, Hans-Joachim %A Doose, Sören %C 600 TECHNOLOGY SQUARE, 5TH FLOOR, CAMBRIDGE, MA 02139 USA %D 2010 %I CELL PRESS %J BIOPHYSICAL JOURNAL %N 12 %P 3044-3053 %T Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy %U http://dx.doi.org/10.1016/j.bpj.2010.03.040 %V 98 %X Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10(3) M-1 s(-1) and 10(-4) s(-1), respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.
• In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET. Seidel, Thorsten; Seefeldt, Britta; Sauer, Markus; Dietz, Karl-Josef. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 272–279. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Seidel2010, abstract = {Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Seidel, Thorsten and Seefeldt, Britta and Sauer, Markus and Dietz, Karl-Josef}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {09}, number = {4, Sp. Iss. SI}, pages = {272-279}, publisher = {ELSEVIER SCIENCE BV}, title = {In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Seidel2010 %A Seidel, Thorsten %A Seefeldt, Britta %A Sauer, Markus %A Dietz, Karl-Josef %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 272-279 %T In vivo analysis of the 2-Cys peroxiredoxin oligomeric state by two-step FRET %U http://dx.doi.org/10.1016/j.jbiotec.2010.06.016 %V 149 %X Fluorescence resonance energy transfer (FRET) analysis in biological systems has reached broad application along with the fast improvement of fluorescent proteins. This work shows the advancement of the commonly used single-step FRET between two fluorophores to a two-step FRET analysis with three fluorophores in vivo. In addition to CFP and YFP the DsRed derivative mCherry was genetically fused in frame to the coding region of the plastidic 2-Cys peroxiredoxin and co-expressed in plant cells resulting in detectable radiationless energy transfer from CFP via YFP to mCherry. The use of control constructs such as fused fluorophore pairs of CFP, YFP and mCherry, but also YFP:mCherry:CFP and REACh:mCherry:CFP allowed for the generation of a reference matrix for two-step FRET calculations. The occurrence of two-step FRET proves that the obligate 2-Cys peroxiredoxin dimers assemble to higher mass oligomers presumably decamers in vivo. This finding together with previous reports on structural dynamics and functional switching of 2-Cys peroxiredoxin might indicate a conformation linked redox-signalling function of the 2-Cys Prx. Although three different fusion proteins had to be imported by the chloroplast two-step FRET was significant within the 2-Cys peroxiredoxin complex. In addition to the proof of oligomerisation in vivo, the results demonstrate the large potential of the method for investigating tripartite protein interactions in subcellular compartments and in general cell biology. (C) 2010 Elsevier B.V. All rights reserved.
• The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces. Arbely, Eyal; Neuweiler, Hannes; Sharpe, Timothy D.; Johnson, Christopher M.; Fersht, Alan R. In PROTEIN SCIENCE, 19(9), S. 1704–1713. JOHN WILEY & SONS INC, 111 RIVER ST, HOBOKEN, NJ 07030 USA, 2010.
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  Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.

  @article{Arbely2010a, abstract = {Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.}, address = {111 RIVER ST, HOBOKEN, NJ 07030 USA}, author = {Arbely, Eyal and Neuweiler, Hannes and Sharpe, Timothy D. and Johnson, Christopher M. and Fersht, Alan R.}, journal = {PROTEIN SCIENCE}, keywords = {hannes}, month = {09}, number = 9, pages = {1704-1713}, publisher = {JOHN WILEY \& SONS INC}, title = {The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces}, type = {Article}, volume = 19, year = 2010 }

  %0 Journal Article %1 Arbely2010a %A Arbely, Eyal %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Johnson, Christopher M. %A Fersht, Alan R. %C 111 RIVER ST, HOBOKEN, NJ 07030 USA %D 2010 %I JOHN WILEY & SONS INC %J PROTEIN SCIENCE %N 9 %P 1704-1713 %T The human peripheral subunit-binding domain folds rapidly while overcoming repulsive Coulomb forces %U http://dx.doi.org/10.1002/pro.453 %V 19 %X Peripheral subunit binding domains (PSBDs) are integral parts of large multienzyme complexes involved in carbohydrate metabolism. PSBDs facilitate shuttling of prosthetic groups between different catalytic subunits. Their protein surface is characterized by a high density of positive charges required for binding to subunits within the complex. Here, we investigated folding thermodynamics and kinetics of the human PSBD (HSBD) using circular dichroism and tryptophan fluorescence experiments. HSBD was only marginally stable under physiological solvent conditions but folded within microseconds via a barrier-limited apparent two-state transition, analogous to its bacterial homologues. The high positive surface-charge density of HSBD leads to repulsive Coulomb forces that modulate protein stability and folding kinetics, and appear to even induce native-state movement. The electrostatic strain was alleviated at high solution-ionic-strength by Debye-Huckel screening. Differences in ionic-strength dependent characteristics among PSBD homologues could be explained by differences in their surface charge distributions. The findings highlight the trade-off between protein function and stability during protein evolution.
• BioImaging: Contributions from biology, physics and informatics. Goelzhaeuser, A.; Nattkemper, T.; Niehaus, K.; Puehler, A.; Sauer, M. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 227–228. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  @article{Goelzhaeuser2010, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Goelzhaeuser, A. and Nattkemper, T. and Niehaus, K. and Puehler, A. and Sauer, M.}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {09}, number = {4, Sp. Iss. SI}, pages = {227-228}, publisher = {ELSEVIER SCIENCE BV}, title = {BioImaging: Contributions from biology, physics and informatics}, type = {Editorial Material}, volume = 149, year = 2010 }

  %0 Journal Article %1 Goelzhaeuser2010 %A Goelzhaeuser, A. %A Nattkemper, T. %A Niehaus, K. %A Puehler, A. %A Sauer, M. %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 227-228 %T BioImaging: Contributions from biology, physics and informatics %U http://dx.doi.org/10.1016/j.jbiotec.2010.08.015 %V 149
• Live-cell super-resolution imaging with trimethoprim conjugates. Wombacher, Richard; Heidbreder, Meike; van de Linde, Sebastian; Sheetz, Michael P.; Heilemann, Mike; Cornish, Virginia W.; Sauer, Markus. In NATURE METHODS, 7(9), S. 717-U72. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2010.
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  The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.

  @article{Wombacher2010, abstract = {The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Wombacher, Richard and Heidbreder, Meike and van de Linde, Sebastian and Sheetz, Michael P. and Heilemann, Mike and Cornish, Virginia W. and Sauer, Markus}, journal = {NATURE METHODS}, keywords = {mike}, month = {09}, number = 9, pages = {717-U72}, publisher = {NATURE PUBLISHING GROUP}, title = {Live-cell super-resolution imaging with trimethoprim conjugates}, type = {Article}, volume = 7, year = 2010 }

  %0 Journal Article %1 Wombacher2010 %A Wombacher, Richard %A Heidbreder, Meike %A van de Linde, Sebastian %A Sheetz, Michael P. %A Heilemann, Mike %A Cornish, Virginia W. %A Sauer, Markus %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2010 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 9 %P 717-U72 %T Live-cell super-resolution imaging with trimethoprim conjugates %U http://dx.doi.org/10.1038/NMETH.1489 %V 7 %X The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at similar to 20 nm resolution.
• Fluorescently labeled 1 nm thin nanomembranes. Nottbohm, Christoph T.; Sopher, Ran; Heilemann, Mike; Sauer, Markus; Goelzhaeuser, Armin. In JOURNAL OF BIOTECHNOLOGY, 149(4, Sp. Iss. SI), S. 267–271. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Nottbohm2010, abstract = {Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Nottbohm, Christoph T. and Sopher, Ran and Heilemann, Mike and Sauer, Markus and Goelzhaeuser, Armin}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {mike}, month = {09}, number = {4, Sp. Iss. SI}, pages = {267-271}, publisher = {ELSEVIER SCIENCE BV}, title = {Fluorescently labeled 1 nm thin nanomembranes}, type = {Article}, volume = 149, year = 2010 }

  %0 Journal Article %1 Nottbohm2010 %A Nottbohm, Christoph T. %A Sopher, Ran %A Heilemann, Mike %A Sauer, Markus %A Goelzhaeuser, Armin %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 4, Sp. Iss. SI %P 267-271 %T Fluorescently labeled 1 nm thin nanomembranes %U http://dx.doi.org/10.1016/j.jbiotec.2010.01.018 %V 149 %X Fluorescent labeling of self-assembled monolayers (SAMs) has a great potential for chemical and biotechnological sensing. However, its use is limited by the quenching of the fluorescence in the proximity of the conducting substrates. We show that this quenching can be overcome by the labeling of a cross-linked aromatic SAM (nanosheet) and its subsequent transfer onto a non-conducting substrate. We demonstrate the successful labeling of nanosheets with a fluorophore (tetramethylrhodamine) and its subsequent transfer to oxidized silicon, where they are detected by optical as well as fluorescence microscopy. Fluorescently labeled freestanding nanosheets, i.e. nanomembranes were obtained by a similar transfer of the nanosheets to TEM grids. (C) 2010 Elsevier B.V. All rights reserved.
• Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots. Heidbreder, Meike; Endesfelder, Ulrike; van de Linde, Sebastian; Hennig, Simon; Widera, Darius; Kaltschmidt, Barbara; Kaltschmidt, Christian; Heilemann, Mike. In BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 1803(10), S. 1224–1229. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  We introduce semiconductor quantum dot-based fluorescence imaging with similar to 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Heidbreder2010, abstract = {We introduce semiconductor quantum dot-based fluorescence imaging with similar to 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Heidbreder, Meike and Endesfelder, Ulrike and van de Linde, Sebastian and Hennig, Simon and Widera, Darius and Kaltschmidt, Barbara and Kaltschmidt, Christian and Heilemann, Mike}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH}, keywords = {mike}, month = 10, number = 10, pages = {1224-1229}, publisher = {ELSEVIER SCIENCE BV}, title = {Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots}, type = {Article}, volume = 1803, year = 2010 }

  %0 Journal Article %1 Heidbreder2010 %A Heidbreder, Meike %A Endesfelder, Ulrike %A van de Linde, Sebastian %A Hennig, Simon %A Widera, Darius %A Kaltschmidt, Barbara %A Kaltschmidt, Christian %A Heilemann, Mike %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH %N 10 %P 1224-1229 %T Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots %U http://dx.doi.org/10.1016/j.bbamcr.2010.06.004 %V 1803 %X We introduce semiconductor quantum dot-based fluorescence imaging with similar to 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells. (C) 2010 Elsevier B.V. All rights reserved.
• Carboxyl pK(a) Values and Acid Denaturation of BBL. Arbely, Eyal; Rutherford, Trevor J.; Neuweiler, Hannes; Sharpe, Timothy D.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 403(2), S. 313–327. ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2010.
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  The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.

  @article{Arbely2010, abstract = {The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Arbely, Eyal and Rutherford, Trevor J. and Neuweiler, Hannes and Sharpe, Timothy D. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = 10, number = 2, pages = {313-327}, publisher = {ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD}, title = {Carboxyl pK(a) Values and Acid Denaturation of BBL}, type = {Article}, volume = 403, year = 2010 }

  %0 Journal Article %1 Arbely2010 %A Arbely, Eyal %A Rutherford, Trevor J. %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2010 %I ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 2 %P 313-327 %T Carboxyl pK(a) Values and Acid Denaturation of BBL %U http://dx.doi.org/10.1016/j.jmb.2010.08.052 %V 403 %X The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK(a) values of all the carboxylic residues in this pH range. We employed C-13 direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pKa values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pKa values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pKa, and, using thermodynamic cycles, we could calculate their pK(a)s in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured C-13(alpha) chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state. (C) 2010 Elsevier Ltd. All rights reserved.
• Monitoring multiple distances within a single molecule using switchable FRET. Uphoff, Stephan; Holden, Seamus J.; Reste, Ludovic Le; Periz, Javier; van de Linde, Sebastian; Heilemann, Mike; Kapanidis, Achillefs N. In NATURE METHODS, 7(10), S. 831-U90. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2010.
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  The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called `switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.

  @article{Uphoff2010, abstract = {The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called `switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Uphoff, Stephan and Holden, Seamus J. and Reste, Ludovic Le and Periz, Javier and van de Linde, Sebastian and Heilemann, Mike and Kapanidis, Achillefs N.}, journal = {NATURE METHODS}, keywords = {mike}, month = 10, number = 10, pages = {831-U90}, publisher = {NATURE PUBLISHING GROUP}, title = {Monitoring multiple distances within a single molecule using switchable FRET}, type = {Article}, volume = 7, year = 2010 }

  %0 Journal Article %1 Uphoff2010 %A Uphoff, Stephan %A Holden, Seamus J. %A Reste, Ludovic Le %A Periz, Javier %A van de Linde, Sebastian %A Heilemann, Mike %A Kapanidis, Achillefs N. %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2010 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 10 %P 831-U90 %T Monitoring multiple distances within a single molecule using switchable FRET %U http://dx.doi.org/10.1038/NMETH.1502 %V 7 %X The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called `switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.
• Kinetics of chain motions within a protein-folding intermediate. Neuweiler, Hannes; Banachewicz, Wiktor; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107(51), S. 22106–22110. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2010.
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  Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.

  @article{Neuweiler2010, abstract = {Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, Hannes and Banachewicz, Wiktor and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 12, number = 51, pages = {22106-22110}, publisher = {NATL ACAD SCIENCES}, title = {Kinetics of chain motions within a protein-folding intermediate}, type = {Article}, volume = 107, year = 2010 }

  %0 Journal Article %1 Neuweiler2010 %A Neuweiler, Hannes %A Banachewicz, Wiktor %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2010 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 51 %P 22106-22110 %T Kinetics of chain motions within a protein-folding intermediate %U http://dx.doi.org/10.1073/pnas.1011666107 %V 107 %X Small proteins can fold remarkably rapidly, even in mu s. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 mu s, but the final docking of preformed helix1 in I requires approximately 20 mu s. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2- and 20-mu s time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-mu s and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-mu s phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.
• Comparative monitoring of temporal and spatial changes in tree water status using the non-invasive leaf patch clamp pressure probe and the pressure bomb. Rueger, S.; Ehrenberger, W.; Arend, M.; Gessner, P.; Zimmermann, G.; Zimmermann, D.; Bentrup, F. W.; Nadler, A.; Raveh, E.; Sukhorukov, V. L.; Zimmermann, U. In AGRICULTURAL WATER MANAGEMENT, 98(2), S. 283–290. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2010.
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  Real-time monitoring of plant water status under field conditions remains difficult to quantify. Here we give evidence that the magnetic-based leaf patch clamp pressure (LPCP) probe is a non-invasive and online-measuring method that can elucidate short- and long-term temporal and spatial dynamics of leaf water status of trees with high precision in real time. Measurements were controlled remotely by telemetry and data transfer to the Internet. Concomitant measurements using the pressure chamber technique (frequently applied for leaf water status monitoring) showed that both techniques yield in principle the same results despite of the high sampling variability of the pressure chamber data. There was a very good correlation between the output pressure signals of the LPCP probe and the balancing pressure values (on average r(2) = 0.90 +/- 0.05; n = 8), i.e. the external pressure at which water appears at the cut end of a leaf under pressure chamber conditions. Simultaneously performed direct measurements of leaf cell turgor pressure using the well-established cell turgor pressure probe technique evidenced that both techniques measure relative changes in leaf turgor pressure. The output pressure signals of the LPCP probe and the balancing pressure values were inversely correlated to turgor pressure. Consistent with this, the balancing pressure values and the cell turgor pressure values could be fitted quite well by the same firm theoretical backing derived recently for the LPCP probe (Zimmermann et al., 2008). This finding suggests that use of the LPCP probe technique in agricultural water management can be built up on the knowledge accumulated on spot leaf or stem water potential measurements. (C) 2010 Elsevier B.V. All rights reserved.

  @article{Rueger2010, abstract = {Real-time monitoring of plant water status under field conditions remains difficult to quantify. Here we give evidence that the magnetic-based leaf patch clamp pressure (LPCP) probe is a non-invasive and online-measuring method that can elucidate short- and long-term temporal and spatial dynamics of leaf water status of trees with high precision in real time. Measurements were controlled remotely by telemetry and data transfer to the Internet. Concomitant measurements using the pressure chamber technique (frequently applied for leaf water status monitoring) showed that both techniques yield in principle the same results despite of the high sampling variability of the pressure chamber data. There was a very good correlation between the output pressure signals of the LPCP probe and the balancing pressure values (on average r(2) = 0.90 +/- 0.05; n = 8), i.e. the external pressure at which water appears at the cut end of a leaf under pressure chamber conditions. Simultaneously performed direct measurements of leaf cell turgor pressure using the well-established cell turgor pressure probe technique evidenced that both techniques measure relative changes in leaf turgor pressure. The output pressure signals of the LPCP probe and the balancing pressure values were inversely correlated to turgor pressure. Consistent with this, the balancing pressure values and the cell turgor pressure values could be fitted quite well by the same firm theoretical backing derived recently for the LPCP probe (Zimmermann et al., 2008). This finding suggests that use of the LPCP probe technique in agricultural water management can be built up on the knowledge accumulated on spot leaf or stem water potential measurements. (C) 2010 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Rueger, S. and Ehrenberger, W. and Arend, M. and Gessner, P. and Zimmermann, G. and Zimmermann, D. and Bentrup, F. W. and Nadler, A. and Raveh, E. and Sukhorukov, V. L. and Zimmermann, U.}, journal = {AGRICULTURAL WATER MANAGEMENT}, keywords = {vladimir}, month = 12, number = 2, pages = {283-290}, publisher = {ELSEVIER SCIENCE BV}, title = {Comparative monitoring of temporal and spatial changes in tree water status using the non-invasive leaf patch clamp pressure probe and the pressure bomb}, type = {Article}, volume = 98, year = 2010 }

  %0 Journal Article %1 Rueger2010 %A Rueger, S. %A Ehrenberger, W. %A Arend, M. %A Gessner, P. %A Zimmermann, G. %A Zimmermann, D. %A Bentrup, F. W. %A Nadler, A. %A Raveh, E. %A Sukhorukov, V. L. %A Zimmermann, U. %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2010 %I ELSEVIER SCIENCE BV %J AGRICULTURAL WATER MANAGEMENT %N 2 %P 283-290 %T Comparative monitoring of temporal and spatial changes in tree water status using the non-invasive leaf patch clamp pressure probe and the pressure bomb %U http://dx.doi.org/10.1016/j.agwat.2010.08.022 %V 98 %X Real-time monitoring of plant water status under field conditions remains difficult to quantify. Here we give evidence that the magnetic-based leaf patch clamp pressure (LPCP) probe is a non-invasive and online-measuring method that can elucidate short- and long-term temporal and spatial dynamics of leaf water status of trees with high precision in real time. Measurements were controlled remotely by telemetry and data transfer to the Internet. Concomitant measurements using the pressure chamber technique (frequently applied for leaf water status monitoring) showed that both techniques yield in principle the same results despite of the high sampling variability of the pressure chamber data. There was a very good correlation between the output pressure signals of the LPCP probe and the balancing pressure values (on average r(2) = 0.90 +/- 0.05; n = 8), i.e. the external pressure at which water appears at the cut end of a leaf under pressure chamber conditions. Simultaneously performed direct measurements of leaf cell turgor pressure using the well-established cell turgor pressure probe technique evidenced that both techniques measure relative changes in leaf turgor pressure. The output pressure signals of the LPCP probe and the balancing pressure values were inversely correlated to turgor pressure. Consistent with this, the balancing pressure values and the cell turgor pressure values could be fitted quite well by the same firm theoretical backing derived recently for the LPCP probe (Zimmermann et al., 2008). This finding suggests that use of the LPCP probe technique in agricultural water management can be built up on the knowledge accumulated on spot leaf or stem water potential measurements. (C) 2010 Elsevier B.V. All rights reserved.

• Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution. Hennig, Simon; van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus. In NANO LETTERS, 9(6), S. 2466–2470. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2009.
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  We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.

  @article{Hennig2009, abstract = {We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Hennig, Simon and van de Linde, Sebastian and Heilemann, Mike and Sauer, Markus}, journal = {NANO LETTERS}, keywords = {mike}, month = {06}, number = 6, pages = {2466-2470}, publisher = {AMER CHEMICAL SOC}, title = {Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution}, type = {Article}, volume = 9, year = 2009 }

  %0 Journal Article %1 Hennig2009 %A Hennig, Simon %A van de Linde, Sebastian %A Heilemann, Mike %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2009 %I AMER CHEMICAL SOC %J NANO LETTERS %N 6 %P 2466-2470 %T Quantum Dot Triexciton Imaging with Three-Dimensional Subdiffraction Resolution %U http://dx.doi.org/10.1021/nl9012387 %V 9 %X We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton. As a pure physical approach, the resolution enhancement is independent from the nanoenvironment and demonstrated to work in living cells.
• Super-Resolution Imaging with Small Organic Fluorophores. Heilemann, Mike; van de Linde, Sebastian; Mukherjee, Anindita; Sauer, Markits. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 48(37), S. 6903–6908. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.

  @article{Heilemann2009b, abstract = {What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and van de Linde, Sebastian and Mukherjee, Anindita and Sauer, Markits}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 37, pages = {6903-6908}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Super-Resolution Imaging with Small Organic Fluorophores}, type = {Article}, volume = 48, year = 2009 }

  %0 Journal Article %1 Heilemann2009b %A Heilemann, Mike %A van de Linde, Sebastian %A Mukherjee, Anindita %A Sauer, Markits %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 37 %P 6903-6908 %T Super-Resolution Imaging with Small Organic Fluorophores %U http://dx.doi.org/10.1002/anie.200902073 %V 48 %X What's your color? A universal and facile method for reversible photoswitching of commercially available Alexa Fluor and ATTO dyes spanning the entire visible range yields an optical resolution of approximately 20 nm. The intriguing simplicity of the method facilitates applications and opens avenues for multicolor super-resolution imaging in fixed and living cells.
• Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification. Heilemann, Mike; Dedecker, Peter; Hofkens, Johan; Sauer, Markus. In LASER & PHOTONICS REVIEWS, 3(1-2), S. 180–202. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.

  @article{Heilemann2009a, abstract = {Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and Dedecker, Peter and Hofkens, Johan and Sauer, Markus}, journal = {LASER \& PHOTONICS REVIEWS}, keywords = {mike}, month = {03}, number = {1-2}, pages = {180-202}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification}, type = {Review}, volume = 3, year = 2009 }

  %0 Journal Article %1 Heilemann2009a %A Heilemann, Mike %A Dedecker, Peter %A Hofkens, Johan %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J LASER & PHOTONICS REVIEWS %N 1-2 %P 180-202 %T Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification %U http://dx.doi.org/10.1002/lpor.200810043 %V 3 %X Optical microscopes, often referred to as `light microscopes', use visible light and a system of lenses to provide us with magnified images of small samples. Combined with highly sensitive fluorescence detection techniques and efficient fluorescent probes they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. They are all based on the selective switching of fluoropbores between a fluorescent and a nonfluorescent state and are therefore generalized under the denotation ``Photoswitching Microscopy{''}. Here we review recent progress in subdiffraction-resolution fluorescence imaging microscopy using various photoswitchable fluorophores and strategies. Special emphasis will be placed on the design and development of photoswitches and the requirements photoswitches have to fulfill for successful use in photoswitching microscopy. Moreover, we demonstrate how photoswitches can be used advantageously for molecular quantification, i.e. the determination of densities and absolute numbers of proteins located in specific subceflular compartments and discuss concepts how standard organic fluorophores can be used successfully for photoswitching microscopy.
• Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes. Neuweiler, Hannes; Sharpe, Timothy D.; Johnson, Christopher M.; Teufel, Daniel P.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 387(4), S. 975–985. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2009.
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  Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2009a, abstract = {Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Sharpe, Timothy D. and Johnson, Christopher M. and Teufel, Daniel P. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {04}, number = 4, pages = {975-985}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes}, type = {Article}, volume = 387, year = 2009 }

  %0 Journal Article %1 Neuweiler2009a %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Johnson, Christopher M. %A Teufel, Daniel P. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2009 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 4 %P 975-985 %T Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes %U http://dx.doi.org/10.1016/j.jmb.2008.12.056 %V 387 %X Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of ail alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics. (C) 2009 Elsevier Ltd. All rights reserved.
• Physicochemical features of ultra-high viscosity alginates. Storz, Henning; Mueller, Kilian J.; Ehrhart, Friederike; Gomez, Ivan; Shirley, Stephen G.; Gessner, Petra; Zimmermann, Gertraud; Weyand, Esther; Sukhorukov, Vladimir L.; Forst, Thomas; Weber, Matthias M.; Zimmermann, Heiko; Kulicke, Werner-Michael; Zimmermann, Ulrich. In CARBOHYDRATE RESEARCH, 344(8), S. 985–995. ELSEVIER SCI LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND, 2009.
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  The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHVN) and Lessonia trabeculata (UHVT) were analyzed. Fluorescence and H-1 NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHVT, but low for UHVN. Intrinsic viscosity {[}eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas {[}eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M-w up to 1.1 x 10(6) g/mol) and of the z-average gyration radius (< R-G >(z) up to 191 nm). The M-w and < R-G >(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M-w dependency of {[}eta] and < RG >(z) could be described by {[}eta] = 0.059 x M-w(0.78) and < R-G >(z) = 0.103 x M-w(x). (UHVN: x= 0.52: UHVT: x = 0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M-w and < R-G >(z) Values of UHVT- and UHVN-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates. (C) 2009 Elsevier Ltd. All rights reserved.

  @article{Storz2009, abstract = {The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHVN) and Lessonia trabeculata (UHVT) were analyzed. Fluorescence and H-1 NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHVT, but low for UHVN. Intrinsic viscosity {[}eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas {[}eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M-w up to 1.1 x 10(6) g/mol) and of the z-average gyration radius (< R-G >(z) up to 191 nm). The M-w and < R-G >(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M-w dependency of {[}eta] and < RG >(z) could be described by {[}eta] = 0.059 x M-w(0.78) and < R-G >(z) = 0.103 x M-w(x). (UHVN: x= 0.52: UHVT: x = 0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M-w and < R-G >(z) Values of UHVT- and UHVN-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates. (C) 2009 Elsevier Ltd. All rights reserved.}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, author = {Storz, Henning and Mueller, Kilian J. and Ehrhart, Friederike and Gomez, Ivan and Shirley, Stephen G. and Gessner, Petra and Zimmermann, Gertraud and Weyand, Esther and Sukhorukov, Vladimir L. and Forst, Thomas and Weber, Matthias M. and Zimmermann, Heiko and Kulicke, Werner-Michael and Zimmermann, Ulrich}, journal = {CARBOHYDRATE RESEARCH}, keywords = {vladimir}, month = {05}, number = 8, pages = {985-995}, publisher = {ELSEVIER SCI LTD}, title = {Physicochemical features of ultra-high viscosity alginates}, type = {Article}, volume = 344, year = 2009 }

  %0 Journal Article %1 Storz2009 %A Storz, Henning %A Mueller, Kilian J. %A Ehrhart, Friederike %A Gomez, Ivan %A Shirley, Stephen G. %A Gessner, Petra %A Zimmermann, Gertraud %A Weyand, Esther %A Sukhorukov, Vladimir L. %A Forst, Thomas %A Weber, Matthias M. %A Zimmermann, Heiko %A Kulicke, Werner-Michael %A Zimmermann, Ulrich %C THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND %D 2009 %I ELSEVIER SCI LTD %J CARBOHYDRATE RESEARCH %N 8 %P 985-995 %T Physicochemical features of ultra-high viscosity alginates %U http://dx.doi.org/10.1016/j.carres.2009.02.016 %V 344 %X The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHVN) and Lessonia trabeculata (UHVT) were analyzed. Fluorescence and H-1 NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHVT, but low for UHVN. Intrinsic viscosity {[}eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas {[}eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M-w up to 1.1 x 10(6) g/mol) and of the z-average gyration radius (< R-G >(z) up to 191 nm). The M-w and < R-G >(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M-w dependency of {[}eta] and < RG >(z) could be described by {[}eta] = 0.059 x M-w(0.78) and < R-G >(z) = 0.103 x M-w(x). (UHVN: x= 0.52: UHVT: x = 0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M-w and < R-G >(z) Values of UHVT- and UHVN-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates. (C) 2009 Elsevier Ltd. All rights reserved.
• Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging. van de Linde, Sebastian; Endesfelder, Ulrike; Mukherjee, Anindita; Schuettpelz, Mark; Wiebusch, Gerd; Wolter, Steve; Heilemann, Mike; Sauer, Markus. In PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 8(4), S. 465–469. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2009.
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  We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.

  @article{Linde2009, abstract = {We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {van de Linde, Sebastian and Endesfelder, Ulrike and Mukherjee, Anindita and Schuettpelz, Mark and Wiebusch, Gerd and Wolter, Steve and Heilemann, Mike and Sauer, Markus}, journal = {PHOTOCHEMICAL \& PHOTOBIOLOGICAL SCIENCES}, keywords = {mike}, number = 4, pages = {465-469}, publisher = {ROYAL SOC CHEMISTRY}, title = {Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging}, type = {Article}, volume = 8, year = 2009 }

  %0 Journal Article %1 Linde2009 %A van de Linde, Sebastian %A Endesfelder, Ulrike %A Mukherjee, Anindita %A Schuettpelz, Mark %A Wiebusch, Gerd %A Wolter, Steve %A Heilemann, Mike %A Sauer, Markus %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2009 %I ROYAL SOC CHEMISTRY %J PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES %N 4 %P 465-469 %T Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging %U http://dx.doi.org/10.1039/b822533h %V 8 %X We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quanti. cation of proteins in the inner mitochondrial membrane with similar to 20 nm optical resolution.
• Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: Implications for radiation treatment. Djuzenova, Cholpon S.; Krasnyanska, Julia; Kiesel, Martin; Stingl, Lavinia; Zimmermann, Ulrich; Flentje, Michael; Sukhorukov, Vladimir L. In MOLECULAR MEDICINE REPORTS, 2(4), S. 633–640. SPANDIDOS PUBL LTD, POB 18179, ATHENS, 116 10, GREECE, 2009.
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  2-Deoxy-D-glucose (2DG), a well-known inhibitor of anaerobic glycolysis, is expected to exert cytotoxic and radiosensitizing effects. In order to test this hypothesis, the response of four tumor cell lines (U87-MG, GaMG, A549 and HT1080) to 2DG was analyzed for cell proliferation, changes in cell volume and nucleus size, as well as for radiation-induced DNA fragmentation, measured by the alkaline Comet assay. Two methods were used for loading cells with 2DG. The Ion-term method included cell cultivation in the presence of 5 mM 2DG for 24 h, while rapid intracellular delivery of 2DG was achieved by exposing the cells for 20 min to a hypotonic solution containing 100 mM 2DG. Irrespective of the loading method, 2DG inhibited the growth of HT1080 and A549 cells. In contrast, two glioblastoma lines (U87 and GaMG) were resistant to 2DG. In three of the four cell lines (all except HT1080), long-term treatment with 2DG reduced radiation-induced DNA fragmentation in conjunction with 2DG-mediated nucleus shrinkage (probably via chromatin condensation) in non-irradiated cells. Complementary volumetric experiments revealed the avid hypotonic uptake of 2DG by all tumor lines. Nonetheless, only HT1080 cells exhibited a significant increase in radiation-induced DNA fragmentation upon hypotonic loading with 2DG, associated with marked nucleus expansion in non-irradiated samples. Our data suggest that, dependant on cell type as well as on medium composition and tonicity, sugar treatment can induce the compaction or expansion of chromatin, thus decreasing or increasing radiation-induced DNA fragmentation. These results raise interesting questions for further studies on the mechanistic links between the sugar-modulated cell volume changes, chromatin structure and radiosensitivity of tumor and normal cells.

  @article{Djuzenova2009, abstract = {2-Deoxy-D-glucose (2DG), a well-known inhibitor of anaerobic glycolysis, is expected to exert cytotoxic and radiosensitizing effects. In order to test this hypothesis, the response of four tumor cell lines (U87-MG, GaMG, A549 and HT1080) to 2DG was analyzed for cell proliferation, changes in cell volume and nucleus size, as well as for radiation-induced DNA fragmentation, measured by the alkaline Comet assay. Two methods were used for loading cells with 2DG. The Ion-term method included cell cultivation in the presence of 5 mM 2DG for 24 h, while rapid intracellular delivery of 2DG was achieved by exposing the cells for 20 min to a hypotonic solution containing 100 mM 2DG. Irrespective of the loading method, 2DG inhibited the growth of HT1080 and A549 cells. In contrast, two glioblastoma lines (U87 and GaMG) were resistant to 2DG. In three of the four cell lines (all except HT1080), long-term treatment with 2DG reduced radiation-induced DNA fragmentation in conjunction with 2DG-mediated nucleus shrinkage (probably via chromatin condensation) in non-irradiated cells. Complementary volumetric experiments revealed the avid hypotonic uptake of 2DG by all tumor lines. Nonetheless, only HT1080 cells exhibited a significant increase in radiation-induced DNA fragmentation upon hypotonic loading with 2DG, associated with marked nucleus expansion in non-irradiated samples. Our data suggest that, dependant on cell type as well as on medium composition and tonicity, sugar treatment can induce the compaction or expansion of chromatin, thus decreasing or increasing radiation-induced DNA fragmentation. These results raise interesting questions for further studies on the mechanistic links between the sugar-modulated cell volume changes, chromatin structure and radiosensitivity of tumor and normal cells.}, address = {POB 18179, ATHENS, 116 10, GREECE}, author = {Djuzenova, Cholpon S. and Krasnyanska, Julia and Kiesel, Martin and Stingl, Lavinia and Zimmermann, Ulrich and Flentje, Michael and Sukhorukov, Vladimir L.}, journal = {MOLECULAR MEDICINE REPORTS}, keywords = {vladimir}, month = {07}, number = 4, pages = {633-640}, publisher = {SPANDIDOS PUBL LTD}, title = {Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: Implications for radiation treatment}, type = {Article}, volume = 2, year = 2009 }

  %0 Journal Article %1 Djuzenova2009 %A Djuzenova, Cholpon S. %A Krasnyanska, Julia %A Kiesel, Martin %A Stingl, Lavinia %A Zimmermann, Ulrich %A Flentje, Michael %A Sukhorukov, Vladimir L. %C POB 18179, ATHENS, 116 10, GREECE %D 2009 %I SPANDIDOS PUBL LTD %J MOLECULAR MEDICINE REPORTS %N 4 %P 633-640 %T Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: Implications for radiation treatment %U http://dx.doi.org/10.3892/mmr_00000149 %V 2 %X 2-Deoxy-D-glucose (2DG), a well-known inhibitor of anaerobic glycolysis, is expected to exert cytotoxic and radiosensitizing effects. In order to test this hypothesis, the response of four tumor cell lines (U87-MG, GaMG, A549 and HT1080) to 2DG was analyzed for cell proliferation, changes in cell volume and nucleus size, as well as for radiation-induced DNA fragmentation, measured by the alkaline Comet assay. Two methods were used for loading cells with 2DG. The Ion-term method included cell cultivation in the presence of 5 mM 2DG for 24 h, while rapid intracellular delivery of 2DG was achieved by exposing the cells for 20 min to a hypotonic solution containing 100 mM 2DG. Irrespective of the loading method, 2DG inhibited the growth of HT1080 and A549 cells. In contrast, two glioblastoma lines (U87 and GaMG) were resistant to 2DG. In three of the four cell lines (all except HT1080), long-term treatment with 2DG reduced radiation-induced DNA fragmentation in conjunction with 2DG-mediated nucleus shrinkage (probably via chromatin condensation) in non-irradiated cells. Complementary volumetric experiments revealed the avid hypotonic uptake of 2DG by all tumor lines. Nonetheless, only HT1080 cells exhibited a significant increase in radiation-induced DNA fragmentation upon hypotonic loading with 2DG, associated with marked nucleus expansion in non-irradiated samples. Our data suggest that, dependant on cell type as well as on medium composition and tonicity, sugar treatment can induce the compaction or expansion of chromatin, thus decreasing or increasing radiation-induced DNA fragmentation. These results raise interesting questions for further studies on the mechanistic links between the sugar-modulated cell volume changes, chromatin structure and radiosensitivity of tumor and normal cells.
• Direct observation of ultrafast folding and denatured state dynamics in single protein molecules. Neuweiler, Hannes; Johnson, Christopher M.; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(44), S. 18569–18574. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2009.
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  Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.

  @article{Neuweiler2009, abstract = {Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, Hannes and Johnson, Christopher M. and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 11, number = 44, pages = {18569-18574}, publisher = {NATL ACAD SCIENCES}, title = {Direct observation of ultrafast folding and denatured state dynamics in single protein molecules}, type = {Article}, volume = 106, year = 2009 }

  %0 Journal Article %1 Neuweiler2009 %A Neuweiler, Hannes %A Johnson, Christopher M. %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2009 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 44 %P 18569-18574 %T Direct observation of ultrafast folding and denatured state dynamics in single protein molecules %U http://dx.doi.org/10.1073/pnas.0910860106 %V 106 %X Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than approximate to 100 mu s and distances greater than approximate to 2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-mu s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperature-jump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type ({''}D-phys''). D-phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ``speed limit'' of folding. D-phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.
• Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics. Huang, Fang; Ying, Liming; Neuweiler, Hannes; Fersht, Alan R. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(52), S. E140. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2009.
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  @article{Huang2009, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Huang, Fang and Ying, Liming and Neuweiler, Hannes and Fersht, Alan R.}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 12, number = 52, pages = {E140}, publisher = {NATL ACAD SCIENCES}, title = {Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics}, type = {Letter}, volume = 106, year = 2009 }

  %0 Journal Article %1 Huang2009 %A Huang, Fang %A Ying, Liming %A Neuweiler, Hannes %A Fersht, Alan R. %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2009 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 52 %P E140 %T Reply to Campos et al.: Direct observation versus ambiguous kinetics and thermodynamics %U http://dx.doi.org/10.1073/pnas.0913321107 %V 106
• The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family. Neuweiler, Hannes; Sharpe, Timothy D.; Rutherford, Trevor J.; Johnson, Christopher M.; Allen, Mark D.; Ferguson, Neil; Fersht, Alan R. In JOURNAL OF MOLECULAR BIOLOGY, 390(5), S. 1060–1073. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2009.
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  Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2009b, abstract = {Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Sharpe, Timothy D. and Rutherford, Trevor J. and Johnson, Christopher M. and Allen, Mark D. and Ferguson, Neil and Fersht, Alan R.}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {07}, number = 5, pages = {1060-1073}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family}, type = {Article}, volume = 390, year = 2009 }

  %0 Journal Article %1 Neuweiler2009b %A Neuweiler, Hannes %A Sharpe, Timothy D. %A Rutherford, Trevor J. %A Johnson, Christopher M. %A Allen, Mark D. %A Ferguson, Neil %A Fersht, Alan R. %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2009 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 5 %P 1060-1073 %T The Folding Mechanism of BBL: Plasticity of Transition-State Structure Observed within an Ultrafast Folding Protein Family %U http://dx.doi.org/10.1016/j.jmb.2009.05.011 %V 390 %X Studies on members of protein families with similar structures but divergent sequences provide insights into the effects of sequence composition on the mechanism of folding. Members of the peripheral subunit-binding domain (PSBD) family fold ultrafast and approach the smallest size for cooperatively folding proteins. Phi-Value analysis of the PSBDs E3BD and POB reveals folding via nucleation-condensation through structurally very similar, polarized transition states. Here, we present a Phi-value analysis of the family member BBL and found that it also folds by a nucleation-condensation mechanism. The mean Phi values of BBL, E3BD, and POB were near identical, indicating similar fractions of non-covalent interactions being formed in the transition state. Despite the overall conservation of folding mechanism in this protein family, however, the pattern of Phi values determined for BBL revealed a larger dispersion of the folding nucleus across the entire structure, and the transition state was less polarized. The observed plasticity of transition-state structure can be rationalized by the different helix-forming propensities of PSBD sequences. The very strong helix propensity in the first helix of BBL, relative to E3BD and POB, appears to recruit more structure formation in that helix in the transition state at the expense of weaker interactions in the second helix. Differences in sequence composition can modulate transition-state structure of even the smallest natural protein domains. (C) 2009 Elsevier Ltd. All rights reserved.
• Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion. Sukhorukov, Vladimir L.; Imes, Dennis; Woellhaf, Michael W.; Andronic, Joseph; Kiesel, Martin; Shirakashi, Ryo; Zimmermann, Ulrich; Zimmermann, Heiko. In BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1788(9), S. 1841–1850. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2009.
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  The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R-h Of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of similar to 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R-h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues. (C) 2009 Elsevier B.V. All rights reserved.

  @article{Sukhorukov2009, abstract = {The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R-h Of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of similar to 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R-h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues. (C) 2009 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Sukhorukov, Vladimir L. and Imes, Dennis and Woellhaf, Michael W. and Andronic, Joseph and Kiesel, Martin and Shirakashi, Ryo and Zimmermann, Ulrich and Zimmermann, Heiko}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES}, keywords = {vladimir}, month = {09}, number = 9, pages = {1841-1850}, publisher = {ELSEVIER SCIENCE BV}, title = {Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion}, type = {Article}, volume = 1788, year = 2009 }

  %0 Journal Article %1 Sukhorukov2009 %A Sukhorukov, Vladimir L. %A Imes, Dennis %A Woellhaf, Michael W. %A Andronic, Joseph %A Kiesel, Martin %A Shirakashi, Ryo %A Zimmermann, Ulrich %A Zimmermann, Heiko %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2009 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES %N 9 %P 1841-1850 %T Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion %U http://dx.doi.org/10.1016/j.bbamem.2009.06.016 %V 1788 %X The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R-h Of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of similar to 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R-h = 0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues. (C) 2009 Elsevier B.V. All rights reserved.
• Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules. Doose, Sören; Neuweiler, Hannes; Sauer, Markus. In CHEMPHYSCHEM, 10(9-10, Sp. Iss. SI), S. 1389–1398. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2009.
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  Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

  @article{Doose2009, abstract = {Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören and Neuweiler, Hannes and Sauer, Markus}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = {07}, number = {9-10, Sp. Iss. SI}, pages = {1389-1398}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules}, type = {Review}, volume = 10, year = 2009 }

  %0 Journal Article %1 Doose2009 %A Doose, Sören %A Neuweiler, Hannes %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2009 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 9-10, Sp. Iss. SI %P 1389-1398 %T Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules %U http://dx.doi.org/10.1002/cphc.200900238 %V 10 %X Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report-on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence life-time of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.

• Effects on capacitance by overexpression of membrane proteins. Zimmermann, D.; Zhou, A.; Kiesel, M.; Feldbauer, K.; Terpitz, U.; Haase, W.; Schneider-Hohendorf, T.; Bamberg, E.; Sukhorukov, V. L. In BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 369(4), S. 1022–1026. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2008.
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  Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.

  @article{Zimmermann2008, abstract = {Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Zimmermann, D. and Zhou, A. and Kiesel, M. and Feldbauer, K. and Terpitz, U. and Haase, W. and Schneider-Hohendorf, T. and Bamberg, E. and Sukhorukov, V. L.}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, keywords = {terpitz}, month = {05}, number = 4, pages = {1022-1026}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Effects on capacitance by overexpression of membrane proteins}, type = {Article}, volume = 369, year = 2008 }

  %0 Journal Article %1 Zimmermann2008 %A Zimmermann, D. %A Zhou, A. %A Kiesel, M. %A Feldbauer, K. %A Terpitz, U. %A Haase, W. %A Schneider-Hohendorf, T. %A Bamberg, E. %A Sukhorukov, V. L. %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2008 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %N 4 %P 1022-1026 %T Effects on capacitance by overexpression of membrane proteins %U http://dx.doi.org/10.1016/j.bbrc.2008.02.153 %V 369 %X Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4) channels/mu m(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C-m). The C-m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C-m of 1.15 +/- 0.08 mu F/cm(2) in ChR2-expressing; cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (039 +/- 0.02 mu F/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C-m (0.85 +/- 0.07 mu F/cm(2)). as compared to non-expressing control cells (0.70 +/- 0.03 mu F/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins. (C) 2008 Elsevier Inc. All rights reserved.
• A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Vogelsang, Jan; Kasper, Robert; Steinhauer, Christian; Person, Britta; Heilemann, Mike; Sauer, Markus; Tinnefeld, Philip. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 47(29), S. 5465–5469. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.

  @article{Vogelsang2008, abstract = {On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Vogelsang, Jan and Kasper, Robert and Steinhauer, Christian and Person, Britta and Heilemann, Mike and Sauer, Markus and Tinnefeld, Philip}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 29, pages = {5465-5469}, publisher = {WILEY-V C H VERLAG GMBH}, title = {A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes}, type = {Article}, volume = 47, year = 2008 }

  %0 Journal Article %1 Vogelsang2008 %A Vogelsang, Jan %A Kasper, Robert %A Steinhauer, Christian %A Person, Britta %A Heilemann, Mike %A Sauer, Markus %A Tinnefeld, Philip %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 29 %P 5465-5469 %T A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes %U http://dx.doi.org/10.1002/anie.200801518 %V 47 %X On the ROXS: Photobleaching represents one of the main limitations of modern fluorescence microscopy. A reducing and oxidizing system (ROXS) has been introduced that recovers triplet states as well as charge-separated states through electron-transfer reactions (see picture). Thus the blinking and photobleaching of fluorophores is strikingly reduced.
• Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Heilemann, Mike; van de Linde, Sebastian; Schuttpelz, Mark; Kasper, Robert; Seefeldt, Britta; Mukherjee, Anindita; Tinnefeld, Philip; Sauer, Markus. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 47(33), S. 6172–6176. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.

  @article{Heilemann2008, abstract = {Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heilemann, Mike and van de Linde, Sebastian and Schuttpelz, Mark and Kasper, Robert and Seefeldt, Britta and Mukherjee, Anindita and Tinnefeld, Philip and Sauer, Markus}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {mike}, number = 33, pages = {6172-6176}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes}, type = {Article}, volume = 47, year = 2008 }

  %0 Journal Article %1 Heilemann2008 %A Heilemann, Mike %A van de Linde, Sebastian %A Schuttpelz, Mark %A Kasper, Robert %A Seefeldt, Britta %A Mukherjee, Anindita %A Tinnefeld, Philip %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 33 %P 6172-6176 %T Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes %U http://dx.doi.org/10.1002/anie.200802376 %V 47 %X Eagle eyes: dSTORM uses conventional photoswitchable fluorescent dyes that can be reversibly cycled between a fluorescent and a dark state by irradiation with light of different wavelengths (see picture). This elegant approach can visualize cellular structures with a resolution of approximately 20 nm, far beyond the diffraction limit of light, without the need of an activator molecule.
• A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions. Zimmermann, D.; Kiesel, M.; Terpitz, U.; Zhou, A.; Reuss, R.; Kraus, J.; Schenk, W. A.; Bamberg, E.; Sukhorukov, V. L. In JOURNAL OF MEMBRANE BIOLOGY, 221(2), S. 107–121. SPRINGER, 233 SPRING STREET, NEW YORK, NY 10013 USA, 2008.
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  Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.

  @article{Zimmermann2008a, abstract = {Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.}, address = {233 SPRING STREET, NEW YORK, NY 10013 USA}, author = {Zimmermann, D. and Kiesel, M. and Terpitz, U. and Zhou, A. and Reuss, R. and Kraus, J. and Schenk, W. A. and Bamberg, E. and Sukhorukov, V. L.}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {terpitz}, month = {01}, number = 2, pages = {107-121}, publisher = {SPRINGER}, title = {A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions}, type = {Article}, volume = 221, year = 2008 }

  %0 Journal Article %1 Zimmermann2008a %A Zimmermann, D. %A Kiesel, M. %A Terpitz, U. %A Zhou, A. %A Reuss, R. %A Kraus, J. %A Schenk, W. A. %A Bamberg, E. %A Sukhorukov, V. L. %C 233 SPRING STREET, NEW YORK, NY 10013 USA %D 2008 %I SPRINGER %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 107-121 %T A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions %U http://dx.doi.org/10.1007/s00232-007-9090-4 %V 221 %X Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB-) and {[}W-2(CO)(10)(S2CH)](-), the putative lipophilic anion B(CF3)(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB- and B(CF3)(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB- and B(CF3)(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.
• Reconfigurable, braced, three-dimensional DNA nanostructures. Goodman, Russell P.; Heilemann, Mike; Doose, Sören; Erben, Christoph M.; Kapanidis, Achillefs N.; Turberfield, Andrew J. In NATURE NANOTECHNOLOGY, 3(2), S. 93–96. NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND, 2008.
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  DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.

  @article{Goodman2008, abstract = {DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.}, address = {MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND}, author = {Goodman, Russell P. and Heilemann, Mike and Doose, Sören and Erben, Christoph M. and Kapanidis, Achillefs N. and Turberfield, Andrew J.}, journal = {NATURE NANOTECHNOLOGY}, keywords = {mike}, month = {02}, number = 2, pages = {93-96}, publisher = {NATURE PUBLISHING GROUP}, title = {Reconfigurable, braced, three-dimensional DNA nanostructures}, type = {Article}, volume = 3, year = 2008 }

  %0 Journal Article %1 Goodman2008 %A Goodman, Russell P. %A Heilemann, Mike %A Doose, Sören %A Erben, Christoph M. %A Kapanidis, Achillefs N. %A Turberfield, Andrew J. %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2008 %I NATURE PUBLISHING GROUP %J NATURE NANOTECHNOLOGY %N 2 %P 93-96 %T Reconfigurable, braced, three-dimensional DNA nanostructures %U http://dx.doi.org/10.1038/nnano.2008.3 %V 3 %X DNA nanotechnology makes use of the exquisite self-recognition of DNA in order to build on a molecular scale(1). Although static structures may find applications in structural biology(2-4) and computer science 5, many applications in nanomedicine and nanorobotics require the additional capacity for controlled three-dimensional movement(6). DNA architectures can span three dimensions(4,7-10) and DNA devices are capable of movement(10-16), but active control of well-defined three-dimensional structures has not been achieved. We demonstrate the operation of reconfigurable DNA tetrahedra whose shapes change precisely and reversibly in response to specific molecular signals. Shape changes are confirmed by gel electrophoresis and by bulk and single-molecule Forster resonance energy transfer measurements. DNA tetrahedra are natural building blocks for three-dimensional construction 9; they may be synthesized rapidly with high yield of a single stereoisomer, and their triangulated architecture conveys structural stability. The introduction of shape-changing structural modules opens new avenues for the manipulation of matter on the nanometre scale.
• Fluorescent proteins for single-molecule fluorescence applications. Seefeldt, Britta; Kasper, Robert; Seidel, Thorsten; Tinnefeld, Philip; Dietz, Karl-Josef; Heilemann, Mike; Sauer, Markus. In JOURNAL OF BIOPHOTONICS, 1(1), S. 74–82. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  @article{Seefeldt2008, abstract = {We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH \& Co. KGaA, Weinheim}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Seefeldt, Britta and Kasper, Robert and Seidel, Thorsten and Tinnefeld, Philip and Dietz, Karl-Josef and Heilemann, Mike and Sauer, Markus}, journal = {JOURNAL OF BIOPHOTONICS}, keywords = {mike}, month = {02}, number = 1, pages = {74-82}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Fluorescent proteins for single-molecule fluorescence applications}, type = {Article}, volume = 1, year = 2008 }

  %0 Journal Article %1 Seefeldt2008 %A Seefeldt, Britta %A Kasper, Robert %A Seidel, Thorsten %A Tinnefeld, Philip %A Dietz, Karl-Josef %A Heilemann, Mike %A Sauer, Markus %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J JOURNAL OF BIOPHOTONICS %N 1 %P 74-82 %T Fluorescent proteins for single-molecule fluorescence applications %U http://dx.doi.org/10.1002/jbio.200710024 %V 1 %X We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. {[}GRAPHICS] mCherry exhibits a relatively high photostability and brightness under single-molecule fluorescence imaging conditions. (C) 2008 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
• Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis. Seidel, Thorsten; Schnitzer, Daniel; Golldack, Dortje; Sauer, Markus; Dietz, Karl-Josef. In BMC CELL BIOLOGY, 9. BIOMED CENTRAL LTD, CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND, 2008.
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  Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.

  @article{Seidel2008, abstract = {Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.}, address = {CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND}, author = {Seidel, Thorsten and Schnitzer, Daniel and Golldack, Dortje and Sauer, Markus and Dietz, Karl-Josef}, journal = {BMC CELL BIOLOGY}, keywords = {sauer}, month = {05}, publisher = {BIOMED CENTRAL LTD}, title = {Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis}, type = {Article}, volume = 9, year = 2008 }

  %0 Journal Article %1 Seidel2008 %A Seidel, Thorsten %A Schnitzer, Daniel %A Golldack, Dortje %A Sauer, Markus %A Dietz, Karl-Josef %C CURRENT SCIENCE GROUP, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND %D 2008 %I BIOMED CENTRAL LTD %J BMC CELL BIOLOGY %T Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis %U http://dx.doi.org/10.1186/1471-2121-9-28 %V 9 %X Background: The V-ATPase ( VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms. Results: The transmembrane topology and stoichiometry of the proteolipid VHA-c{''} as well as the stoichiometry of the membrane integral subunit VHA-e within the V-ATPase complex were investigated by in vivo fluorescence resonance energy transfer ( FRET). VHA-c{''}, VHA-e1 and VHA-e2, VHA-a, VHA-c3, truncated variants of VHA-c3 and a chimeric VHA-c/VHA-c{''} hybrid were fused to cyan ( CFP) and yellow fluorescent protein ( YFP), respectively. The constructs were employed for transfection experiments with Arabidopsis thaliana mesophyll protoplasts. Subcellular localization and FRET analysis by confocal laser scanning microscopy ( CLSM) demonstrated that (i.) the N- and C-termini of VHA-c{''} are localised in the vacuolar lumen, ( ii.) one copy of VHA-c{''} is present within the VHA-complex, and ( iii.) VHA-c{''} is localised at the ER and associated Golgi bodies. ( iv.) A similar localisation was observed for VHA-e2, whereas ( v.) the subcellular localisation of VHA-e1 indicated the trans Golgi network ( TGN)-specifity of this subunit. Conclusion: The plant proteolipid ring is a highly flexible protein subcomplex, tolerating the incorporation of truncated and hybrid proteolipid subunits, respectively. Whereas the membrane integral subunit VHA-e is present in two copies within the complex, the proteolipid subunit VHA-c{''} takes part in complex formation with only one copy. However, neither VHA-c{''} isoform 1 nor any of the two VHA-e isoforms were identified at the tonoplast. This suggest a function in endomembrane specific VHA-assembly or targeting rather than proton transport.
• Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins. Terpitz, Ulrich; Raimunda, Daniel; Westhoff, Markus; Sukhorukov, Vladimir L.; Beauge, Luis; Bamberg, Ernst; Zimmermann, Dirk. In BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1778(6), S. 1493–1500. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2008.
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  Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.

  @article{Terpitz2008, abstract = {Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Terpitz, Ulrich and Raimunda, Daniel and Westhoff, Markus and Sukhorukov, Vladimir L. and Beauge, Luis and Bamberg, Ernst and Zimmermann, Dirk}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES}, keywords = {terpitz}, month = {06}, number = 6, pages = {1493-1500}, publisher = {ELSEVIER SCIENCE BV}, title = {Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins}, type = {Article}, volume = 1778, year = 2008 }

  %0 Journal Article %1 Terpitz2008 %A Terpitz, Ulrich %A Raimunda, Daniel %A Westhoff, Markus %A Sukhorukov, Vladimir L. %A Beauge, Luis %A Bamberg, Ernst %A Zimmermann, Dirk %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2008 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES %N 6 %P 1493-1500 %T Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins %U http://dx.doi.org/10.1016/j.bbamem.2008.03.015 %V 1778 %X Giant protoplasts of Saccharomyces cerevisiae of 10-35 mu m in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and ``patchability{''} of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 +/- 0.07 mu F/cm(2)) and conductance (23-44 mu S/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. (C) 2008 Elsevier B.V. All rights reserved.
• Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy. Schuettpelz, Mark; Schoening, Jan C.; Doose, Sören; Neuweiler, Hannes; Peters, Elisabeth; Staiger, Dorothee; Sauer, Markus. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 130(29), S. 9507–9513. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2008.
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  The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

  @article{Schuettpelz2008, abstract = {The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Schuettpelz, Mark and Schoening, Jan C. and Doose, Sören and Neuweiler, Hannes and Peters, Elisabeth and Staiger, Dorothee and Sauer, Markus}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {07}, number = 29, pages = {9507-9513}, publisher = {AMER CHEMICAL SOC}, title = {Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 130, year = 2008 }

  %0 Journal Article %1 Schuettpelz2008 %A Schuettpelz, Mark %A Schoening, Jan C. %A Doose, Sören %A Neuweiler, Hannes %A Peters, Elisabeth %A Staiger, Dorothee %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2008 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 29 %P 9507-9513 %T Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ja801994z %V 130 %X The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.
• Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides. Doose, Sören. In CHEMPHYSCHEM, 9(18), S. 2687–2689. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2008.
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  @article{Doose2008, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören}, journal = {CHEMPHYSCHEM}, keywords = {doose}, month = 12, number = 18, pages = {2687-2689}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides}, type = {Editorial Material}, volume = 9, year = 2008 }

  %0 Journal Article %1 Doose2008 %A Doose, Sören %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2008 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 18 %P 2687-2689 %T Importance of Backbone and Solvent Properties for Conformational Dynamics in Polypeptides %U http://dx.doi.org/10.1002/cphc.200800545 %V 9
• Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores. van de Linde, Sebastian; Sauer, Markus; Heilemann, Mike. In JOURNAL OF STRUCTURAL BIOLOGY, 164(3), S. 250–254. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2008.
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  Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.

  @article{Linde2008a, abstract = {Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {van de Linde, Sebastian and Sauer, Markus and Heilemann, Mike}, journal = {JOURNAL OF STRUCTURAL BIOLOGY}, keywords = {mike}, month = 12, number = 3, pages = {250-254}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores}, type = {Article}, volume = 164, year = 2008 }

  %0 Journal Article %1 Linde2008a %A van de Linde, Sebastian %A Sauer, Markus %A Heilemann, Mike %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2008 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J JOURNAL OF STRUCTURAL BIOLOGY %N 3 %P 250-254 %T Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores %U http://dx.doi.org/10.1016/j.jsb.2008.08.002 %V 164 %X Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of similar to 20 nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. (C) 2008 Elsevier Inc. All rights reserved.
• Photoswitching microscopy with standard fluorophores. van de Linde, S.; Kasper, R.; Heilemann, M.; Sauer, M. In APPLIED PHYSICS B-LASERS AND OPTICS, 93(4), S. 725–731. SPRINGER, 233 SPRING ST, NEW YORK, NY 10013 USA, 2008.
  • [ Abstract ]
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  We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

  @article{Linde2008, abstract = {We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.}, address = {233 SPRING ST, NEW YORK, NY 10013 USA}, author = {van de Linde, S. and Kasper, R. and Heilemann, M. and Sauer, M.}, journal = {APPLIED PHYSICS B-LASERS AND OPTICS}, keywords = {mike}, month = 12, number = 4, pages = {725-731}, publisher = {SPRINGER}, title = {Photoswitching microscopy with standard fluorophores}, type = {Article}, volume = 93, year = 2008 }

  %0 Journal Article %1 Linde2008 %A van de Linde, S. %A Kasper, R. %A Heilemann, M. %A Sauer, M. %C 233 SPRING ST, NEW YORK, NY 10013 USA %D 2008 %I SPRINGER %J APPLIED PHYSICS B-LASERS AND OPTICS %N 4 %P 725-731 %T Photoswitching microscopy with standard fluorophores %U http://dx.doi.org/10.1007/s00340-008-3250-9 %V 93 %X We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2-10 mM) of reducing, thiol-containing compounds such as beta-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of similar to 20 nm. The method requires wide-field illumination with only a single laser beam for read-out and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.

• Physical and biological properties of barium cross-linked alginate membranes. Zimmermann, Heiko; Waehlisch, Felix; Baier, Claudia; Westhoff, Markus; Reuss, Randolph; Zimmermann, Dirk; Behringer, Marcus; Ehrhart, Friederike; Katsen-Globa, Alisa; Giese, Christoph; Marx, Uwe; Sukhorukov, Vladimir L.; Vasquez, Julio A.; Jakob, Peter; Shirley, Stephen G.; Zimmermann, Ulrich. In BIOMATERIALS, 28(7), S. 1327–1345. ELSEVIER SCI LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND, 2007.
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  We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the ``crystal gun{''} {[}Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L-p, from PEG-induced flows ranges between 2.4 x 10(-12) and 6.5 x 10(-12) m Pa-1 s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3 x 10(-10) in Pa-1 s(-1) at 4 kPa. Mechanical introduction of 200 mu m-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2 h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes. (c) 2006 Elsevier Ltd. All rights reserved.

  @article{Zimmermann2007, abstract = {We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the ``crystal gun{''} {[}Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L-p, from PEG-induced flows ranges between 2.4 x 10(-12) and 6.5 x 10(-12) m Pa-1 s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3 x 10(-10) in Pa-1 s(-1) at 4 kPa. Mechanical introduction of 200 mu m-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2 h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes. (c) 2006 Elsevier Ltd. All rights reserved.}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, author = {Zimmermann, Heiko and Waehlisch, Felix and Baier, Claudia and Westhoff, Markus and Reuss, Randolph and Zimmermann, Dirk and Behringer, Marcus and Ehrhart, Friederike and Katsen-Globa, Alisa and Giese, Christoph and Marx, Uwe and Sukhorukov, Vladimir L. and Vasquez, Julio A. and Jakob, Peter and Shirley, Stephen G. and Zimmermann, Ulrich}, journal = {BIOMATERIALS}, keywords = {vladimir}, month = {03}, number = 7, pages = {1327-1345}, publisher = {ELSEVIER SCI LTD}, title = {Physical and biological properties of barium cross-linked alginate membranes}, type = {Article}, volume = 28, year = 2007 }

  %0 Journal Article %1 Zimmermann2007 %A Zimmermann, Heiko %A Waehlisch, Felix %A Baier, Claudia %A Westhoff, Markus %A Reuss, Randolph %A Zimmermann, Dirk %A Behringer, Marcus %A Ehrhart, Friederike %A Katsen-Globa, Alisa %A Giese, Christoph %A Marx, Uwe %A Sukhorukov, Vladimir L. %A Vasquez, Julio A. %A Jakob, Peter %A Shirley, Stephen G. %A Zimmermann, Ulrich %C THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND %D 2007 %I ELSEVIER SCI LTD %J BIOMATERIALS %N 7 %P 1327-1345 %T Physical and biological properties of barium cross-linked alginate membranes %U http://dx.doi.org/10.1016/j.biomaterials.2006.11.032 %V 28 %X We describe the manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability. Clinical grade, ultra-high viscosity alginate is gelled by diffusion of Ba2+ followed by use of the ``crystal gun{''} {[}Zimmermann H. et al., Fabrication of homogeneously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003;24:2083-96]. Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on manufacture and storage details. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG 6000, which may be related to a Hagen-Poiseuille flow. Hydraulic conductivity, L-p, from PEG-induced flows ranges between 2.4 x 10(-12) and 6.5 x 10(-12) m Pa-1 s(-1). Hydraulic conductivity measured with hydrostatic pressure up to 6 kPa is 2-3 orders of magnitude higher and decreases with increasing pressure to about 3 x 10(-10) in Pa-1 s(-1) at 4 kPa. Mechanical introduction of 200 mu m-diameter pores increases hydraulic conductivity dramatically without loss of mechanical stability or flexibility. NMR imaging with Cu2+ as contrast agent shows a layered structure in membranes cross-linked for 2 h. Phase contrast and atomic force microscopy in liquid environment reveals surface protrusions and cavities correlating with steps of the production process. Murine L929 cells adhere strongly to the rough surface of crystal-bombarded membranes. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol allowing cell culture on the membranes. (c) 2006 Elsevier Ltd. All rights reserved.
• Periodic acceptor excitation spectroscopy of single molecules. Doose, Sören; Heilemann, Mike; Michalet, Xavier; Weiss, Shimon; Kapanidis, Achillefs. In European Biophysics Journal, 36(6), S. 669–674. Springer Berlin / Heidelberg, 2007.
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  Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

  @article{Doose2007d, abstract = {Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.}, author = {Doose, Sören and Heilemann, Mike and Michalet, Xavier and Weiss, Shimon and Kapanidis, Achillefs}, journal = {European Biophysics Journal}, keywords = {mike}, note = {10.1007/s00249-007-0133-7}, pages = {669-674}, publisher = {Springer Berlin / Heidelberg}, title = {Periodic acceptor excitation spectroscopy of single molecules}, volume = 36, year = 2007 }

  %0 Journal Article %1 Doose2007d %A Doose, Sören %A Heilemann, Mike %A Michalet, Xavier %A Weiss, Shimon %A Kapanidis, Achillefs %D 2007 %I Springer Berlin / Heidelberg %J European Biophysics Journal %P 669-674 %T Periodic acceptor excitation spectroscopy of single molecules %U http://dx.doi.org/10.1007/s00249-007-0133-7 %V 36 %X Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.
• Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis. Schulze, Philipp; Schuettpelz, Mark; Sauer, Markus; Belder, Detlev. In LAB ON A CHIP, 7(12), S. 1841–1844. ROYAL SOC CHEMISTRY, THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND, 2007.
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  Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.

  @article{Schulze2007, abstract = {Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.}, address = {THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND}, author = {Schulze, Philipp and Schuettpelz, Mark and Sauer, Markus and Belder, Detlev}, journal = {LAB ON A CHIP}, keywords = {sauer}, number = 12, pages = {1841-1844}, publisher = {ROYAL SOC CHEMISTRY}, title = {Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis}, type = {Article}, volume = 7, year = 2007 }

  %0 Journal Article %1 Schulze2007 %A Schulze, Philipp %A Schuettpelz, Mark %A Sauer, Markus %A Belder, Detlev %C THOMAS GRAHAM HOUSE, SCIENCE PARK, MILTON RD, CAMBRIDGE CB4 0WF, CAMBS, ENGLAND %D 2007 %I ROYAL SOC CHEMISTRY %J LAB ON A CHIP %N 12 %P 1841-1844 %T Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis %U http://dx.doi.org/10.1039/b710762e %V 7 %X Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.
• A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer. Agaylan, Ashraf; Binder, Daniel; Sauer, Markus; Neuweiler, Hannes; Meyer, Oliver; Kiesewetter, Holger; Salama, Abdulgabar. In Cancer, 110(11), S. 2502–2506. Wiley Subscription Services, Inc., A Wiley Company, 2007.
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  Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.

  @article{CNCR:CNCR23057, abstract = {Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.}, author = {Agaylan, Ashraf and Binder, Daniel and Sauer, Markus and Neuweiler, Hannes and Meyer, Oliver and Kiesewetter, Holger and Salama, Abdulgabar}, journal = {Cancer}, keywords = {hannes}, number = 11, pages = {2502–2506}, publisher = {Wiley Subscription Services, Inc., A Wiley Company}, title = {A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer}, volume = 110, year = 2007 }

  %0 Journal Article %1 CNCR:CNCR23057 %A Agaylan, Ashraf %A Binder, Daniel %A Sauer, Markus %A Neuweiler, Hannes %A Meyer, Oliver %A Kiesewetter, Holger %A Salama, Abdulgabar %D 2007 %I Wiley Subscription Services, Inc., A Wiley Company %J Cancer %N 11 %P 2502--2506 %R 10.1002/cncr.23057 %T A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer %U http://dx.doi.org/10.1002/cncr.23057 %V 110 %X Abstract BACKGROUND.Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).METHODS.Superparamagnetic particles were coated with different peptides spanning the entire p53 protein. These particles were incubated with serum samples from healthy blood donors (n = 180), from patients without malignancies (n = 27), and from patients with various forms of lung cancer (n = 166). The particles were washed and placed into the reaction chamber of a gel card. After centrifugation, agglutination results were read visually. Positive reactions were defined by a layer of particles on top of the gel or agglutinated particles dispersed through the gel matrix.RESULTS.Depending on the peptide used, p53 autoantibodies were detected in from 17.5% to 35% of the investigated patients with lung cancer. By using a commercially available enzyme-linked immunoadsorbent assay (ELISA) kit, p53 autoantibodies were detected in only 3% of those patients. P53 protein and p53 protein-antibody complexes were not detected in patients with lung cancer (n = 20).CONCLUSIONS.The newly developed assay was easy to perform and had sensitivity superior to that of the currently available p53 ELISAs. Cancer 2007. © 2007 American Cancer Society.
• Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy. Neuweiler, Hannes; Lollmann, Marc; Doose, Sören; Sauer, Markus. In JOURNAL OF MOLECULAR BIOLOGY, 365(3), S. 856–869. ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND, 2007.
  • [ Abstract ]
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  Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.

  @article{Neuweiler2007, abstract = {Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.}, address = {24-28 OVAL RD, LONDON NW1 7DX, ENGLAND}, author = {Neuweiler, Hannes and Lollmann, Marc and Doose, Sören and Sauer, Markus}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, keywords = {hannes}, month = {01}, number = 3, pages = {856-869}, publisher = {ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD}, title = {Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 365, year = 2007 }

  %0 Journal Article %1 Neuweiler2007 %A Neuweiler, Hannes %A Lollmann, Marc %A Doose, Sören %A Sauer, Markus %C 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND %D 2007 %I ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD %J JOURNAL OF MOLECULAR BIOLOGY %N 3 %P 856-869 %T Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1016/j.jmb.2006.10.021 %V 365 %X Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides, have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides. consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in ``off{''} and ``on{''} switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypepticle chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in k unfolded proteins can still proceed quite efficiently in spite of hindered L diffusion caused by high macromolecular content. (c) 2006 Elsevier Ltd. All rights reserved.
• Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix. Doose, Sören; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(44), S. 17400–17405. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2007.
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  Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

  @article{Doose2007c, abstract = {Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Doose, Sören and Neuweiler, Hannes and Barsch, Hannes and Sauer, Markus}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 10, number = 44, pages = {17400-17405}, publisher = {NATL ACAD SCIENCES}, title = {Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix}, type = {Article}, volume = 104, year = 2007 }

  %0 Journal Article %1 Doose2007c %A Doose, Sören %A Neuweiler, Hannes %A Barsch, Hannes %A Sauer, Markus %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2007 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 44 %P 17400-17405 %T Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix %U http://dx.doi.org/10.1073/pnas.0705605104 %V 104 %X Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Forster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics Of Poly-L-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.
• Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern. Dessie, Salilew-Wondim; Rings, Franca; Holker, Michael; Gilles, Markus; Jennen, Danyel; Tholen, Ernst; Havlicek, Vitezslav; Besenfelder, Urban; Sukhorukov, Vladimir L.; Zimmermann, Ulrich; Endter, Joerg M.; Sirard, Marc-Andre; Schellander, Karl; Tesfaye, Dawit. In REPRODUCTION, 133(5), S. 931–946. BIO SCIENTIFICA LTD, EURO HOUSE, 22 APEX COURT WOODLANDS, BRADLEY STOKE, BRISTOL BS32 4JT, ENGLAND, 2007.
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  Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase 11 oocytes with (PB+) and without (PB-) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mu m electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of postactivation, the blastocyst rate of very slow dielectrophoretic PB+ and PB- oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P<0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, clielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.

  @article{Dessie2007, abstract = {Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase 11 oocytes with (PB+) and without (PB-) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mu m electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of postactivation, the blastocyst rate of very slow dielectrophoretic PB+ and PB- oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P<0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, clielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.}, address = {EURO HOUSE, 22 APEX COURT WOODLANDS, BRADLEY STOKE, BRISTOL BS32 4JT, ENGLAND}, author = {Dessie, Salilew-Wondim and Rings, Franca and Holker, Michael and Gilles, Markus and Jennen, Danyel and Tholen, Ernst and Havlicek, Vitezslav and Besenfelder, Urban and Sukhorukov, Vladimir L. and Zimmermann, Ulrich and Endter, Joerg M. and Sirard, Marc-Andre and Schellander, Karl and Tesfaye, Dawit}, journal = {REPRODUCTION}, keywords = {vladimir}, month = {05}, number = 5, pages = {931-946}, publisher = {BIO SCIENTIFICA LTD}, title = {Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern}, type = {Article}, volume = 133, year = 2007 }

  %0 Journal Article %1 Dessie2007 %A Dessie, Salilew-Wondim %A Rings, Franca %A Holker, Michael %A Gilles, Markus %A Jennen, Danyel %A Tholen, Ernst %A Havlicek, Vitezslav %A Besenfelder, Urban %A Sukhorukov, Vladimir L. %A Zimmermann, Ulrich %A Endter, Joerg M. %A Sirard, Marc-Andre %A Schellander, Karl %A Tesfaye, Dawit %C EURO HOUSE, 22 APEX COURT WOODLANDS, BRADLEY STOKE, BRISTOL BS32 4JT, ENGLAND %D 2007 %I BIO SCIENTIFICA LTD %J REPRODUCTION %N 5 %P 931-946 %T Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern %U http://dx.doi.org/10.1530/REP-06-0277 %V 133 %X Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase 11 oocytes with (PB+) and without (PB-) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mu m electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of postactivation, the blastocyst rate of very slow dielectrophoretic PB+ and PB- oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P<0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, clielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.
• Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment. Clifford, John N.; Bell, Toby D. M.; Tinnefeld, Philip; Heilemann, Mike; Melnikov, Sergey M.; ichi Hotta, Jun; Sliwa, Michel; Dedecker, Peter; Sauer, Markus; Hofkens, Johan; Yeow, Edwin K. L. In JOURNAL OF PHYSICAL CHEMISTRY B, 111(25), S. 6987–6991. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2007.
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  The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.

  @article{Clifford2007, abstract = {The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Clifford, John N. and Bell, Toby D. M. and Tinnefeld, Philip and Heilemann, Mike and Melnikov, Sergey M. and ichi Hotta, Jun and Sliwa, Michel and Dedecker, Peter and Sauer, Markus and Hofkens, Johan and Yeow, Edwin K. L.}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {mike}, month = {06}, number = 25, pages = {6987-6991}, publisher = {AMER CHEMICAL SOC}, title = {Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment}, type = {Article}, volume = 111, year = 2007 }

  %0 Journal Article %1 Clifford2007 %A Clifford, John N. %A Bell, Toby D. M. %A Tinnefeld, Philip %A Heilemann, Mike %A Melnikov, Sergey M. %A ichi Hotta, Jun %A Sliwa, Michel %A Dedecker, Peter %A Sauer, Markus %A Hofkens, Johan %A Yeow, Edwin K. L. %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2007 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 25 %P 6987-6991 %R 10.1021/jp072864d %T Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment %U http://dx.doi.org/10.1021/jp072864d %V 111 %X The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.
• Polymer properties of polythymine as revealed by translational diffusion. Doose, Sören; Barsch, Hannes; Sauer, Markus. In BIOPHYSICAL JOURNAL, 93(4), S. 1224–1234. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2007.
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  Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.

  @article{Doose2007b, abstract = {Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Doose, Sören and Barsch, Hannes and Sauer, Markus}, journal = {BIOPHYSICAL JOURNAL}, keywords = {sauer}, month = {08}, number = 4, pages = {1224-1234}, publisher = {BIOPHYSICAL SOCIETY}, title = {Polymer properties of polythymine as revealed by translational diffusion}, type = {Article}, volume = 93, year = 2007 }

  %0 Journal Article %1 Doose2007b %A Doose, Sören %A Barsch, Hannes %A Sauer, Markus %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2007 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 4 %P 1224-1234 %T Polymer properties of polythymine as revealed by translational diffusion %U http://dx.doi.org/10.1529/biophysj.107.107342 %V 93 %X Biopolymers, such as single- stranded DNA ( ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA ( polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.5 and 0.7 depending on ionic strength I. With Rh being proportional to the square root of the persistence length L-p, we found that L-p approximate to l(m), with m = - 0.22 +/- 0.01 for polythymine with 100 residues. For comparison, we performed molecular dynamics ( MD) simulations with a force field that accounts for short- range interactions in vacuum, and determined the characteristic polymer properties end- to- end distance R, radius of gyration S, and persistence length L-p of various labeled and nonlabeled polythymine derivatives. We found excellent agreement for the length dependence of simulated S and experimental R-h measured at 100 mM NaCl, revealing that electrostatic interactions are completely shielded in aqueous solution at such ionic strength. MD simulations further showed that polythymine with.; 30 residues can be described as a semiflexible polymer with negligible influence of the fluorescent label; and that static flexibility is limited by geometrical and steric constraints as expressed by an intrinsic persistence length of; 1.7 nm. These results provide a benchmark for theories and MD simulations describing the influence of electrostatic interactions on polyelectrolyte properties, and thus help to develop a complete and accurate description of ssDNA.
• Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range. Vogelsang, Jan; Doose, Sören; Sauer, Markus; Tinnefeld, Philip. In ANALYTICAL CHEMISTRY, 79(19), S. 7367–7375. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2007.
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  In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.

  @article{Vogelsang2007, abstract = {In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Vogelsang, Jan and Doose, Sören and Sauer, Markus and Tinnefeld, Philip}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 10, number = 19, pages = {7367-7375}, publisher = {AMER CHEMICAL SOC}, title = {Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range}, type = {Article}, volume = 79, year = 2007 }

  %0 Journal Article %1 Vogelsang2007 %A Vogelsang, Jan %A Doose, Sören %A Sauer, Markus %A Tinnefeld, Philip %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2007 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 19 %P 7367-7375 %T Single-Molecule Fluorescence Resonance Energy Transfer in Nanopipets: Improving Distance Resolution and Concentration Range %U http://dx.doi.org/10.1021/ac071176n %V 79 %X In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of similar to 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (< 10(9) M-1) using minute sample amounts (< 5 mu L). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.
• Optical amplification from single excitons in collodial quantum dots. Doose, Sören. In SMALL, 3(11), S. 1856–1858. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2007.
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  @article{Doose2007, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, Sören}, journal = {SMALL}, keywords = {doose}, month = 11, number = 11, pages = {1856-1858}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Optical amplification from single excitons in collodial quantum dots}, type = {Editorial Material}, volume = 3, year = 2007 }

  %0 Journal Article %1 Doose2007 %A Doose, Sören %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2007 %I WILEY-V C H VERLAG GMBH %J SMALL %N 11 %P 1856-1858 %T Optical amplification from single excitons in collodial quantum dots %U http://dx.doi.org/10.1002/smll.200700478 %V 3
• Mechanisms of electrically mediated cytosolic Ca2+ transients in Aequorin-Transformed tobacco cells. Sukhorukov, V. L.; Endter, J. M.; Zimmermann, D.; Shirakashi, R.; Fehrmann, S.; Kiesel, M.; Reuss, R.; Becker, D.; Hedrich, R.; Bamberg, E.; Roitsch, Th.; Zimmermann, U. In BIOPHYSICAL JOURNAL, 93(9), S. 3324–3337. BIOPHYSICAL SOC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2007.
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  Cytosolic Ca2+ changes induced by electric. field pulses of 50-mu s duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca2+-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca2+ concentration reaching a peak value within < 100-200 ms. Peaking of {[}Ca2+](cyt) was followed by a biphasic decay due to removal of Ca2+ (e. g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of; similar to 0.5 and 3-5 s, respectively. Experiments with various external Ca2+ concentrations and conductivities showed that the fast decay arises from Ca2+ fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca2+ fluxes through the tonoplast. The amplitude of the {[}Ca2+](cyt) transients increased with increasing. field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical. field strength of similar to 450 V/cm, whereas breakdown of the tonoplast required similar to 580 V/cm. The above. finndings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying. field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca2+ and presumably other vacuolar ingredients.

  @article{Sukhorukov2007, abstract = {Cytosolic Ca2+ changes induced by electric. field pulses of 50-mu s duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca2+-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca2+ concentration reaching a peak value within < 100-200 ms. Peaking of {[}Ca2+](cyt) was followed by a biphasic decay due to removal of Ca2+ (e. g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of; similar to 0.5 and 3-5 s, respectively. Experiments with various external Ca2+ concentrations and conductivities showed that the fast decay arises from Ca2+ fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca2+ fluxes through the tonoplast. The amplitude of the {[}Ca2+](cyt) transients increased with increasing. field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical. field strength of similar to 450 V/cm, whereas breakdown of the tonoplast required similar to 580 V/cm. The above. finndings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying. field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca2+ and presumably other vacuolar ingredients.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Sukhorukov, V. L. and Endter, J. M. and Zimmermann, D. and Shirakashi, R. and Fehrmann, S. and Kiesel, M. and Reuss, R. and Becker, D. and Hedrich, R. and Bamberg, E. and Roitsch, Th. and Zimmermann, U.}, journal = {BIOPHYSICAL JOURNAL}, keywords = {vladimir}, month = 11, number = 9, pages = {3324-3337}, publisher = {BIOPHYSICAL SOC}, title = {Mechanisms of electrically mediated cytosolic Ca2+ transients in Aequorin-Transformed tobacco cells}, type = {Article}, volume = 93, year = 2007 }

  %0 Journal Article %1 Sukhorukov2007 %A Sukhorukov, V. L. %A Endter, J. M. %A Zimmermann, D. %A Shirakashi, R. %A Fehrmann, S. %A Kiesel, M. %A Reuss, R. %A Becker, D. %A Hedrich, R. %A Bamberg, E. %A Roitsch, Th. %A Zimmermann, U. %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2007 %I BIOPHYSICAL SOC %J BIOPHYSICAL JOURNAL %N 9 %P 3324-3337 %T Mechanisms of electrically mediated cytosolic Ca2+ transients in Aequorin-Transformed tobacco cells %U http://dx.doi.org/10.1529/biophysj.107.110783 %V 93 %X Cytosolic Ca2+ changes induced by electric. field pulses of 50-mu s duration and 200-800 V/cm strength were monitored by measuring chemiluminescence in aequorin-transformed BY-2 tobacco cells. In Ca2+-substituted media, electropulsing led to a very fast initial increase of the cytosolic Ca2+ concentration reaching a peak value within < 100-200 ms. Peaking of {[}Ca2+](cyt) was followed by a biphasic decay due to removal of Ca2+ (e. g., by binding and/or sequestration in the cytosol). The decay had fast and slow components, characterized by time constants of; similar to 0.5 and 3-5 s, respectively. Experiments with various external Ca2+ concentrations and conductivities showed that the fast decay arises from Ca2+ fluxes through the plasmalemma, whereas the slow decay must be assigned to Ca2+ fluxes through the tonoplast. The amplitude of the {[}Ca2+](cyt) transients increased with increasing. field strength, whereas the time constants of the decay kinetics remained invariant. Breakdown of the plasmalemma was achieved at a critical. field strength of similar to 450 V/cm, whereas breakdown of the tonoplast required similar to 580 V/cm. The above. finndings could be explained by the transient potential profiles generated across the two membranes in response to an exponentially decaying. field pulse. The dielectric data required for calculation of the tonoplast and plasmalemma potentials were derived from electrorotation experiments on isolated vacuolated and evacuolated BY-2 protoplasts. The electrorotation response of vacuolated protoplasts could be described in terms of a three-shell model (i.e., by assuming that the capacitances of tonoplast and plasmalemma are arranged in series). Among other things, the theoretical analysis together with the experimental data show that genetic manipulations of plant cells by electrotransfection or electrofusion must be performed in low-conductivity media to minimize release of vacuolar Ca2+ and presumably other vacuolar ingredients.

• Application of multiline two-photon microscopy to functional in vivo imaging. Kurtz, R; Fricke, M; Kalb, J; Tinnefeld, P; Sauer, M. In JOURNAL OF NEUROSCIENCE METHODS, 151(2), S. 276–286. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2006.
  • [ Abstract ]
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  High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.

  @article{Kurtz2006, abstract = {High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Kurtz, R and Fricke, M and Kalb, J and Tinnefeld, P and Sauer, M}, journal = {JOURNAL OF NEUROSCIENCE METHODS}, keywords = {sauer}, month = {03}, number = 2, pages = {276-286}, publisher = {ELSEVIER SCIENCE BV}, title = {Application of multiline two-photon microscopy to functional in vivo imaging}, type = {Article}, volume = 151, year = 2006 }

  %0 Journal Article %1 Kurtz2006 %A Kurtz, R %A Fricke, M %A Kalb, J %A Tinnefeld, P %A Sauer, M %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2006 %I ELSEVIER SCIENCE BV %J JOURNAL OF NEUROSCIENCE METHODS %N 2 %P 276-286 %T Application of multiline two-photon microscopy to functional in vivo imaging %U http://dx.doi.org/10.1016/j.jneumeth.2005.12.003 %V 151 %X High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motionsensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and `spine'-like structures. Calcium dynamics depended on branch size, but `spines' did not systematically differ from their `parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode. (c) 2005 Elsevier B.V. All rights reserved.
• The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy. Kim, J; Doose, S; Neuweiler, H; Sauer, M. In NUCLEIC ACIDS RESEARCH, 34(9), S. 2516–2527. OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2006.
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  Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.

  @article{Kim2006, abstract = {Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.}, address = {GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND}, author = {Kim, J and Doose, S and Neuweiler, H and Sauer, M}, journal = {NUCLEIC ACIDS RESEARCH}, keywords = {hannes}, number = 9, pages = {2516-2527}, publisher = {OXFORD UNIV PRESS}, title = {The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy}, type = {Article}, volume = 34, year = 2006 }

  %0 Journal Article %1 Kim2006 %A Kim, J %A Doose, S %A Neuweiler, H %A Sauer, M %C GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND %D 2006 %I OXFORD UNIV PRESS %J NUCLEIC ACIDS RESEARCH %N 9 %P 2516-2527 %T The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy %U http://dx.doi.org/10.1093/nar/gkl221 %V 34 %X Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC-dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC-dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC-dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.
• UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions. Schüttpelz, Mark; Müller, Christian; Neuweiler, Hannes; Sauer, Markus. In Analytical Chemistry, 78(3), S. 663–669. 2006.
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  Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays

  @article{doi:10.1021/ac051938j, abstract = {Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays}, author = {Schüttpelz, Mark and Müller, Christian and Neuweiler, Hannes and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {hannes}, note = {PMID: 16448037}, number = 3, pages = {663-669}, title = {UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions}, volume = 78, year = 2006 }

  %0 Journal Article %1 doi:10.1021/ac051938j %A Schüttpelz, Mark %A Müller, Christian %A Neuweiler, Hannes %A Sauer, Markus %D 2006 %J Analytical Chemistry %N 3 %P 663-669 %R 10.1021/ac051938j %T UV Fluorescence Lifetime Imaging Microscopy: A Label-Free Method for Detection and Quantification of Protein Interactions %U http://pubs.acs.org/doi/abs/10.1021/ac051938j %V 78 %X Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 ± 0.6) × 109 M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays
• Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy. Marmé, Nicole; Friedrich, Achim; Müller, Matthias; Nolte, Oliver; Wolfrum, Jürgen; Hoheisel, Jörg D.; Sauer, Markus; Knemeyer, Jens-Peter. In Nucl. Acids Res., 34(13), S. e90. 2006.
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  We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s

  @article{marméidentification, abstract = {We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s}, author = {Marmé, Nicole and Friedrich, Achim and Müller, Matthias and Nolte, Oliver and Wolfrum, Jürgen and Hoheisel, Jörg D. and Sauer, Markus and Knemeyer, Jens-Peter}, journal = {Nucl. Acids Res.}, keywords = {sauer}, number = 13, pages = {e90}, title = {Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy}, volume = 34, year = 2006 }

  %0 Journal Article %1 marméidentification %A Marmé, Nicole %A Friedrich, Achim %A Müller, Matthias %A Nolte, Oliver %A Wolfrum, Jürgen %A Hoheisel, Jörg D. %A Sauer, Markus %A Knemeyer, Jens-Peter %D 2006 %J Nucl. Acids Res. %N 13 %P e90 %T Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy %U http://nar.oxfordjournals.org/content/34/13/e90 %V 34 %X We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10−8 M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10−11 M solutions of PCR amplicons from M.tuberculosis in only 100 s
• Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy. Tsay, JM; Doose, S; Weiss, S. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(5), S. 1639–1647. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.

  @article{Tsay2006, abstract = {CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tsay, JM and Doose, S and Weiss, S}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {doose}, month = {02}, number = 5, pages = {1639-1647}, publisher = {AMER CHEMICAL SOC}, title = {Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy}, type = {Article}, volume = 128, year = 2006 }

  %0 Journal Article %1 Tsay2006 %A Tsay, JM %A Doose, S %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 5 %P 1639-1647 %T Rotational and translational diffusion of peptide-coated CdSe/CdS/ZnS nanorods studied by fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ja056162i %V 128 %X CdSe/CdS/ZnS nanorods (NRs) of three aspect ratios were coated with phytochelatin-related peptides and studied using fluorescence correlation spectroscopy (FCS). Theoretical predictions of the NRs' rotational diffusion contribution to the correlation curves were experimentally confirmed. We monitored rotational and translational diffusion of NRs and extracted hydrodynamic radii from the extracted diffusion constants. Translational and rotational diffusion constants (D-trans and D-rot) for NRs were in good agreement with Tirado and Garcia de la Torre's as well as with Broersma's theories when accounting for the ligand dimensions. NRs fall in the size range where rotational diffusion can be monitored with higher sensitivity than translational diffusion due to a steeper length dependence, D-rot similar to L-3 versus D-trans similar to L-1. By titrating peptide-coated NRs with bovine serum albumin, we monitored (nonspecific) binding through rotational diffusion and showed that D-rot is an advantageous observable for monitoring binding. Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to be useful for observing binding and conformational dynamics in biological systems.
• Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals. Biebricher, A; Sauer, M; Tinnefeld, P. In JOURNAL OF PHYSICAL CHEMISTRY B, 110(11), S. 5174–5178. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.

  @article{Biebricher2006, abstract = {Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Biebricher, A and Sauer, M and Tinnefeld, P}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = {03}, number = 11, pages = {5174-5178}, publisher = {AMER CHEMICAL SOC}, title = {Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals}, type = {Letter}, volume = 110, year = 2006 }

  %0 Journal Article %1 Biebricher2006 %A Biebricher, A %A Sauer, M %A Tinnefeld, P %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 11 %P 5174-5178 %T Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals %U http://dx.doi.org/10.1021/jp060660b %V 110 %X Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect. Correlation of the different photophysical parameters revealed an intrinsic nonradiative rate and enabled the disentangling of intrinsic and extrinsic nonradiative rate constants. Moreover, it allowed us to assess the PL quantum yield of single NCs. Finally, the presented technique was successfully applied to demonstrate that the addition of antiblinking reagents such as mercaptoethylamine accelerates the observed fluctuations between different photophysical states.
• A biophysical approach to the optimisation of dendritic-tumour cell electrofusion. Sukhorukov, Vladimir L.; Reuss, Randolph; Endter, Joerg M.; Fehrmann, Steffen; Katsen-Globa, Alisa; Gessner, Petra; Steinbach, Andrea; Mueller, Kilian J.; Karpas, Abraham; Zimmermann, Ulrich; Zimmermann, Heiko. In BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 346(3), S. 829–839. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2006.
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  Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and elect to rotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%. as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells. (c) 2006 Elsevier Inc. All rights reserved.

  @article{Sukhorukov2006, abstract = {Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and elect to rotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19\%. as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells. (c) 2006 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Sukhorukov, Vladimir L. and Reuss, Randolph and Endter, Joerg M. and Fehrmann, Steffen and Katsen-Globa, Alisa and Gessner, Petra and Steinbach, Andrea and Mueller, Kilian J. and Karpas, Abraham and Zimmermann, Ulrich and Zimmermann, Heiko}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, keywords = {vladimir}, month = {08}, number = 3, pages = {829-839}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {A biophysical approach to the optimisation of dendritic-tumour cell electrofusion}, type = {Article}, volume = 346, year = 2006 }

  %0 Journal Article %1 Sukhorukov2006 %A Sukhorukov, Vladimir L. %A Reuss, Randolph %A Endter, Joerg M. %A Fehrmann, Steffen %A Katsen-Globa, Alisa %A Gessner, Petra %A Steinbach, Andrea %A Mueller, Kilian J. %A Karpas, Abraham %A Zimmermann, Ulrich %A Zimmermann, Heiko %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2006 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %N 3 %P 829-839 %T A biophysical approach to the optimisation of dendritic-tumour cell electrofusion %U http://dx.doi.org/10.1016/j.bbrc.2006.05.193 %V 346 %X Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and elect to rotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%. as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells. (c) 2006 Elsevier Inc. All rights reserved.
• Advances in fluorescence imaging with quantum dot bio-probes. Pinaud, F; Michalet, X; Bentolila, LA; Tsay, JM; Doose, S; Li, JJ; Iyer, G; Weiss, S. In BIOMATERIALS, 27(9), S. 1679–1687. ELSEVIER SCI LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND, 2006.
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  After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.

  @article{Pinaud2006, abstract = {After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND}, author = {Pinaud, F and Michalet, X and Bentolila, LA and Tsay, JM and Doose, S and Li, JJ and Iyer, G and Weiss, S}, journal = {BIOMATERIALS}, keywords = {doose}, month = {03}, number = 9, pages = {1679-1687}, publisher = {ELSEVIER SCI LTD}, title = {Advances in fluorescence imaging with quantum dot bio-probes}, type = {Article}, volume = 27, year = 2006 }

  %0 Journal Article %1 Pinaud2006 %A Pinaud, F %A Michalet, X %A Bentolila, LA %A Tsay, JM %A Doose, S %A Li, JJ %A Iyer, G %A Weiss, S %C THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLAND %D 2006 %I ELSEVIER SCI LTD %J BIOMATERIALS %N 9 %P 1679-1687 %T Advances in fluorescence imaging with quantum dot bio-probes %U http://dx.doi.org/10.1016/j.biomaterials.2005.11.018 %V 27 %X After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed. the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo. (C) 2005 Elsevier Ltd. All rights reserved.
• Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system. Zimmermann, D.; Terpitz, U.; Zhou, A.; Reuss, R.; Mueller, K.; Sukhorukov, V. L.; Gessner, P.; Nagel, G.; Zimmermann, U.; Bamberg, E. In BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 348(2), S. 673–681. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2006.
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  Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.

  @article{Zimmermann2006, abstract = {Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Zimmermann, D. and Terpitz, U. and Zhou, A. and Reuss, R. and Mueller, K. and Sukhorukov, V. L. and Gessner, P. and Nagel, G. and Zimmermann, U. and Bamberg, E.}, journal = {BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, keywords = {terpitz}, month = {09}, number = 2, pages = {673-681}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system}, type = {Article}, volume = 348, year = 2006 }

  %0 Journal Article %1 Zimmermann2006 %A Zimmermann, D. %A Terpitz, U. %A Zhou, A. %A Reuss, R. %A Mueller, K. %A Sukhorukov, V. L. %A Gessner, P. %A Nagel, G. %A Zimmermann, U. %A Bamberg, E. %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2006 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS %N 2 %P 673-681 %T Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system %U http://dx.doi.org/10.1016/j.bbrc.2006.07.112 %V 348 %X Giant HEK293 cells of 30-65 mu m in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1 +/- 0.1 mu F/cm(2) for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 mu m) was found to be between 12.8 and 16.1 mu S/cm(2), which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system. (c) 2006 Elsevier Inc. All rights reserved.
• p53 family members in myogenic differentiation and rhabdomyosarcoma development. Cam, Hakan; Griesmann, Heidi; Beitzinger, Michaela; Hofmann, Lars; Beinoraviciute-Kellner, Rasa; Sauer, Markus; Huettinger-Kirchhof, Nicole; Oswald, Claudia; Friedl, Peter; Gattenloehner, Stefan; Burek, Christof; Rosenwald, Andreas; Stiewe, Thorsten. In CANCER CELL, 10(4), S. 281–293. CELL PRESS, 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA, 2006.
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  The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.

  @article{Cam2006, abstract = {The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.}, address = {1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA}, author = {Cam, Hakan and Griesmann, Heidi and Beitzinger, Michaela and Hofmann, Lars and Beinoraviciute-Kellner, Rasa and Sauer, Markus and Huettinger-Kirchhof, Nicole and Oswald, Claudia and Friedl, Peter and Gattenloehner, Stefan and Burek, Christof and Rosenwald, Andreas and Stiewe, Thorsten}, journal = {CANCER CELL}, keywords = {sauer}, month = 10, number = 4, pages = {281-293}, publisher = {CELL PRESS}, title = {p53 family members in myogenic differentiation and rhabdomyosarcoma development}, type = {Article}, volume = 10, year = 2006 }

  %0 Journal Article %1 Cam2006 %A Cam, Hakan %A Griesmann, Heidi %A Beitzinger, Michaela %A Hofmann, Lars %A Beinoraviciute-Kellner, Rasa %A Sauer, Markus %A Huettinger-Kirchhof, Nicole %A Oswald, Claudia %A Friedl, Peter %A Gattenloehner, Stefan %A Burek, Christof %A Rosenwald, Andreas %A Stiewe, Thorsten %C 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA %D 2006 %I CELL PRESS %J CANCER CELL %N 4 %P 281-293 %T p53 family members in myogenic differentiation and rhabdomyosarcoma development %U http://dx.doi.org/10.1016/j.ccr.2006.08.024 %V 10 %X The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. Delta Np73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. Delta Np73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.
• Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant. Terpitz, Ulrich; Kothe, Erika. In JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK, 80(2), S. 187–193. DRUCKEREI LIDDY HALM, BACKHAUSSTRASSE 9B, 37081 GOTTINGEN, GERMANY, 2006.
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  In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.

  @article{Terpitz2006, abstract = {In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.}, address = {BACKHAUSSTRASSE 9B, 37081 GOTTINGEN, GERMANY}, author = {Terpitz, Ulrich and Kothe, Erika}, journal = {JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK}, keywords = {terpitz}, month = 11, number = 2, pages = {187-193}, publisher = {DRUCKEREI LIDDY HALM}, title = {Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant}, type = {Article}, volume = 80, year = 2006 }

  %0 Journal Article %1 Terpitz2006 %A Terpitz, Ulrich %A Kothe, Erika %C BACKHAUSSTRASSE 9B, 37081 GOTTINGEN, GERMANY %D 2006 %I DRUCKEREI LIDDY HALM %J JOURNAL OF APPLIED BOTANY AND FOOD QUALITY-ANGEWANDTE BOTANIK %N 2 %P 187-193 %T Diversity and adaptation of soil fungi in an ecosystem with contamination originating from a phosphate fertilizer plant %V 80 %X In the vicinity of a former phosphate fertilizer production plant, high phosphate contents with up to 463 mg phosphate per kilogram of soil dry matter were found 13 years after closing the plant while pH is only slightly elevated at pH 7 to 8. The deposited phosphate is seen to be moved to deeper soil horizons. The effect on soil microbiota was analyzed with respect to soil respiration, fungal biomass and cultivation of benomyl resistant and ligninolytic fungi. Increasing numbers and diversity of soil fungi were found with distance from the former emittent. This was confirmed by investigation of mycorrhization rates of mycotrophic, ectomycorrhizal birch trees. We tested for adaptation by growth and phosphate acquisition on soil extract media. The best growth was seen on the media containing highest phosphate concentrations showing no in vitro growth inhibition. In contrast to previous findings, however, more polyphosphate granula were seen on soil extract media from distant sites, although phosphate concentration was lowest in these media.
• Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires. Heilemann, Mike; Kasper, Robert; Tinnefeld, Philip; Sauer, Markus. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 128(51), S. 16864–16875. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.

  @article{Heilemann2006, abstract = {Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, Mike and Kasper, Robert and Tinnefeld, Philip and Sauer, Markus}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {mike}, month = 12, number = 51, pages = {16864-16875}, publisher = {AMER CHEMICAL SOC}, title = {Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires}, type = {Article}, volume = 128, year = 2006 }

  %0 Journal Article %1 Heilemann2006 %A Heilemann, Mike %A Kasper, Robert %A Tinnefeld, Philip %A Sauer, Markus %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 51 %P 16864-16875 %T Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-Based photonic wires %U http://dx.doi.org/10.1021/ja065585x %V 128 %X Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time. In this article, we describe our efforts to synthesize and study DNA-based molecular photonic wires that carry several chromophores arranged in an energetic downhill cascade and exploit fluorescence resonance energy transfer to convey excited-state energy. The focus of this work is to understand and control the heterogeneity of such complex systems, applying single-molecule fluorescence spectroscopy (SMFS) to dissect the different sources of heterogeneity, i.e., chemical heterogeneity and inhomogeneous broadening induced by the nanoenvironment. We demonstrate that the homogeneity of excited-state energy transport in DNA-based photonic wires is dramatically improved by immobilizing photonic wires in aqueous solution without perturbation by the surface. In addition, our study shows that the in situ construction of wire molecules, i.e., the stepwise hybridization of differently labeled oligonucleotides on glass cover slides, further decreases the observed heterogeneity in overall energy-transfer efficiency. The developed strategy enables efficient energy transfer between up to five chromophores in the majority of molecules investigated along a distance of similar to 14 nm. Finally, we used multiparameter SMFS to analyze the energy flow in photonic wires in more detail and to assign residual heterogeneity under optimized conditions in solution to different leakages and competing energy-transfer processes.
• DNA-based molecular wires: Multiple emission pathways of individual constructs. Sanchez-Mosteiro, Gabriel; van Dijk, Erik M. H. P.; Hernando, Jordi; Heilemann, Mike; Tinnefeld, Philip; Sauer, Markus; Koberlin, Felix; Patting, Matthias; Wahl, Michael; Erdmann, Rainer; van Hulst, Niek F.; Garcia-Parajo, Maria F. In JOURNAL OF PHYSICAL CHEMISTRY B, 110(51), S. 26349–26353. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2006.
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  The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.

  @article{Sanchez-Mosteiro2006, abstract = {The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100\% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Sanchez-Mosteiro, Gabriel and van Dijk, Erik M. H. P. and Hernando, Jordi and Heilemann, Mike and Tinnefeld, Philip and Sauer, Markus and Koberlin, Felix and Patting, Matthias and Wahl, Michael and Erdmann, Rainer and van Hulst, Niek F. and Garcia-Parajo, Maria F.}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {mike}, month = 12, number = 51, pages = {26349-26353}, publisher = {AMER CHEMICAL SOC}, title = {DNA-based molecular wires: Multiple emission pathways of individual constructs}, type = {Article}, volume = 110, year = 2006 }

  %0 Journal Article %1 Sanchez-Mosteiro2006 %A Sanchez-Mosteiro, Gabriel %A van Dijk, Erik M. H. P. %A Hernando, Jordi %A Heilemann, Mike %A Tinnefeld, Philip %A Sauer, Markus %A Koberlin, Felix %A Patting, Matthias %A Wahl, Michael %A Erdmann, Rainer %A van Hulst, Niek F. %A Garcia-Parajo, Maria F. %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2006 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 51 %P 26349-26353 %T DNA-based molecular wires: Multiple emission pathways of individual constructs %U http://dx.doi.org/10.1021/jp064701f %V 110 %X The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes. Dynamical and multiple pathways for the photon emission resulting from conformational freedom of the wire are readily uncovered. These results provide the basis for guiding the synthesis of DNA-based supramolecular arrays with improved photon transport at the nanometer scale.
• Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation. Kiesel, M; Reuss, R; Endter, J; Zimmermann, D; Zimmermann, H; Shirakashi, R; Bamberg, E; Zimmermann, U; Sukhorukov, VL. In BIOPHYSICAL JOURNAL, 90(12), S. 4720–4729. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2006.
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  Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane ( probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C-m(mu F/cm(2)). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C-C (pF) in strongly hypotonic solutions.

  @article{Kiesel2006, abstract = {Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane ( probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C-m(mu F/cm(2)). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C-C (pF) in strongly hypotonic solutions.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Kiesel, M and Reuss, R and Endter, J and Zimmermann, D and Zimmermann, H and Shirakashi, R and Bamberg, E and Zimmermann, U and Sukhorukov, VL}, journal = {BIOPHYSICAL JOURNAL}, keywords = {vladimir}, month = {06}, number = 12, pages = {4720-4729}, publisher = {BIOPHYSICAL SOCIETY}, title = {Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation}, type = {Article}, volume = 90, year = 2006 }

  %0 Journal Article %1 Kiesel2006 %A Kiesel, M %A Reuss, R %A Endter, J %A Zimmermann, D %A Zimmermann, H %A Shirakashi, R %A Bamberg, E %A Zimmermann, U %A Sukhorukov, VL %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2006 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 12 %P 4720-4729 %T Swelling-activated pathways in human T-lymphocytes studied by cell volumetry and electrorotation %U http://dx.doi.org/10.1529/biophysj.105.078725 %V 90 %X Small organic solutes, including sugar derivatives, amino acids, etc., contribute significantly to the osmoregulation of mammalian cells. The present study explores the mechanisms of swelling-activated membrane permeability for electrolytes and neutral carbohydrates in Jurkat cells. Electrorotation was used to analyze the relationship between the hypotonically induced changes in the electrically accessible surface area of the plasma membrane ( probed by the capacitance) and its permeability to the monomeric sugar alcohol sorbitol, the disaccharide trehalose, and electrolyte. Time-resolved capacitance and volumetric measurements were performed in parallel using media of different osmolalities containing either sorbitol or trehalose as the major solute. Under mild hypotonic stress in 200 mOsm sorbitol or trehalose solutions, the cells accomplished regulatory volume decrease by releasing cytosolic electrolytes presumably through pathways activated by the swelling-mediated retraction of microvilli. This is suggested by a rapid decrease of the area-specific membrane capacitance C-m(mu F/cm(2)). The cell membrane was impermeable to both carbohydrates in 200 mOsm media. Whereas trehalose permeability remained also very poor in 100 mOsm medium, extreme swelling of cells in a strongly hypotonic solution (100 mOsm) led to a dramatic increase in sorbitol permeability as evidenced by regulatory volume decrease inhibition. The different osmotic thresholds for activation of electrolyte release and sorbitol influx suggest the involvement of separate swelling-activated pathways. Whereas the electrolyte efflux seemed to utilize pathways preexisting in the plasma membrane, putative sorbitol channels might be inserted into the membrane from cytosolic vesicles via swelling-mediated exocytosis, as indicated by a substantial increase in the whole-cell capacitance C-C (pF) in strongly hypotonic solutions.

• Exploring life by single-molecule fluorescence spectroscopy. Neuweiler, H; Sauer, M. In ANALYTICAL CHEMISTRY, 77(9), S. 178A-185A. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  @article{Neuweiler2005a, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {hannes}, month = {05}, number = 9, pages = {178A-185A}, publisher = {AMER CHEMICAL SOC}, title = {Exploring life by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Neuweiler2005a %A Neuweiler, H %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 9 %P 178A-185A %T Exploring life by single-molecule fluorescence spectroscopy %V 77
• Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology. Tinnefeld, Philip; Sauer, Markus. In Angewandte Chemie International Edition, 44(18), S. 2642–2671. WILEY-VCH Verlag, 2005.
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  In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.

  @article{ANIE:ANIE200300647, abstract = {In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.}, author = {Tinnefeld, Philip and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, number = 18, pages = {2642–2671}, publisher = {WILEY-VCH Verlag}, title = {Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology}, volume = 44, year = 2005 }

  %0 Journal Article %1 ANIE:ANIE200300647 %A Tinnefeld, Philip %A Sauer, Markus %D 2005 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 18 %P 2642--2671 %R 10.1002/anie.200300647 %T Branching Out of Single-Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology %U http://dx.doi.org/10.1002/anie.200300647 %V 44 %X In the last decade emerging single-molecule fluorescence-spectroscopy tools have been developed and adapted to analyze individual molecules under various conditions. Single-molecule-sensitive optical techniques are now well established and help to increase our understanding of complex problems in different disciplines ranging from materials science to cell biology. Previous dreams, such as the monitoring of the motility and structural changes of single motor proteins in living cells or the detection of single-copy genes and the determination of their distance from polymerase molecules in transcription factories in the nucleus of a living cell, no longer constitute unsolvable problems. In this Review we demonstrate that single-molecule fluorescence spectroscopy has become an independent discipline capable of solving problems in molecular biology. We outline the challenges and future prospects for optical single-molecule techniques which can be used in combination with smart labeling strategies to yield quantitative three-dimensional information about the dynamic organization of living cells.
• Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy. Scheffler, S; Sauer, M; Neuweiler, H. In ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS, 219(5), S. 665–678. R OLDENBOURG VERLAG, LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY, 2005.
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  We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.

  @article{Scheffler2005, abstract = {We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.}, address = {LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY}, author = {Scheffler, S and Sauer, M and Neuweiler, H}, journal = {ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY \& CHEMICAL PHYSICS}, keywords = {hannes}, number = 5, pages = {665-678}, publisher = {R OLDENBOURG VERLAG}, title = {Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy}, type = {Article}, volume = 219, year = 2005 }

  %0 Journal Article %1 Scheffler2005 %A Scheffler, S %A Sauer, M %A Neuweiler, H %C LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY %D 2005 %I R OLDENBOURG VERLAG %J ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS %N 5 %P 665-678 %T Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy %U http://dx.doi.org/10.1524/zpch.219.5.665.64323 %V 219 %X We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.
• Quantum dots for live cells, in vivo imaging, and diagnostics. Michalet, X; Pinaud, FF; Bentolila, LA; Tsay, JM; Doose, S; Li, JJ; Sundaresan, G; Wu, AM; Gambhir, SS; Weiss, S. In SCIENCE, 307(5709), S. 538–544. AMER ASSOC ADVANCEMENT SCIENCE, 1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA, 2005.
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  Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

  @article{Michalet2005, abstract = {Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.}, address = {1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA}, author = {Michalet, X and Pinaud, FF and Bentolila, LA and Tsay, JM and Doose, S and Li, JJ and Sundaresan, G and Wu, AM and Gambhir, SS and Weiss, S}, journal = {SCIENCE}, keywords = {doose}, month = {01}, number = 5709, pages = {538-544}, publisher = {AMER ASSOC ADVANCEMENT SCIENCE}, title = {Quantum dots for live cells, in vivo imaging, and diagnostics}, type = {Review}, volume = 307, year = 2005 }

  %0 Journal Article %1 Michalet2005 %A Michalet, X %A Pinaud, FF %A Bentolila, LA %A Tsay, JM %A Doose, S %A Li, JJ %A Sundaresan, G %A Wu, AM %A Gambhir, SS %A Weiss, S %C 1200 NEW YORK AVE, NW, WASHINGTON, DC 20005 USA %D 2005 %I AMER ASSOC ADVANCEMENT SCIENCE %J SCIENCE %N 5709 %P 538-544 %T Quantum dots for live cells, in vivo imaging, and diagnostics %U http://dx.doi.org/10.1126/science.1104274 %V 307 %X Research on fluorescent semiconductor nanocrystais (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.
• Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation. Tsay, JM; Doose, S; Pinaud, F; Weiss, S. In JOURNAL OF PHYSICAL CHEMISTRY B, 109(5), S. 1669–1674. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.

  @article{Tsay2005, abstract = {The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tsay, JM and Doose, S and Pinaud, F and Weiss, S}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {doose}, month = {02}, number = 5, pages = {1669-1674}, publisher = {AMER CHEMICAL SOC}, title = {Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation}, type = {Article}, volume = 109, year = 2005 }

  %0 Journal Article %1 Tsay2005 %A Tsay, JM %A Doose, S %A Pinaud, F %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 5 %P 1669-1674 %T Enhancing the photoluminescence of peptide-coated nanocrystals with shell composition and UV irradiation %U http://dx.doi.org/10.1021/jp046828f %V 109 %X The composition and structure of inorganic shells grown over CdSe semiconductor nanocrystal dots and rods were optimized to yield enhanced photoluminescence properties after ligand exchange followed by coating with phytochelatin-related peptides. We show that, in addition to the peptides imparting superior colloidal properties and providing biofunctionality in a single-step reaction, the improved shells and pretreatment with UV irradiation resulted in high quantum yields for the nanocrystals in water. Moreover, peptide coating caused a noticeable red-shift in the absorption and emission spectra for one of the tested shells, suggesting that exciton-molecular orbital (X-MO) coupling might take place in these hybrid inorganic-organic composite materials.
• Design of molecular photonic wires based on multistep electronic excitation transfer. Tinnefeld, P; Heilemann, M; Sauer, M. In CHEMPHYSCHEM, 6(2), S. 217–222. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
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  Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.

  @article{Tinnefeld2005, abstract = {Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Tinnefeld, P and Heilemann, M and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {02}, number = 2, pages = {217-222}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Design of molecular photonic wires based on multistep electronic excitation transfer}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Tinnefeld2005 %A Tinnefeld, P %A Heilemann, M %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 2 %P 217-222 %T Design of molecular photonic wires based on multistep electronic excitation transfer %U http://dx.doi.org/10.1002/cphc.200400513 %V 6 %X Light-harvesting complexes, one of nature's supreme examples of nonoscole engineering, have inspired researchers to construct optical devices, such as photonic wires, which ore optimised for efficient transfer of excited-state energy over large distances. The control parameters for the design and the advantages of single-molecule fluorescence spectroscopy for the study of such complex systems are discussed with respect to energy-transfer mechanisms, chromophore selection and arrangement as well as static and dynamic heterogeneity.
• Carbocyanine dyes as efficient reversible single-molecule optical switch. Heilemann, M; Margeat, E; Kasper, R; Sauer, M; Tinnefeld, P. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 127(11), S. 3801–3806. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of > 90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.

  @article{Heilemann2005, abstract = {We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher \β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of \> 90\% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, M and Margeat, E and Kasper, R and Sauer, M and Tinnefeld, P}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {mike}, month = {03}, number = 11, pages = {3801-3806}, publisher = {AMER CHEMICAL SOC}, title = {Carbocyanine dyes as efficient reversible single-molecule optical switch}, type = {Article}, volume = 127, year = 2005 }

  %0 Journal Article %1 Heilemann2005 %A Heilemann, M %A Margeat, E %A Kasper, R %A Sauer, M %A Tinnefeld, P %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 11 %P 3801-3806 %T Carbocyanine dyes as efficient reversible single-molecule optical switch %U http://dx.doi.org/10.1021/ja044686x %V 127 %X We demonstrate that commercially available unmodified carbocyanine dyes such as Cy5 (usually excited at 633 nm) can be used as efficient reversible single-molecule optical switch, whose fluorescent state after apparent photobleaching can be restored at room temperature upon irradiation at shorter wavelengths. Ensemble photobleaching and recovery experiments of Cy5 in aqueous solution irradiating first at 633 nm, then at 337, 488, or 532 nm, demonstrate that restoration of absorption and fluorescence strongly depends on efficient oxygen removal and the addition of the triplet quencher β-mercaptoethylamine. Single-molecule fluorescence experiments show that individual immobilized Cy5 molecules can be switched optically in milliseconds by applying alternating excitation at 633 and 488 nm between a fluorescent and nonfluorescent state up to 100 times with a reliability of > 90% at room temperature. Because of their intriguing performance, carbocyanine dyes volunteer as a simple alternative for ultrahigh-density optical data storage. Measurements on single donor/acceptor (tetramethylrhodamine/Cy5) labeled oligonucleotides point out that the described light-driven switching behavior imposes fundamental limitations on the use of carbocyanine dyes as energy transfer acceptors for the study of biological processes.
• Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy. Doose, S; Tsay, JM; Pinaud, F; Weiss, S. In ANALYTICAL CHEMISTRY, 77(7), S. 2235–2242. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.

  @article{Doose2005, abstract = {A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Doose, S and Tsay, JM and Pinaud, F and Weiss, S}, journal = {ANALYTICAL CHEMISTRY}, keywords = {doose}, month = {04}, number = 7, pages = {2235-2242}, publisher = {AMER CHEMICAL SOC}, title = {Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Doose2005 %A Doose, S %A Tsay, JM %A Pinaud, F %A Weiss, S %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 7 %P 2235-2242 %T Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy %U http://dx.doi.org/10.1021/ac050035n %V 77 %X A number of different surface chemistries have been developed in recent years to render semiconductor nanocrystals (NCs) stable in water and biocompatible. However, most of these surface modifications affect NCs' photophysical properties, calling for a method to simultaneously monitor colloidal and fluorescence properties. Fluorescence correlation spectroscopy (FCS) combined with ensemble spectroscopic methods and Monte Carlo simulations were used to interpret and derive photophysical as well as colloidal properties of four different NC surface treatments. Using a novel FCS scheme with alternating laser excitation at two different intensities, we first ruled out influences from optical gradient forces (optical trapping). We then compared concentration of emitting particles, brightness per particle, saturation intensity, blinking (intermittency), hydrodynamic radius, and propensity for aggregation of the different bioconjugated NCs. This approach was successfully applied during the development and optimization of peptide-coated NCs.
• High-resolution colocalization of single molecules within the resolution gap of far-field microscopy. Heinlein, T; Biebricher, A; Schluter, P; Roth, CM; Herten, DP; Wolfrum, J; Heilemann, M; Muller, C; Tinnefeld, P; Sauer, M. In CHEMPHYSCHEM, 6(5), S. 949–955. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
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  To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

  @article{Heinlein2005, abstract = {To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Heinlein, T and Biebricher, A and Schluter, P and Roth, CM and Herten, DP and Wolfrum, J and Heilemann, M and Muller, C and Tinnefeld, P and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {mike}, month = {05}, number = 5, pages = {949-955}, publisher = {WILEY-V C H VERLAG GMBH}, title = {High-resolution colocalization of single molecules within the resolution gap of far-field microscopy}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Heinlein2005 %A Heinlein, T %A Biebricher, A %A Schluter, P %A Roth, CM %A Herten, DP %A Wolfrum, J %A Heilemann, M %A Muller, C %A Tinnefeld, P %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 5 %P 949-955 %T High-resolution colocalization of single molecules within the resolution gap of far-field microscopy %U http://dx.doi.org/10.1002/cphc.200400622 %V 6 %X To obtain detailed information about the three-dimensional (30) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 6301650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 6301650) that can be efficiently excited by a single pulsed diode loser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.
• Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation. Zimmermann, H; Zimmermann, D; Reuss, R; Feilen, PJ; Manz, B; Katsen, A; Weber, M; Ihmig, FR; Ehrhart, F; Gessner, P; Behringer, M; Steinbach, A; Wegner, LH; Sukhorukov, VL; Vasquez, JA; Schneider, S; Weber, MM; Volke, F; Wolf, R; Zimmermann, U. In JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE, 16(6), S. 491–501. SPRINGER, VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS, 2005.
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  The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice. (C) 2005 Springer Science + Business Media, Inc.

  @article{Zimmermann2005, abstract = {The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice. (C) 2005 Springer Science + Business Media, Inc.}, address = {VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS}, author = {Zimmermann, H and Zimmermann, D and Reuss, R and Feilen, PJ and Manz, B and Katsen, A and Weber, M and Ihmig, FR and Ehrhart, F and Gessner, P and Behringer, M and Steinbach, A and Wegner, LH and Sukhorukov, VL and Vasquez, JA and Schneider, S and Weber, MM and Volke, F and Wolf, R and Zimmermann, U}, journal = {JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE}, keywords = {vladimir}, month = {06}, number = 6, pages = {491-501}, publisher = {SPRINGER}, title = {Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation}, type = {Proceedings Paper}, volume = 16, year = 2005 }

  %0 Journal Article %1 Zimmermann2005 %A Zimmermann, H %A Zimmermann, D %A Reuss, R %A Feilen, PJ %A Manz, B %A Katsen, A %A Weber, M %A Ihmig, FR %A Ehrhart, F %A Gessner, P %A Behringer, M %A Steinbach, A %A Wegner, LH %A Sukhorukov, VL %A Vasquez, JA %A Schneider, S %A Weber, MM %A Volke, F %A Wolf, R %A Zimmermann, U %C VAN GODEWIJCKSTRAAT 30, 3311 GZ DORDRECHT, NETHERLANDS %D 2005 %I SPRINGER %J JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE %N 6 %P 491-501 %T Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation %U http://dx.doi.org/10.1007/s10856-005-0523-2 %V 16 %X The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice. (C) 2005 Springer Science + Business Media, Inc.
• Surviving high-intensity field pulses: Strategies for improving robustness and performance of electrotransfection and electrofusion. Sukhorukov, VL; Reuss, R; Zimmermann, D; Held, C; Muller, KJ; Kiesel, M; Gessner, P; Steinbach, A; Schenk, WA; Bamberg, E; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 206(3), S. 187–201. SPRINGER, 233 SPRING STREET, NEW YORK, NY 10013 USA, 2005.
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  Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

  @article{Sukhorukov2005, abstract = {Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.}, address = {233 SPRING STREET, NEW YORK, NY 10013 USA}, author = {Sukhorukov, VL and Reuss, R and Zimmermann, D and Held, C and Muller, KJ and Kiesel, M and Gessner, P and Steinbach, A and Schenk, WA and Bamberg, E and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {08}, number = 3, pages = {187-201}, publisher = {SPRINGER}, title = {Surviving high-intensity field pulses: Strategies for improving robustness and performance of electrotransfection and electrofusion}, type = {Article}, volume = 206, year = 2005 }

  %0 Journal Article %1 Sukhorukov2005 %A Sukhorukov, VL %A Reuss, R %A Zimmermann, D %A Held, C %A Muller, KJ %A Kiesel, M %A Gessner, P %A Steinbach, A %A Schenk, WA %A Bamberg, E %A Zimmermann, U %C 233 SPRING STREET, NEW YORK, NY 10013 USA %D 2005 %I SPRINGER %J JOURNAL OF MEMBRANE BIOLOGY %N 3 %P 187-201 %T Surviving high-intensity field pulses: Strategies for improving robustness and performance of electrotransfection and electrofusion %U http://dx.doi.org/10.1007/s00232-005-0791-2 %V 206 %X Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.
• Self-quenching DNA probes based on dye dimerization for identification of mycobacteria. Knemeyer, JP; Marme, N; Hafner, B; Habl, G; Schafer, G; Muller, M; Nolte, O; Sauer, M; Wolfrum, J. In INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY, 85(9-11), S. 625–637. TAYLOR & FRANCIS LTD, 4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND, 2005.
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  This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.

  @article{Knemeyer2005, abstract = {This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.}, address = {4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND}, author = {Knemeyer, JP and Marme, N and Hafner, B and Habl, G and Schafer, G and Muller, M and Nolte, O and Sauer, M and Wolfrum, J}, journal = {INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = {08}, number = {9-11}, pages = {625-637}, publisher = {TAYLOR \& FRANCIS LTD}, title = {Self-quenching DNA probes based on dye dimerization for identification of mycobacteria}, type = {Proceedings Paper}, volume = 85, year = 2005 }

  %0 Journal Article %1 Knemeyer2005 %A Knemeyer, JP %A Marme, N %A Hafner, B %A Habl, G %A Schafer, G %A Muller, M %A Nolte, O %A Sauer, M %A Wolfrum, J %C 4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND %D 2005 %I TAYLOR & FRANCIS LTD %J INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY %N 9-11 %P 625-637 %T Self-quenching DNA probes based on dye dimerization for identification of mycobacteria %U http://dx.doi.org/10.1080/03067310500146094 %V 85 %X This paper presents new self-quenching DNA probes that exploit the efficient fluorescence quenching by the formation of dye dimers. The probes consist of a hairpin-structured oligonucleotide that is labeled with two identical fluorescence dyes that are able to form non-fluorescent H-type dimers while the hairpin is closed. We used the oxazine derivative MR 121 that has a sufficient dimerization tendency and can be excited by a pulsed diode laser emitting at 635 nm. Upon hybridization to the target DNA, the dyes are separated and a 12-fold increase or the florescence intensity occurs. The probe was used for the specific detection of Mycobacterium xenopi in a model system. Specific target DNA and a control target, differing by six nucleotides were amplified by polymerase chain reaction (PCR). A confocal fluorescence microscope was used to observe the fluorescence bursts of individual probe molecules in the presence of target PCR product and controls. By experiment and by the respective simulation we demonstrated that the secondary structure of the target DNA hinders the hybridization to the DNA probe at room temperature. Based on these data a successful hybridization procedure was developed and allowing the detection of nanomolar concentrations of M i cobacterium xenopi specific target at room temperature, using single-molecule detection techniques.
• Defocused imaging of quantum-dot angular distribution of radiation. Patra, D; Gregor, I; Enderlein, J; Sauer, M. In APPLIED PHYSICS LETTERS, 87(10). AMER INST PHYSICS, CIRCULATION & FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA, 2005.
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  We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.

  @article{Patra2005, abstract = {We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.}, address = {CIRCULATION \& FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA}, author = {Patra, D and Gregor, I and Enderlein, J and Sauer, M}, journal = {APPLIED PHYSICS LETTERS}, keywords = {sauer}, month = {09}, number = 10, publisher = {AMER INST PHYSICS}, title = {Defocused imaging of quantum-dot angular distribution of radiation}, type = {Article}, volume = 87, year = 2005 }

  %0 Journal Article %1 Patra2005 %A Patra, D %A Gregor, I %A Enderlein, J %A Sauer, M %C CIRCULATION & FULFILLMENT DIV, 2 HUNTINGTON QUADRANGLE, STE 1 N O 1, MELVILLE, NY 11747-4501 USA %D 2005 %I AMER INST PHYSICS %J APPLIED PHYSICS LETTERS %N 10 %T Defocused imaging of quantum-dot angular distribution of radiation %U http://dx.doi.org/10.1063/1.2037194 %V 87 %X We have applied defocused imaging of single fluorescent quantum dots to study the angular distribution of their emission. It is found that quantum dots exhibit an angular distribution best described by a superposition of emission of three perpendicular dipoles. A theory of the defocused images of such emitters is presented and compared with the measurements. Furthermore, it is shown that standard fluorescence anisotropy measurements are not able to uncover such complex emission behavior. (c) 2005 American Institute of Physics.
• A close look at fluorescence quenching of organic dyes by tryptophan. Doose, S; Neuweiler, H; Sauer, M. In CHEMPHYSCHEM, 6(11), S. 2277–2285. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2005.
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  Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.

  @article{Doose2005a, abstract = {Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30\% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.}, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Doose, S and Neuweiler, H and Sauer, M}, journal = {CHEMPHYSCHEM}, keywords = {hannes}, month = 11, number = 11, pages = {2277-2285}, publisher = {WILEY-V C H VERLAG GMBH}, title = {A close look at fluorescence quenching of organic dyes by tryptophan}, type = {Article}, volume = 6, year = 2005 }

  %0 Journal Article %1 Doose2005a %A Doose, S %A Neuweiler, H %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2005 %I WILEY-V C H VERLAG GMBH %J CHEMPHYSCHEM %N 11 %P 2277-2285 %T A close look at fluorescence quenching of organic dyes by tryptophan %U http://dx.doi.org/10.1002/cphc.200500191 %V 6 %X Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M-1, 2) static quenching with a bimolecular association constant K-s of 61 M-1, and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M-1. The latter two are characterized as nonfluorescent complexes, formed with approximate to 30% efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 2030 kJmol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.
• Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes. Stohr, K; Hafner, B; Nolte, O; Wolfrum, J; Sauer, M; Herten, DP. In ANALYTICAL CHEMISTRY, 77(22), S. 7195–7203. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2005.
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  Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.

  @article{Stohr2005, abstract = {Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Stohr, K and Hafner, B and Nolte, O and Wolfrum, J and Sauer, M and Herten, DP}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 11, number = 22, pages = {7195-7203}, publisher = {AMER CHEMICAL SOC}, title = {Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes}, type = {Article}, volume = 77, year = 2005 }

  %0 Journal Article %1 Stohr2005 %A Stohr, K %A Hafner, B %A Nolte, O %A Wolfrum, J %A Sauer, M %A Herten, DP %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2005 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 22 %P 7195-7203 %T Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes %U http://dx.doi.org/10.1021/ac051447z %V 77 %X Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides beating a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of similar to 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.
• A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. Neuweiler, H; Doose, S; Sauer, M. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(46), S. 16650–16655. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2005.
  • [ Abstract ]
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  The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.

  @article{Neuweiler2005, abstract = {The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Neuweiler, H and Doose, S and Sauer, M}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {hannes}, month = 11, number = 46, pages = {16650-16655}, publisher = {NATL ACAD SCIENCES}, title = {A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate}, type = {Article}, volume = 102, year = 2005 }

  %0 Journal Article %1 Neuweiler2005 %A Neuweiler, H %A Doose, S %A Sauer, M %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2005 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 46 %P 16650-16655 %T A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate %U http://dx.doi.org/10.1073/pnas.0507351102 %V 102 %X The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.
• Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis. Kapanidis, AN; Margeat, E; Laurence, TA; Doose, S; Ho, SO; Mukhopadhyay, J; Kortkhonjia, E; Mekler, V; Ebright, RH; Weiss, S. In MOLECULAR CELL, 20(3), S. 347–356. CELL PRESS, 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA, 2005.
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  We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70%-90%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50%-60%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.

  @article{Kapanidis2005, abstract = {We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70\%-90\%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50\%-60\%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.}, address = {1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA}, author = {Kapanidis, AN and Margeat, E and Laurence, TA and Doose, S and Ho, SO and Mukhopadhyay, J and Kortkhonjia, E and Mekler, V and Ebright, RH and Weiss, S}, journal = {MOLECULAR CELL}, keywords = {doose}, month = 11, number = 3, pages = {347-356}, publisher = {CELL PRESS}, title = {Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis}, type = {Article}, volume = 20, year = 2005 }

  %0 Journal Article %1 Kapanidis2005 %A Kapanidis, AN %A Margeat, E %A Laurence, TA %A Doose, S %A Ho, SO %A Mukhopadhyay, J %A Kortkhonjia, E %A Mekler, V %A Ebright, RH %A Weiss, S %C 1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA %D 2005 %I CELL PRESS %J MOLECULAR CELL %N 3 %P 347-356 %T Retention of transcription initiation factor sigma(70) in transcription elongation: Single-molecule analysis %U http://dx.doi.org/10.1016/j.molcel.2005.10.012 %V 20 %X We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma(70) contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, similar to 70%-90%, of early elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region increases sigma(70) retention in early elongation complexes. The results establish that a significant fraction, similar to 50%-60%, of mature elongation complexes retain sigma(70) and that a determinant for sigma(70) recognition in the initial transcribed region does not appreciably affect sigma 70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma(70) the half-life of sigma(70) retention is long relative to the timescale of elongation, suggesting that some complexes may retain sigma(70) throughout elongation.
• Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging. Sauer, M. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(27), S. 9433–9434. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2005.
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  @article{Sauer2005, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Sauer, M}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {sauer}, month = {07}, number = 27, pages = {9433-9434}, publisher = {NATL ACAD SCIENCES}, title = {Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging}, type = {Editorial Material}, volume = 102, year = 2005 }

  %0 Journal Article %1 Sauer2005 %A Sauer, M %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2005 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 27 %P 9433-9434 %T Reversible molecular photoswitches: A key technology for nanoscience and fluorescence imaging %U http://dx.doi.org/10.1073/pnas.0504264102 %V 102

• Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy. Tinnefeld, Philip; Hofkens, Johan; Herten, Dirk-Peter; Masuo, Sadahiro; Vosch, Tom; Cotlet, Mircea; Habuchi, Satoshi; Müllen, Klaus; Schryver, Frans C. De; Sauer, Markus. In ChemPhysChem, 5(11), S. 1786–1790. WILEY-VCH Verlag, 2004.
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  Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems

  @article{CPHC:CPHC200400325, abstract = {Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems}, author = {Tinnefeld, Philip and Hofkens, Johan and Herten, Dirk-Peter and Masuo, Sadahiro and Vosch, Tom and Cotlet, Mircea and Habuchi, Satoshi and Müllen, Klaus and Schryver, Frans C. De and Sauer, Markus}, journal = {ChemPhysChem}, keywords = {sauer}, number = 11, pages = {1786–1790}, publisher = {WILEY-VCH Verlag}, title = {Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy}, volume = 5, year = 2004 }

  %0 Journal Article %1 CPHC:CPHC200400325 %A Tinnefeld, Philip %A Hofkens, Johan %A Herten, Dirk-Peter %A Masuo, Sadahiro %A Vosch, Tom %A Cotlet, Mircea %A Habuchi, Satoshi %A Müllen, Klaus %A Schryver, Frans C. De %A Sauer, Markus %D 2004 %I WILEY-VCH Verlag %J ChemPhysChem %N 11 %P 1786--1790 %R 10.1002/cphc.200400325 %T Higher-Excited-State Photophysical Pathways in Multichromophoric Systems Revealed by Single-Molecule Fluorescence Spectroscopy %U http://dx.doi.org/10.1002/cphc.200400325 %V 5 %X Singlets annihilated: A single-molecule fluorescence spectroscopic study of multichromophoric peryleneimide-substituted polyphenylene-core dendrimers under various conditions indicates that increased intersystem crossing (see graphic) as a consequence of an annihilation process might represent a general motif for increased photobleaching of the first intensity levels in multichromophoric systems
• Optical single molecule techniques for analytical and biological applications. Dirk-Peter, HERTEN; Andreas, BIEBRICHER; Mike, HEILEMANN; Thomas, HEINLEIN; Christian, MÜLLER; Pia, SCHLÜTER; Philip, TINNEFELD; D., WESTON Kenneth; Markus, SAUER; Jürgen, WOLFRUM. In Recent Res. Devel. Applied Phys., Vol. 7 (2004), Part II, S. 345–368. 2004.
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  @article{Dirk-Peter2004, author = {Dirk-Peter, HERTEN and Andreas, BIEBRICHER and Mike, HEILEMANN and Thomas, HEINLEIN and Christian, MÜLLER and Pia, SCHLÜTER and Philip, TINNEFELD and D., WESTON Kenneth and Markus, SAUER and Jürgen, WOLFRUM}, journal = {Recent Res. Devel. Applied Phys.}, keywords = {mike}, pages = {345-368}, title = {Optical single molecule techniques for analytical and biological applications}, volume = {Vol. 7 (2004), Part II}, year = 2004 }

  %0 Journal Article %1 Dirk-Peter2004 %A Dirk-Peter, HERTEN %A Andreas, BIEBRICHER %A Mike, HEILEMANN %A Thomas, HEINLEIN %A Christian, MÜLLER %A Pia, SCHLÜTER %A Philip, TINNEFELD %A D., WESTON Kenneth %A Markus, SAUER %A Jürgen, WOLFRUM %D 2004 %J Recent Res. Devel. Applied Phys. %P 345-368 %T Optical single molecule techniques for analytical and biological applications %V Vol. 7 (2004), Part II
• Chemical and Biological Investigations of beta-Oligoarginines. Seebach, Dieter; Namoto, Kenji; Mahajan, Yogesh R.; Bindschädler, Pascal; Sustmann, Reiner; Kirsch, Michael; Ryder, Neil S.; Weiss, Markus; Sauer, Markus; Roth, Christian; Werner, Sabine; Beer, Hans-Dietmar; Munding, Christine; Walde, Peter; Voser, Matthias. In Chemistry & Biodiversity, 1(1), S. 65–97. WILEY-VCH Verlag, 2004.
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  In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.

  @article{CBDV:CBDV200490014, abstract = {In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.}, author = {Seebach, Dieter and Namoto, Kenji and Mahajan, Yogesh R. and Bindschädler, Pascal and Sustmann, Reiner and Kirsch, Michael and Ryder, Neil S. and Weiss, Markus and Sauer, Markus and Roth, Christian and Werner, Sabine and Beer, Hans-Dietmar and Munding, Christine and Walde, Peter and Voser, Matthias}, journal = {Chemistry & Biodiversity}, keywords = {sauer}, number = 1, pages = {65–97}, publisher = {WILEY-VCH Verlag}, title = {Chemical and Biological Investigations of beta-Oligoarginines}, volume = 1, year = 2004 }

  %0 Journal Article %1 CBDV:CBDV200490014 %A Seebach, Dieter %A Namoto, Kenji %A Mahajan, Yogesh R. %A Bindschädler, Pascal %A Sustmann, Reiner %A Kirsch, Michael %A Ryder, Neil S. %A Weiss, Markus %A Sauer, Markus %A Roth, Christian %A Werner, Sabine %A Beer, Hans-Dietmar %A Munding, Christine %A Walde, Peter %A Voser, Matthias %D 2004 %I WILEY-VCH Verlag %J Chemistry & Biodiversity %N 1 %P 65--97 %R 10.1002/cbdv.200490014 %T Chemical and Biological Investigations of beta-Oligoarginines %U http://dx.doi.org/10.1002/cbdv.200490014 %V 1 %X In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous β-homoarginines are central in our investigations of β-peptides (Fig. 1). The preparation of β2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-β3hArg(Boc)2-OH is used for manual solid-phase synthesis of β3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, β3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2–4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-β2hArg-OH and (S)-H-β3hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the β3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free β-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 μM; Table 1), and no significant antibiotic activity (MIC≥64 μg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant β3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain β-oligoarginine amides (8 and 10 residues; Figs. 4–6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37° and 4° with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the β-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-μM concentration of the fluorescence-labelled β-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the β-octaarginine derivative show migration of the β-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled β-octa- and β-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the β-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a β-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the ‘protein transduction’ by β-oligoarginines are discussed.
• Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer. Marmé, Nicole; Knemeyer, Jens-Peter; Wolfrum, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, 43(29), S. 3798–3801. WILEY-VCH Verlag, 2004.
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  Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range

  @article{ANIE:ANIE200453835, abstract = {Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range}, author = {Marmé, Nicole and Knemeyer, Jens-Peter and Wolfrum, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {sauer}, number = 29, pages = {3798–3801}, publisher = {WILEY-VCH Verlag}, title = {Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer}, volume = 43, year = 2004 }

  %0 Journal Article %1 ANIE:ANIE200453835 %A Marmé, Nicole %A Knemeyer, Jens-Peter %A Wolfrum, Jürgen %A Sauer, Markus %D 2004 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 29 %P 3798--3801 %R 10.1002/anie.200453835 %T Highly Sensitive Protease Assay Using Fluorescence Quenching of Peptide Probes Based on Photoinduced Electron Transfer %U http://dx.doi.org/10.1002/anie.200453835 %V 43 %X Making the cut and then detecting it: Short fluorophore-labeled peptide substrates (see scheme) show strong fluorescence quenching due to electron transfer from a tryptophan residue (blue) to the dye (gray, red). Upon specific cleavage of the peptide by proteolytic enzymes, quenching is prevented and the fluorescence intensity increases. The method can be used to assay proteolytic enzymes with detection limits in the picomolar range
• Single photon emission from a dendrimer containing eight perylene diimide chromophores. Bell, TDM; Habuchi, S; Masuo, S; Osterling, I; Mullen, K; Tinnefeld, P; Sauer, M; van der Auweraer, M; Hofkens, J; Schryver, FC De. In AUSTRALIAN JOURNAL OF CHEMISTRY, 57(12), S. 1169–1173. C S I R O PUBLISHING, 150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA, 2004.
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  A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.

  @article{Bell2004, abstract = {A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.}, address = {150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA}, author = {Bell, TDM and Habuchi, S and Masuo, S and Osterling, I and Mullen, K and Tinnefeld, P and Sauer, M and van der Auweraer, M and Hofkens, J and Schryver, FC De}, journal = {AUSTRALIAN JOURNAL OF CHEMISTRY}, keywords = {sauer}, number = 12, pages = {1169-1173}, publisher = {C S I R O PUBLISHING}, title = {Single photon emission from a dendrimer containing eight perylene diimide chromophores}, type = {Proceedings Paper}, volume = 57, year = 2004 }

  %0 Journal Article %1 Bell2004 %A Bell, TDM %A Habuchi, S %A Masuo, S %A Osterling, I %A Mullen, K %A Tinnefeld, P %A Sauer, M %A van der Auweraer, M %A Hofkens, J %A Schryver, FC De %C 150 OXFORD ST, PO BOX 1139, COLLINGWOOD, VICTORIA 3066, AUSTRALIA %D 2004 %I C S I R O PUBLISHING %J AUSTRALIAN JOURNAL OF CHEMISTRY %N 12 %P 1169-1173 %T Single photon emission from a dendrimer containing eight perylene diimide chromophores %U http://dx.doi.org/10.1071/CH04133 %V 57 %X A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching ( coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic ( triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet - singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.
• How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions. Tinnefeld, P.; Buschmann, V.; Weston, K. D.; Biebricher, A.; Herten, D. P.; Piestert, O.; Heinlein, T.; Heilemann, M.; Sauer, M. In Recent Res. Devel. Phys. Chem. 7, 95-125. 2004.
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  A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques

  @article{tinnefeld2004single, abstract = {A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques}, author = {Tinnefeld, P. and Buschmann, V. and Weston, K. D. and Biebricher, A. and Herten, D. P. and Piestert, O. and Heinlein, T. and Heilemann, M. and Sauer, M.}, journal = {Recent Res. Devel. Phys. Chem. 7, 95-125.}, keywords = {mike}, title = {How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions}, year = 2004 }

  %0 Journal Article %1 tinnefeld2004single %A Tinnefeld, P. %A Buschmann, V. %A Weston, K. D. %A Biebricher, A. %A Herten, D. P. %A Piestert, O. %A Heinlein, T. %A Heilemann, M. %A Sauer, M. %D 2004 %J Recent Res. Devel. Phys. Chem. 7, 95-125. %T How single molecule photophysical studies complement ensemble methods for a better understanding of chromophores and chromophore interactions %X A review. Many theor. treatments and exptl. demonstrations of excited state processes originate from the middle of the last century. With the advent of single mol. fluorescence spectroscopy (SMFS) more than a decade ago it is of particular interest to evaluate how these processes are reflected on this ultimate level of sensitivity. The present review deals with two aspects demonstrating the strengths of SMFS: first, the ability to characterize populations and identify subpopulations is demonstrated by studying single chromophores in different environments. Second, the feasibility of studying higher excited state processes such as singlet-singlet annihilation and singlet-triplet annihilation in bichromophoric model systems is presented on the basis of recently developed single mol. techniques
• Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes. Park, Chong-Woo; Knemeyer, Jens-Peter; Marmé, Nicole; Möller, Martin; Spatz, Joachim; Wolfrum, Jürgen; Sauer, Markus. In Chemical Physics, 301(1), S. 105–110. 2004.
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  There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.

  @article{Park2004105, abstract = {There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.}, author = {Park, Chong-Woo and Knemeyer, Jens-Peter and Marmé, Nicole and Möller, Martin and Spatz, Joachim and Wolfrum, Jürgen and Sauer, Markus}, journal = {Chemical Physics}, keywords = {sauer}, number = 1, pages = {105 - 110}, title = {Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes}, volume = 301, year = 2004 }

  %0 Journal Article %1 Park2004105 %A Park, Chong-Woo %A Knemeyer, Jens-Peter %A Marmé, Nicole %A Möller, Martin %A Spatz, Joachim %A Wolfrum, Jürgen %A Sauer, Markus %D 2004 %J Chemical Physics %N 1 %P 105 - 110 %R DOI: 10.1016/j.chemphys.2004.03.006 %T Accurate delivery of single biomolecules by polyethylene glycol coated submicrometer pipettes %U http://www.sciencedirect.com/science/article/B6TFM-4C47PSJ-3/2/01a24087fcf29f6dd86fa8d9234396e6 %V 301 %X There is a great interest to quantitatively manipulate, separate, and deliver low concentrations or even single biomolecules in submicrometer-sized channels. We report the use of cross-linked four-star polyethylene glycol (PEG) as a new coating technique for microchannels. PEG-coating efficiently reduces the electroosmotic flow (EOF) and non-specific adsorption to the glass walls. Our results demonstrate that individual fluorescently labeled hydrophobic cell adhesion proteins (fibronectin molecules) can be drawn through PEG-coated submicrometer pipettes in aqueous solution by electrokinetic forces without the addition of detergents or other additives which potentially deteriorate the activity and specificity of biomolecules.
• Multistep Energy Transfer in Single Molecular Photonic Wires. Heilemann, Mike; Tinnefeld, Philip; Mosteiro, Gabriel Sanchez; Parajo, Maria Garcia; Hulst, Niek F. Van; Sauer, Markus. In Journal of the American Chemical Society, 126(21), S. 6514–6515. 2004.
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  We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events

  @article{doi:10.1021/ja049351u, abstract = {We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events}, author = {Heilemann, Mike and Tinnefeld, Philip and Mosteiro, Gabriel Sanchez and Parajo, Maria Garcia and Hulst, Niek F. Van and Sauer, Markus}, journal = {Journal of the American Chemical Society}, keywords = {mike}, number = 21, pages = {6514-6515}, title = {Multistep Energy Transfer in Single Molecular Photonic Wires}, volume = 126, year = 2004 }

  %0 Journal Article %1 doi:10.1021/ja049351u %A Heilemann, Mike %A Tinnefeld, Philip %A Mosteiro, Gabriel Sanchez %A Parajo, Maria Garcia %A Hulst, Niek F. Van %A Sauer, Markus %D 2004 %J Journal of the American Chemical Society %N 21 %P 6514-6515 %R 10.1021/ja049351u %T Multistep Energy Transfer in Single Molecular Photonic Wires %U http://pubs.acs.org/doi/abs/10.1021/ja049351u %V 126 %X We demonstrate the synthesis and spectroscopic characterization of an unidirectional photonic wire based on four highly efficient fluorescence energy-transfer steps (FRET) between five spectrally different chromophores covalently attached to double-stranded DNA. The DNA-based modular conception enables the introduction of various chromophores at well-defined positions and arbitrary interchromophore distances. While ensemble fluorescence measurements show overall FRET efficiencies between 15 and 30%, single-molecule spectroscopy performed on four spectrally separated detectors easily uncovers subpopulations that exhibit overall FRET efficiencies of up to 90% across a distance of 13.6 nm and a spectral range of 200 nm. Fluorescence trajectories of individual photonic wires show five different fluorescence intensity patterns which can be ascribed to successive photobleaching events
• The effect of disaccharides on the transport of lipophilic ions in cell membranes studied by electrorotation. Reuss, R; Horbaschek, M; Endter, JM; Zimmermann, U; Sukhorukov, VL. In ELECTROSTATICS 2003, (178), H. Morgan (Hrsg.), S. 101–106. IOP PUBLISHING LTD, DIRAC HOUSE, TEMPLE BACK, BRISTOL BS1 6BE, ENGLAND, 2004.
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  The disaccharide trehalose, a natural cryoprotectant, is increasingly being exploited in biomedicine as a very efficient stabilizer of frozen and dry macromolecules, membranes and whole cells. Valuable insight into the mechanisms by which trehalose protects cells can be obtained by studying its effects on the electrical and ion transport properties of cell membranes. In the present study, the binding and translocation of the lipophilic anion dipicrylamine (DPA) across the membrane of Jurkat lymphocytes (suspended in hypotonic trehalose- or sucrose-substituted media) were determined by means of electrorotation. From the rotation spectra of individual cells, not only the passive electrical properties of cell compartments but also the area-specific concentration of DPA adsorbed to the plasma membrane and its translocation rate constants were evaluated. The substitution of sucrose by trehalose increased significantly both the plasma membrane capacitance C-m and the adsorption of DPA to the plasma membrane, whereas the translocation rate of DPA across the membrane was slightly reduced. The high C-m value indicates that hypotonic stress did not cause any noticeable loss of membrane folds and microvilli in the presence of trehalose. The observed changes in the transport of DPA are consistent with the assumption that trehalose increased the dipole potential of the plasma membrane.

  @article{Reuss2004a, abstract = {The disaccharide trehalose, a natural cryoprotectant, is increasingly being exploited in biomedicine as a very efficient stabilizer of frozen and dry macromolecules, membranes and whole cells. Valuable insight into the mechanisms by which trehalose protects cells can be obtained by studying its effects on the electrical and ion transport properties of cell membranes. In the present study, the binding and translocation of the lipophilic anion dipicrylamine (DPA) across the membrane of Jurkat lymphocytes (suspended in hypotonic trehalose- or sucrose-substituted media) were determined by means of electrorotation. From the rotation spectra of individual cells, not only the passive electrical properties of cell compartments but also the area-specific concentration of DPA adsorbed to the plasma membrane and its translocation rate constants were evaluated. The substitution of sucrose by trehalose increased significantly both the plasma membrane capacitance C-m and the adsorption of DPA to the plasma membrane, whereas the translocation rate of DPA across the membrane was slightly reduced. The high C-m value indicates that hypotonic stress did not cause any noticeable loss of membrane folds and microvilli in the presence of trehalose. The observed changes in the transport of DPA are consistent with the assumption that trehalose increased the dipole potential of the plasma membrane.}, address = {DIRAC HOUSE, TEMPLE BACK, BRISTOL BS1 6BE, ENGLAND}, author = {Reuss, R and Horbaschek, M and Endter, JM and Zimmermann, U and Sukhorukov, VL}, booktitle = {ELECTROSTATICS 2003}, editor = {Morgan, H}, keywords = {vladimir}, number = 178, pages = {101-106}, publisher = {IOP PUBLISHING LTD}, series = {INSTITUTE OF PHYSICS CONFERENCE SERIES}, title = {The effect of disaccharides on the transport of lipophilic ions in cell membranes studied by electrorotation}, type = {Proceedings Paper}, year = 2004 }

  %0 Journal Article %1 Reuss2004a %A Reuss, R %A Horbaschek, M %A Endter, JM %A Zimmermann, U %A Sukhorukov, VL %B ELECTROSTATICS 2003 %C DIRAC HOUSE, TEMPLE BACK, BRISTOL BS1 6BE, ENGLAND %D 2004 %E Morgan, H %I IOP PUBLISHING LTD %N 178 %P 101-106 %T The effect of disaccharides on the transport of lipophilic ions in cell membranes studied by electrorotation %U http://dx.doi.org/10.1201/9781420034387.ch16 %X The disaccharide trehalose, a natural cryoprotectant, is increasingly being exploited in biomedicine as a very efficient stabilizer of frozen and dry macromolecules, membranes and whole cells. Valuable insight into the mechanisms by which trehalose protects cells can be obtained by studying its effects on the electrical and ion transport properties of cell membranes. In the present study, the binding and translocation of the lipophilic anion dipicrylamine (DPA) across the membrane of Jurkat lymphocytes (suspended in hypotonic trehalose- or sucrose-substituted media) were determined by means of electrorotation. From the rotation spectra of individual cells, not only the passive electrical properties of cell compartments but also the area-specific concentration of DPA adsorbed to the plasma membrane and its translocation rate constants were evaluated. The substitution of sucrose by trehalose increased significantly both the plasma membrane capacitance C-m and the adsorption of DPA to the plasma membrane, whereas the translocation rate of DPA across the membrane was slightly reduced. The high C-m value indicates that hypotonic stress did not cause any noticeable loss of membrane folds and microvilli in the presence of trehalose. The observed changes in the transport of DPA are consistent with the assumption that trehalose increased the dipole potential of the plasma membrane. %@ 0-7503-0949-0
• Measurement of the permeability and resealing time constant of the electroporated mammalian cell membranes. Shirakashi, R; Sukhorukov, VL; Tanasawa, I; Zimmermann, U. In INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER, 47(21), S. 4517–4524. PERGAMON-ELSEVIER SCIENCE LTD, THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLAND, 2004.
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  In this study a new method is presented for measuring the transient permeability of mammalian cell membranes to sugar and electrolyte molecules based on the volumetric response of cells subjected to electroporation. The time constant of membrane resealing was determined independently by flow cytometry using a fluorescent dye as the reporter molecule. The volumetric and dye uptake data were analyzed with a model relating the cell volume changes to the solute transport across the reversibly permeabilized cell membrane. The experimental approach developed here might be useful for estimating the amount of electroinjected molecules, which are difficult to measure directly. (C) 2004 Elsevier Ltd. All rights reserved.

  @article{Shirakashi2004, abstract = {In this study a new method is presented for measuring the transient permeability of mammalian cell membranes to sugar and electrolyte molecules based on the volumetric response of cells subjected to electroporation. The time constant of membrane resealing was determined independently by flow cytometry using a fluorescent dye as the reporter molecule. The volumetric and dye uptake data were analyzed with a model relating the cell volume changes to the solute transport across the reversibly permeabilized cell membrane. The experimental approach developed here might be useful for estimating the amount of electroinjected molecules, which are difficult to measure directly. (C) 2004 Elsevier Ltd. All rights reserved.}, address = {THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLAND}, author = {Shirakashi, R and Sukhorukov, VL and Tanasawa, I and Zimmermann, U}, journal = {INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER}, keywords = {vladimir}, month = 10, number = 21, pages = {4517-4524}, publisher = {PERGAMON-ELSEVIER SCIENCE LTD}, title = {Measurement of the permeability and resealing time constant of the electroporated mammalian cell membranes}, type = {Article}, volume = 47, year = 2004 }

  %0 Journal Article %1 Shirakashi2004 %A Shirakashi, R %A Sukhorukov, VL %A Tanasawa, I %A Zimmermann, U %C THE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLAND %D 2004 %I PERGAMON-ELSEVIER SCIENCE LTD %J INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER %N 21 %P 4517-4524 %T Measurement of the permeability and resealing time constant of the electroporated mammalian cell membranes %U http://dx.doi.org/10.1016/j.ijheatmasstransfer.2004.04.007 %V 47 %X In this study a new method is presented for measuring the transient permeability of mammalian cell membranes to sugar and electrolyte molecules based on the volumetric response of cells subjected to electroporation. The time constant of membrane resealing was determined independently by flow cytometry using a fluorescent dye as the reporter molecule. The volumetric and dye uptake data were analyzed with a model relating the cell volume changes to the solute transport across the reversibly permeabilized cell membrane. The experimental approach developed here might be useful for estimating the amount of electroinjected molecules, which are difficult to measure directly. (C) 2004 Elsevier Ltd. All rights reserved.
• Implementation of neural networks for the identification of single molecules. Bowen, BP; Scruggs, A; Enderlein, J; Sauer, M; Woodbury, N. In JOURNAL OF PHYSICAL CHEMISTRY A, 108(21), S. 4799–4804. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2004.
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  The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.

  @article{Bowen2004, abstract = {The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Bowen, BP and Scruggs, A and Enderlein, J and Sauer, M and Woodbury, N}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {05}, number = 21, pages = {4799-4804}, publisher = {AMER CHEMICAL SOC}, title = {Implementation of neural networks for the identification of single molecules}, type = {Article}, volume = 108, year = 2004 }

  %0 Journal Article %1 Bowen2004 %A Bowen, BP %A Scruggs, A %A Enderlein, J %A Sauer, M %A Woodbury, N %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2004 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 21 %P 4799-4804 %T Implementation of neural networks for the identification of single molecules %U http://dx.doi.org/10.1021/jp036456v %V 108 %X The effectiveness of neural networks and the optimization of parameters for implementing neural networks were evaluated for use in the identification of single molecules according to their fluorescence lifetime. The best network architecture and training parameters were determined for both ideal and nonideal single-molecule fluorescence data. The effectiveness of the neural network is compared to that of the maximum likelihood estimator on the basis of its ability to correctly identify single molecules. For ideal single-molecule data, it was found that the neural networks and the maximum likelihood estimator perform approximately equally well. For nonideal single-molecule fluorescence data, neural networks were able to correctly identify a larger percentage of single-molecule events than the MLE method.
• Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells. Seidel, T; Kluge, C; Hanitzsch, M; Ross, J; Sauer, M; Dietz, KJ; Golldack, D. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 165–175. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Seidel2004, abstract = {The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Seidel, T and Kluge, C and Hanitzsch, M and Ross, J and Sauer, M and Dietz, KJ and Golldack, D}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {165-175}, publisher = {ELSEVIER SCIENCE BV}, title = {Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Seidel2004 %A Seidel, T %A Kluge, C %A Hanitzsch, M %A Ross, J %A Sauer, M %A Dietz, KJ %A Golldack, D %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 165-175 %T Colocalization and FRET-analysis of subunits c and a of the vacuolar H+-ATPase in living plant cells %U http://dx.doi.org/10.1016/j.jbiotec.2004.04.027 %V 112 %X The proton-translocating plant vacuolar H+-ATPase (VHA) is of prime importance for acidification of intracellular compartments and is essential for processes such as secondary activated transport, maintenance of ion homeostasis, and adaptation to environmental stress. Twelve genes have been identified that encode subunits of the functional V-ATPase complex. In this study, subunits c and a of the V-ATPase from the plant Mesembryanthemum crystallinum were fused to cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and were transiently coexpressed in protoplasts. Two-colour scanning confocal fluorescence microscopy demonstrates that the fusion proteins VHA-c-CFP and VHA-a-YFP are colocalized at the tonoplast, the plasmamembrane, and at endoplasmic membrane structures indicating expression in cytoplasmic vesicles. Furthermore, fluorescence resonance energy transfer (FRET) was used to visualize the interaction of VHA-c and VHA-a in vivo on the nanometer length scale. Excitation of CFP as donor fluorophore caused increased emission of YFP-fluorescence in protoplasts due to FRET. Our results give strong evidence for physical interaction of subunits c and a in living plant cells. (C) 2004 Elsevier B.V. All rights reserved.
• Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules. Kapanidis, AN; Lee, NK; Laurence, TA; Doose, S; Margeat, E; Weiss, S. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101(24), S. 8936–8941. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2004.
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  We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.

  @article{Kapanidis2004, abstract = {We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Kapanidis, AN and Lee, NK and Laurence, TA and Doose, S and Margeat, E and Weiss, S}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {doose}, month = {06}, number = 24, pages = {8936-8941}, publisher = {NATL ACAD SCIENCES}, title = {Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules}, type = {Article}, volume = 101, year = 2004 }

  %0 Journal Article %1 Kapanidis2004 %A Kapanidis, AN %A Lee, NK %A Laurence, TA %A Doose, S %A Margeat, E %A Weiss, S %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2004 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 24 %P 8936-8941 %T Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules %U http://dx.doi.org/10.1073/pnas.0401690101 %V 101 %X We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.
• The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP. Brecht, M; Sewald, K; Schiene, K; Keen, G; Fricke, M; Sauer, M; Niehaus, K. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 151–164. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Brecht2004, abstract = {Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100\% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Brecht, M and Sewald, K and Schiene, K and Keen, G and Fricke, M and Sauer, M and Niehaus, K}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {151-164}, publisher = {ELSEVIER SCIENCE BV}, title = {The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Brecht2004 %A Brecht, M %A Sewald, K %A Schiene, K %A Keen, G %A Fricke, M %A Sauer, M %A Niehaus, K %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 151-164 %T The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP %U http://dx.doi.org/10.1016/j.jbiotec.2004.04.030 %V 112 %X Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier described Ms-Rac1 {[}Mol Gen. Genet. 263 (2000) 761] belong to the class of GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display all conserved regions that are common to GTP-binding proteins. Phylogenetically both are located in the group la, but within this group they are well-separated. Computed structure models of both proteins revealed a high degree of structural conservation. Particularly the switch I and switch II region are 100% conserved between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like G-proteins. Both GTPases differ in structure within the fourth loop and the fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP and by surface plasmon resonance (SPR). By this method the specificity of the G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and Mg2+ on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized. Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected by GTP, EDTA and Mg2+. Thus, the predicted structural differences do not result in different GTP-binding properties of Ms-Rac1 and Ms-Rac4. (C) 2004 Elsevier B.V. All rights reserved.
• Multichromophoric dendrimers as single-photon sources: A single-molecule study. Masuo, S; Vosch, T; Cotlet, M; Tinnefeld, P; Habuchi, S; Bell, TDM; Oesterling, I; Beljonne, D; Champagne, B; Mullen, K; Sauer, M; Hofkens, J; Schryver, FC De. In JOURNAL OF PHYSICAL CHEMISTRY B, 108(43), S. 16686–16696. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2004.
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  Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.

  @article{Masuo2004, abstract = {Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Masuo, S and Vosch, T and Cotlet, M and Tinnefeld, P and Habuchi, S and Bell, TDM and Oesterling, I and Beljonne, D and Champagne, B and Mullen, K and Sauer, M and Hofkens, J and Schryver, FC De}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = 10, number = 43, pages = {16686-16696}, publisher = {AMER CHEMICAL SOC}, title = {Multichromophoric dendrimers as single-photon sources: A single-molecule study}, type = {Article}, volume = 108, year = 2004 }

  %0 Journal Article %1 Masuo2004 %A Masuo, S %A Vosch, T %A Cotlet, M %A Tinnefeld, P %A Habuchi, S %A Bell, TDM %A Oesterling, I %A Beljonne, D %A Champagne, B %A Mullen, K %A Sauer, M %A Hofkens, J %A Schryver, FC De %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2004 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 43 %P 16686-16696 %T Multichromophoric dendrimers as single-photon sources: A single-molecule study %U http://dx.doi.org/10.1021/jp047804b %V 108 %X Single-molecule fluorescence spectroscopy was performed on a number of multi-peryleneimide substituted polyphenylene dendrimers for the purpose of investigating the parameters which influence the efficiency of the dendrimers as single-photon sources at room temperature. Analysis of fluorescence intensity, lifetime, and interphoton arrival time distribution revealed that all measured first-generation dendrimers behave as single-photon emitters when more than one chromophore is excited by a single excitation pulse regardless of the number of constituent chromophores. This is a result of efficient singlet-singlet annihilation, which becomes less efficient in higher-generation dendrimers when the interchromophoric distance increases. The efficiency of the investigated dendrimers as single-photon sources depends on several other parameters, such as the nature of the surrounding polymer matrix, the number of chromophores, and the extent of interchromophoric interactions. These parameters mainly affect the frequency of singlet-triplet annihilation, which in turn dominates the quality of these multichromophoric dendrimers as single-photon sources. The results reported here are important not only for the design of single-photon sources based on single organic molecules but also for a fundamental understanding of natural and artificial multichromophoric systems.
• Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex. Kluge, C; Seidel, T; Bolte, S; Sharma, SS; Hanitzsch, M; Satiat-Jeunemaitre, B; Ross, J; Sauer, M; Golldack, D; Dietz, KJ. In BMC CELL BIOLOGY, 5. BIOMED CENTRAL LTD, MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND, 2004.
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  Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

  @article{Kluge2004, abstract = {Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.}, address = {MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND}, author = {Kluge, C and Seidel, T and Bolte, S and Sharma, SS and Hanitzsch, M and Satiat-Jeunemaitre, B and Ross, J and Sauer, M and Golldack, D and Dietz, KJ}, journal = {BMC CELL BIOLOGY}, keywords = {sauer}, month = {08}, publisher = {BIOMED CENTRAL LTD}, title = {Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex}, type = {Article}, volume = 5, year = 2004 }

  %0 Journal Article %1 Kluge2004 %A Kluge, C %A Seidel, T %A Bolte, S %A Sharma, SS %A Hanitzsch, M %A Satiat-Jeunemaitre, B %A Ross, J %A Sauer, M %A Golldack, D %A Dietz, KJ %C MIDDLESEX HOUSE, 34-42 CLEVELAND ST, LONDON W1T 4LB, ENGLAND %D 2004 %I BIOMED CENTRAL LTD %J BMC CELL BIOLOGY %T Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex %U http://dx.doi.org/10.1186/1471-2121-5-29 %V 5 %X Background: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different ( VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques ( FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: ( i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, ( ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit ( VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.
• Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces. Biebricher, A; Paul, A; Tinnefeld, P; Golzhauser, A; Sauer, M. In JOURNAL OF BIOTECHNOLOGY, 112(1-2), S. 97–107. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2004.
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  We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.

  @article{Biebricher2004, abstract = {We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Biebricher, A and Paul, A and Tinnefeld, P and Golzhauser, A and Sauer, M}, journal = {JOURNAL OF BIOTECHNOLOGY}, keywords = {sauer}, month = {08}, number = {1-2}, pages = {97-107}, publisher = {ELSEVIER SCIENCE BV}, title = {Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces}, type = {Article}, volume = 112, year = 2004 }

  %0 Journal Article %1 Biebricher2004 %A Biebricher, A %A Paul, A %A Tinnefeld, P %A Golzhauser, A %A Sauer, M %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2004 %I ELSEVIER SCIENCE BV %J JOURNAL OF BIOTECHNOLOGY %N 1-2 %P 97-107 %T Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces %U http://dx.doi.org/10.1016/j.jbiotec.2004.03.019 %V 112 %X We used electron-beam lithography to fabricate chemical nanostructures, i.e, amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of similar to2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of similar to1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to similar to2. The S/B-ratio of the fluorescence signals could be further increased to similar to4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes. (C) 2004 Elsevier B.V. All rights reserved.
• Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways. Reuss, R; Ludwig, J; Shirakashi, R; Ehrhart, F; Zimmermann, H; Schneider, S; Weber, MM; Zimmermann, U; Schneider, H; Sukhorukov, VL. In JOURNAL OF MEMBRANE BIOLOGY, 200(2), S. 67–81. SPRINGER, 233 SPRING STREET, NEW YORK, NY 10013 USA, 2004.
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  Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1-2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10-15 min. RVD also occurred in strongly hypotonic 100 mOsni solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose. galactose, mositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity volume-dependent Solute permeabilities and ties. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.

  @article{Reuss2004, abstract = {Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1-2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10-15 min. RVD also occurred in strongly hypotonic 100 mOsni solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose. galactose, mositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity volume-dependent Solute permeabilities and ties. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.}, address = {233 SPRING STREET, NEW YORK, NY 10013 USA}, author = {Reuss, R and Ludwig, J and Shirakashi, R and Ehrhart, F and Zimmermann, H and Schneider, S and Weber, MM and Zimmermann, U and Schneider, H and Sukhorukov, VL}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {07}, number = 2, pages = {67-81}, publisher = {SPRINGER}, title = {Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways}, type = {Article}, volume = 200, year = 2004 }

  %0 Journal Article %1 Reuss2004 %A Reuss, R %A Ludwig, J %A Shirakashi, R %A Ehrhart, F %A Zimmermann, H %A Schneider, S %A Weber, MM %A Zimmermann, U %A Schneider, H %A Sukhorukov, VL %C 233 SPRING STREET, NEW YORK, NY 10013 USA %D 2004 %I SPRINGER %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 67-81 %T Intracellular delivery of carbohydrates into mammalian cells through swelling-activated pathways %U http://dx.doi.org/10.1007/s00232-004-0694-7 %V 200 %X Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1-2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10-15 min. RVD also occurred in strongly hypotonic 100 mOsni solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose. galactose, mositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity volume-dependent Solute permeabilities and ties. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.
• Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level. Neuweiler, H; Sauer, M. In CURRENT PHARMACEUTICAL BIOTECHNOLOGY, 5(3), S. 285–298. BENTHAM SCIENCE PUBL LTD, EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES, 2004.
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  This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

  @article{Neuweiler2004, abstract = {This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.}, address = {EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES}, author = {Neuweiler, H and Sauer, M}, journal = {CURRENT PHARMACEUTICAL BIOTECHNOLOGY}, keywords = {hannes}, month = {06}, number = 3, pages = {285-298}, publisher = {BENTHAM SCIENCE PUBL LTD}, title = {Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level}, type = {Review}, volume = 5, year = 2004 }

  %0 Journal Article %1 Neuweiler2004 %A Neuweiler, H %A Sauer, M %C EXECUTIVE STE Y26, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES %D 2004 %I BENTHAM SCIENCE PUBL LTD %J CURRENT PHARMACEUTICAL BIOTECHNOLOGY %N 3 %P 285-298 %T Using photoinduced charge transfer reactions to study conformational dynamics of biopolymers at the single-molecule level %U http://dx.doi.org/10.2174/1389201043376896 %V 5 %X This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angstroms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

• Direct observation of collective blinking and energy transfer in a bichromophoric system. Tinnefeld, P; Buschmann, V; Weston, KD; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY A, 107(3), S. 323–327. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 --> T-N --> S-1 --> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.

  @article{Tinnefeld2003, abstract = {A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 –> T-N –> S-1 –> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tinnefeld, P and Buschmann, V and Weston, KD and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {01}, number = 3, pages = {323-327}, publisher = {AMER CHEMICAL SOC}, title = {Direct observation of collective blinking and energy transfer in a bichromophoric system}, type = {Letter}, volume = 107, year = 2003 }

  %0 Journal Article %1 Tinnefeld2003 %A Tinnefeld, P %A Buschmann, V %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 3 %P 323-327 %T Direct observation of collective blinking and energy transfer in a bichromophoric system %U http://dx.doi.org/10.1021/jp026565u %V 107 %X A bichromophoric model system-a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5-embedded in poly(vinyl alcohol) (PVA) was used to investigate energy-transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T-1 --> T-N --> S-1 --> S-0) in single Cy5 molecules. We observed that even with the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet-triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.
• The power and prospects of fluorescence microscopies and spectroscopies. Michalet, X; Kapanidis, AN; Laurence, T; Pinaud, F; Doose, S; Pflughoefft, M; Weiss, S. In ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE, 32, S. 161–182. ANNUAL REVIEWS, 4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA, 2003.
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  Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

  @article{Michalet2003, abstract = {Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.}, address = {4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA}, author = {Michalet, X and Kapanidis, AN and Laurence, T and Pinaud, F and Doose, S and Pflughoefft, M and Weiss, S}, journal = {ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE}, keywords = {doose}, pages = {161-182}, publisher = {ANNUAL REVIEWS}, title = {The power and prospects of fluorescence microscopies and spectroscopies}, type = {Review}, volume = 32, year = 2003 }

  %0 Journal Article %1 Michalet2003 %A Michalet, X %A Kapanidis, AN %A Laurence, T %A Pinaud, F %A Doose, S %A Pflughoefft, M %A Weiss, S %C 4139 EL CAMINO WAY, PO BOX 10139, PALO ALTO, CA 94303-0139 USA %D 2003 %I ANNUAL REVIEWS %J ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE %P 161-182 %T The power and prospects of fluorescence microscopies and spectroscopies %U http://dx.doi.org/10.1146/annurev.biophys.32.110601.142525 %V 32 %X Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.
• Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer. Sauer, M. In ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 42(16), S. 1790–1793. WILEY-V C H VERLAG GMBH, PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY, 2003.
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  @article{Sauer2003, address = {PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY}, author = {Sauer, M}, journal = {ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, keywords = {sauer}, number = 16, pages = {1790-1793}, publisher = {WILEY-V C H VERLAG GMBH}, title = {Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer}, type = {Article}, volume = 42, year = 2003 }

  %0 Journal Article %1 Sauer2003 %A Sauer, M %C PO BOX 10 11 61, D-69451 WEINHEIM, GERMANY %D 2003 %I WILEY-V C H VERLAG GMBH %J ANGEWANDTE CHEMIE-INTERNATIONAL EDITION %N 16 %P 1790-1793 %T Single-molecule-sensitive fluorescent sensors based on photoinduced intramolecular charge transfer %U http://dx.doi.org/10.1002/anie.200201611 %V 42
• Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores. Vosch, Tom; Cotlet, Mircea; Hofkens, Johan; Biest, Koen Van Der; Lor, Marc; Weston, Kenneth; Tinnefeld, Philip; Sauer, Markus; Latterini, Loredana; Müllen, Klaus; Schryver, Frans C. De. In The Journal of Physical Chemistry A, 107(36), S. 6920–6931. 2003.
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  Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system

  @article{doi:10.1021/jp034906d, abstract = {Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system}, author = {Vosch, Tom and Cotlet, Mircea and Hofkens, Johan and Biest, Koen Van Der and Lor, Marc and Weston, Kenneth and Tinnefeld, Philip and Sauer, Markus and Latterini, Loredana and Müllen, Klaus and Schryver, Frans C. De}, journal = {The Journal of Physical Chemistry A}, keywords = {sauer}, number = 36, pages = {6920-6931}, title = {Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores}, volume = 107, year = 2003 }

  %0 Journal Article %1 doi:10.1021/jp034906d %A Vosch, Tom %A Cotlet, Mircea %A Hofkens, Johan %A Biest, Koen Van Der %A Lor, Marc %A Weston, Kenneth %A Tinnefeld, Philip %A Sauer, Markus %A Latterini, Loredana %A Müllen, Klaus %A Schryver, Frans C. De %D 2003 %J The Journal of Physical Chemistry A %N 36 %P 6920-6931 %R 10.1021/jp034906d %T Probing Förster Type Energy Pathways in a First Generation Rigid Dendrimer Bearing Two Perylene Imide Chromophores %U http://pubs.acs.org/doi/abs/10.1021/jp034906d %V 107 %X Ensemble and single molecule spectroscopic measurements on a bichromophoric dendrimer system were performed. Although Förster type energy processes such as energy hopping and singlet−singlet annihilation can be observed at the ensemble and single molecule level, only single molecule measurements can visualize the presence of singlet−triplet annihilation. The flux dependent population of the triplet state suggests facilitated formation of the triplet through a higher singlet excited state. The presence of synthesis inherent structural isomers, not to be distinguished at the ensemble level, could be demonstrated by analyzing modulated fluorescence intensity trajectories. These data show the complementarity of both the ensemble and single molecule approaches in the study of detailed photophysics of a multichromophoric system
• From linear genome sequence to three-dimensional organization of the cell nucleus. Politz, Joan; van Driel, Roel; Sauer, Markus; Pombo, Ana. In Genome Biology, 4(3), S. 1–4. BioMed Central, 2003.
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  A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.

  @article{springerlink:10.1186/gb-2003-4-3-310, abstract = {A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.}, author = {Politz, Joan and van Driel, Roel and Sauer, Markus and Pombo, Ana}, journal = {Genome Biology}, keywords = {sauer}, note = {10.1186/gb-2003-4-3-310}, pages = {1-4}, publisher = {BioMed Central}, title = {From linear genome sequence to three-dimensional organization of the cell nucleus}, volume = 4, year = 2003 }

  %0 Journal Article %1 springerlink:10.1186/gb-2003-4-3-310 %A Politz, Joan %A van Driel, Roel %A Sauer, Markus %A Pombo, Ana %D 2003 %I BioMed Central %J Genome Biology %P 1-4 %T From linear genome sequence to three-dimensional organization of the cell nucleus %U http://dx.doi.org/10.1186/gb-2003-4-3-310 %V 4 %X A report on the Jackson Laboratory 'Advances in nanostructural genomics II' meeting, Bar Harbor, USA, 3-6 October 2002.
• Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching. Vaiana, Andrea C.; Schulz, Andreas; Wolfrum, Jürgen; Sauer, Markus; Smith, Jeremy C. In Journal of Computational Chemistry, 24(5), S. 632–639. Wiley Subscription Services, Inc., A Wiley Company, 2003.
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  Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003

  @article{JCC:JCC10190, abstract = {Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003}, author = {Vaiana, Andrea C. and Schulz, Andreas and Wolfrum, Jürgen and Sauer, Markus and Smith, Jeremy C.}, journal = {Journal of Computational Chemistry}, keywords = {sauer}, number = 5, pages = {632–639}, publisher = {Wiley Subscription Services, Inc., A Wiley Company}, title = {Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching}, volume = 24, year = 2003 }

  %0 Journal Article %1 JCC:JCC10190 %A Vaiana, Andrea C. %A Schulz, Andreas %A Wolfrum, Jürgen %A Sauer, Markus %A Smith, Jeremy C. %D 2003 %I Wiley Subscription Services, Inc., A Wiley Company %J Journal of Computational Chemistry %N 5 %P 632--639 %R 10.1002/jcc.10190 %T Molecular mechanics force field parameterization of the fluorescent probe rhodamine 6G using automated frequency matching %U http://dx.doi.org/10.1002/jcc.10190 %V 24 %X Abstract Novel single-molecule fluorescence experimental techniques have prompted a growing need to develop refined computational models of dye-tagged biomolecules. As a necessary first step towards useful molecular simulations of fluorescence-labeled biomolecules, we have derived a force field for the commonly used dye, rhodamine 6G (R6G). A novel automated method is used that includes fitting the molecular mechanics potential to both vibrational frequencies and eigenvector projections derived from quantum chemical calculations. The method is benchmarked on a series of aromatic molecules then applied to derive new parameters for R6G. The force field derived reproduces well the crystal structure of R6G. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 632–639, 2003
• Excitonic behavior of rhodamine dimers: A single-molecule study. Hernando, J; van der Schaaf, M; van Dijk, EMHP; Sauer, M; Garcia-Parajo, MF; van Hulst, NF. In JOURNAL OF PHYSICAL CHEMISTRY A, 107(1), S. 43–52. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.

  @article{Hernando2003, abstract = {The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Hernando, J and van der Schaaf, M and van Dijk, EMHP and Sauer, M and Garcia-Parajo, MF and van Hulst, NF}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {01}, number = 1, pages = {43-52}, publisher = {AMER CHEMICAL SOC}, title = {Excitonic behavior of rhodamine dimers: A single-molecule study}, type = {Article}, volume = 107, year = 2003 }

  %0 Journal Article %1 Hernando2003 %A Hernando, J %A van der Schaaf, M %A van Dijk, EMHP %A Sauer, M %A Garcia-Parajo, MF %A van Hulst, NF %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 1 %P 43-52 %T Excitonic behavior of rhodamine dimers: A single-molecule study %U http://dx.doi.org/10.1021/jp0218995 %V 107 %X The optical behavior of a dimer of tetramethylrhodamine-5-isothiocyanate has been investigated by means of single-molecule measurements. Bulk absorption and fluorescence spectra show the existence of two populations of the dimer molecule that exhibit distinct excitonic interactions (strong and weak coupling). Fluorescence confocal scanning microscopy has been employed to analyze the behavior of the weakly coupled dimers at the single-molecule level. Stepwise photodamage and collective on/off behavior have been observed in real-time fluorescence trajectories of the dimer. By polarization-sensitive detection, we distinguished between two conformationally different subpopulations within the weakly interacting dimers. Correlation between the fluorescence intensity and fluorescence lifetime of the dimer recorded in time has revealed the competition between photobleaching and trap formation as photodamaging processes of the chromophores in the dimer. The results obtained demonstrate the capability of single-molecule techniques to provide detailed insight into the exciton dynamics in multichromophoric systems, which is a key point in understanding the behavior of relevant natural aggregates and, eventually, in allowing for a rational design of molecular photonic devices.
• Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Buschmann, V; Weston, KD; Sauer, M. In BIOCONJUGATE CHEMISTRY, 14(1), S. 195–204. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

  @article{Buschmann2003, abstract = {The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Buschmann, V and Weston, KD and Sauer, M}, journal = {BIOCONJUGATE CHEMISTRY}, keywords = {sauer}, month = {01}, number = 1, pages = {195-204}, publisher = {AMER CHEMICAL SOC}, title = {Spectroscopic study and evaluation of red-absorbing fluorescent dyes}, type = {Article}, volume = 14, year = 2003 }

  %0 Journal Article %1 Buschmann2003 %A Buschmann, V %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J BIOCONJUGATE CHEMISTRY %N 1 %P 195-204 %T Spectroscopic study and evaluation of red-absorbing fluorescent dyes %U http://dx.doi.org/10.1021/bc025600x %V 14 %X The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.
• Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy. Knemeyer, JP; Herten, DP; Sauer, M. In ANALYTICAL CHEMISTRY, 75(9), S. 2147–2153. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.

  @article{Knemeyer2003, abstract = {The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30\% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Knemeyer, JP and Herten, DP and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = {05}, number = 9, pages = {2147-2153}, publisher = {AMER CHEMICAL SOC}, title = {Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy}, type = {Article}, volume = 75, year = 2003 }

  %0 Journal Article %1 Knemeyer2003 %A Knemeyer, JP %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 9 %P 2147-2153 %T Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1021/ac026333r %V 75 %X The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition, rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of similar to1.3 ns and emission maximums of similar to665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(0) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.
• Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA. Adhikary, A; Buschmann, V; Muller, C; Sauer, M. In NUCLEIC ACIDS RESEARCH, 31(8), S. 2178–2186. OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2003.
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  Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.

  @article{Adhikary2003, abstract = {Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.}, address = {GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND}, author = {Adhikary, A and Buschmann, V and Muller, C and Sauer, M}, journal = {NUCLEIC ACIDS RESEARCH}, keywords = {sauer}, month = {04}, number = 8, pages = {2178-2186}, publisher = {OXFORD UNIV PRESS}, title = {Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA}, type = {Article}, volume = 31, year = 2003 }

  %0 Journal Article %1 Adhikary2003 %A Adhikary, A %A Buschmann, V %A Muller, C %A Sauer, M %C GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND %D 2003 %I OXFORD UNIV PRESS %J NUCLEIC ACIDS RESEARCH %N 8 %P 2178-2186 %T Ensemble and single-molecule fluorescence spectroscopic study of the binding modes of the bis-benzimidazole derivative Hoechst 33258 with DNA %U http://dx.doi.org/10.1093/nar/gkg308 %V 31 %X Ensemble and single-molecule fluorescence measurements of 2'-(4-hydroxyphenyl)-5-{[}5-(4-methylpiperazine-1-yl) benzimidazo-2-yl]-benzimidazole (H-258)- calf thymus (CT) DNA complexes at various {[}H-258]/{[}DNA bp] ratios were performed to elucidate the binding of H-258 with DNA. Upon binding to double-stranded CT DNA (CT ds DNA) at a {[}H-258]/{[}DNA bp] ratio of 0.05 the relative fluorescence quantum yield, Phi(f), of H-258 increases from 0.02 to 0.58. The fluorescence decay can be fitted almost by a mono-exponential model with a lifetime of similar to3.6 ns. This indicates that H-258 binds almost quantitatively in the minor groove of DNA at low {[}H-258]/{[}DNA bp] ratios. With increasing {[}H-258]/{[}DNA bp] ratios, e.g. 0.15 and 0.20, the fluorescence quantum yield of H-258 decreases to 0.28 and 0.19, respectively. Fitting of the fluorescence decays measured for higher {[}H-258]/{[}DNA bp] ratios reveals the presence of additional shorter fluorescence lifetime components in the range of 0.5-2.0 ns. Our results suggest that H-258 partially intercalates in G:C sequences at higher {[}H-258]/{[}DNA bp] ratios reflected by a lifetime component of 1.5-2 ns. In addition, stacking or adsorption of H-258 molecules on DNA occurs at higher {[}H-258]/{[}DNA bp] ratios. These molecules exhibit a short fluorescence lifetime of similar to500 ps and are more exposed to the aqueous environment. Fluorescence transients of the intensity and lifetime of single H-258 CT ds DNA demonstrate that weakly (unspecific) bound H-258 molecules exhibit a shorter fluorescence lifetime and a strongly reduced photostability.
• Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing. Schmidt, E; Kromminga, A; Kurschner, M; Zimmermann, H; Katsen, AD; Brocker, EB; Zillikens, D; Zimmermann, U; Sukhorukov, VL. In JOURNAL OF IMMUNOLOGICAL METHODS, 275(1-2), S. 179–190. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2003.
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  Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40degreesC, room temperature (RT), 4-8, -20, and -80 degreesC. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins. (C) 2003 Elsevier Science B.V. All rights reserved.

  @article{Schmidt2003, abstract = {Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40degreesC, room temperature (RT), 4-8, -20, and -80 degreesC. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins. (C) 2003 Elsevier Science B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Schmidt, E and Kromminga, A and Kurschner, M and Zimmermann, H and Katsen, AD and Brocker, EB and Zillikens, D and Zimmermann, U and Sukhorukov, VL}, journal = {JOURNAL OF IMMUNOLOGICAL METHODS}, keywords = {vladimir}, month = {04}, number = {1-2}, pages = {179-190}, publisher = {ELSEVIER SCIENCE BV}, title = {Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing}, type = {Article}, volume = 275, year = 2003 }

  %0 Journal Article %1 Schmidt2003 %A Schmidt, E %A Kromminga, A %A Kurschner, M %A Zimmermann, H %A Katsen, AD %A Brocker, EB %A Zillikens, D %A Zimmermann, U %A Sukhorukov, VL %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2003 %I ELSEVIER SCIENCE BV %J JOURNAL OF IMMUNOLOGICAL METHODS %N 1-2 %P 179-190 %T Trehalose conserves expression of bullous pemphigoid antigen 180 during desiccation and freezing %U http://dx.doi.org/10.1016/S0022-1759(03)00020-6 %V 275 %X Bullous pemphigoid antigen 180 (BP180) is targeted by autoantibodies in a variety of subepidermal blistering skin diseases. We have recently developed a simple, highly specific and sensitive immunofluorescence (IF) assay for the detection of circulating antibodies against BP180. This novel assay involves the expression of full-length (FL) BP180 in Sf21 insect cells that are then examined under IF microscopy after staining with anti-BP180 antibodies. Application of this assay as a routine diagnostic tool requires long-term storage of FL-BP180, which can result in substantial loss of expression. Here, we show that the disaccharide trehalose, a natural cryo- and lyoprotectant, is capable of preserving the FL-BP180 antigen expressed in Sf21 insect cells under various (dry) storage conditions including 40degreesC, room temperature (RT), 4-8, -20, and -80 degreesC. The protective effect was dose-dependent reaching a maximum at about 200 mM trehalose. Trehalose was superior to other sugars or conventional cryoprotective agents (e.g. sucrose, myo-inositol, DMSO) in preventing greatly reduced antigen expression. Trehalose conserved the expression of both extra- and intracellular epitopes of FL-BP180. Interestingly, protection of the intracellular domain was only observed when trehalose was introduced into the cytosol. Trehalose significantly prolonged the storage time of FL-BP180 expressed in Sf21 insect cells, thus permitting the routine use of the IF assay in clinics for the detection of serum antibodies. The method described here has potential applications for the preservation of other transmembrane proteins. (C) 2003 Elsevier Science B.V. All rights reserved.
• Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level. Neuweiler, H; Schulz, A; Bohmer, M; Enderlein, J; Sauer, M. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(18), S. 5324–5330. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.

  @article{Neuweiler2003, abstract = {We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Neuweiler, H and Schulz, A and Bohmer, M and Enderlein, J and Sauer, M}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = {05}, number = 18, pages = {5324-5330}, publisher = {AMER CHEMICAL SOC}, title = {Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Neuweiler2003 %A Neuweiler, H %A Schulz, A %A Bohmer, M %A Enderlein, J %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 18 %P 5324-5330 %T Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level %U http://dx.doi.org/10.1021/ja034040p %V 125 %X We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.
• A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer. Piestert, O; Barsch, H; Buschmann, V; Heinlein, T; Knemeyer, JP; Weston, KD; Sauer, M. In NANO LETTERS, 3(7), S. 979–982. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.

  @article{Piestert2003, abstract = {We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Piestert, O and Barsch, H and Buschmann, V and Heinlein, T and Knemeyer, JP and Weston, KD and Sauer, M}, journal = {NANO LETTERS}, keywords = {sauer}, month = {07}, number = 7, pages = {979-982}, publisher = {AMER CHEMICAL SOC}, title = {A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer}, type = {Article}, volume = 3, year = 2003 }

  %0 Journal Article %1 Piestert2003 %A Piestert, O %A Barsch, H %A Buschmann, V %A Heinlein, T %A Knemeyer, JP %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J NANO LETTERS %N 7 %P 979-982 %T A single-molecule sensitive DNA hairpin system based on intramolecular electron transfer %U http://dx.doi.org/10.1021/nl0341988 %V 3 %X We describe a method for detection of sub-picomolar concentrations of DNA or RNA sequences using novel surface-immobilized DNA hairpins. Within the DNA hairpins a fluorophore is specifically quenched by guanosine residues in the complementary stem sequence via photoinduced intramolecular electron transfer. Upon hybridization to the target sequence, fluorescence is restored due to a conformational reorganization that forces the stem apart. Proper immobilization of the DNA hairpins using blotin/streptavidin binding with minimal perturbation of the surface is required to ensure efficient quenching in the closed state.
• Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins. Heinlein, T; Knemeyer, JP; Piestert, O; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY B, 107(31), S. 7957–7964. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
  • [ Abstract ]
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  Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.

  @article{Heinlein2003, abstract = {Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heinlein, T and Knemeyer, JP and Piestert, O and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY B}, keywords = {sauer}, month = {08}, number = 31, pages = {7957-7964}, publisher = {AMER CHEMICAL SOC}, title = {Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins}, type = {Article}, volume = 107, year = 2003 }

  %0 Journal Article %1 Heinlein2003 %A Heinlein, T %A Knemeyer, JP %A Piestert, O %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY B %N 31 %P 7957-7964 %T Photoinduced electron transfer between fluorescent dyes and guanosine residues in DNA-hairpins %U http://dx.doi.org/10.1021/jp0348068 %V 107 %X Nucleobase-specific quenching interactions of fluorescent dyes can be used in singly labeled hairpin-shaped oligonucleotides to detect hybridization to specific target DNA sequences. In these DNA-hairpins, the dye is attached at the 5'-end and quenched by guanosine residues in the complementary stem. Upon hybridization, a conformational reorganization occurs reflected in an increase in fluorescence intensity. To gain a better insight into the underlying quenching mechanism, we have performed intermolecular quenching experiments with different dyes (rhodamine and oxazine derivatives) and DNA nucleotides. The bimolecular dynamic quenching rate constants k(q,dyn) of similar to1.0-2.0 x 10(9) M-1 s(-1) are relatively small for all dyes investigated though the measured decrease in fluorescence intensity indicates strong static fluorescence quenching. The data give evidence for the formation of weak or nonfluorescent (fluorescence lifetime, tau < 40 ps) ground-state complexes between the fluorophores and guanosine residues. Only within these complexes, that is, upon contact formation, efficient fluorescence quenching via electron transfer occurs. Using a model DNA-hairpin labeled at the 5'-end with the oxazine derivative MR121, we varied the position of guanosine in the complementary stem sequence to reveal the distance dependence of fluorescence quenching. Qualitatively, it is apparent that the double-stranded stem of the DNA-hairpin facilitates efficient electron transfer from the guanosine residue to MR121 with a shallow distance dependence. This result strongly supports the idea that an end-capped conformation with stacking interactions and subsequent DNA mediated electron transfer is required for efficient fluorescence quenching. Our data show that the quenching efficiency can be increased substantially by the attachment of additional overhanging single-stranded nucleotides at the 3'-end and the substitution of guanosine by stronger electron-donating nucleotides, such as 7-deazaguanosine residues. Consideration of the data obtained in this study enables the synthesis of DNA-hairpins solely quenched by guanosine residues and its analogous with a 20-fold increase in fluorescence intensity upon specific binding to the target sequence.
• Electrotransfection of anchorage-dependent mammalian cells. Muller, KJ; Horbaschek, M; Lucas, K; Zimmermann, U; Sukhorukov, VL. In EXPERIMENTAL CELL RESEARCH, 288(2), S. 344–353. ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA, 2003.
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  Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing. (C) 2003 Elsevier Science (USA). All rights reserved.

  @article{Muller2003, abstract = {Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing. (C) 2003 Elsevier Science (USA). All rights reserved.}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, author = {Muller, KJ and Horbaschek, M and Lucas, K and Zimmermann, U and Sukhorukov, VL}, journal = {EXPERIMENTAL CELL RESEARCH}, keywords = {vladimir}, month = {08}, number = 2, pages = {344-353}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, title = {Electrotransfection of anchorage-dependent mammalian cells}, type = {Article}, volume = 288, year = 2003 }

  %0 Journal Article %1 Muller2003 %A Muller, KJ %A Horbaschek, M %A Lucas, K %A Zimmermann, U %A Sukhorukov, VL %C 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA %D 2003 %I ACADEMIC PRESS INC ELSEVIER SCIENCE %J EXPERIMENTAL CELL RESEARCH %N 2 %P 344-353 %T Electrotransfection of anchorage-dependent mammalian cells %U http://dx.doi.org/10.1016/S0014-4827(03)00224-6 %V 288 %X Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing. (C) 2003 Elsevier Science (USA). All rights reserved.
• Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules. Hofkens, J; Cotlet, M; Vosch, T; Tinnefeld, P; Weston, KD; Ego, C; Grimsdale, A; Mullen, K; Beljonne, D; Bredas, JL; Jordens, S; Schweitzer, G; Sauer, M; Schryver, F De. In PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 100(23), S. 13146–13151. NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, 2003.
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  We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.

  @article{Hofkens2003, abstract = {We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.}, address = {2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA}, author = {Hofkens, J and Cotlet, M and Vosch, T and Tinnefeld, P and Weston, KD and Ego, C and Grimsdale, A and Mullen, K and Beljonne, D and Bredas, JL and Jordens, S and Schweitzer, G and Sauer, M and Schryver, F De}, journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, keywords = {sauer}, month = 11, number = 23, pages = {13146-13151}, publisher = {NATL ACAD SCIENCES}, title = {Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules}, type = {Article}, volume = 100, year = 2003 }

  %0 Journal Article %1 Hofkens2003 %A Hofkens, J %A Cotlet, M %A Vosch, T %A Tinnefeld, P %A Weston, KD %A Ego, C %A Grimsdale, A %A Mullen, K %A Beljonne, D %A Bredas, JL %A Jordens, S %A Schweitzer, G %A Sauer, M %A Schryver, F De %C 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA %D 2003 %I NATL ACAD SCIENCES %J PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA %N 23 %P 13146-13151 %T Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules %U http://dx.doi.org/10.1073/pnas.2235805100 %V 100 %X We demonstrate measurements of the efficiency of competing Forster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.
• Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation. Vaiana, AC; Neuweiler, H; Schulz, A; Wolfrum, J; Sauer, M; Smith, JC. In JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 125(47), S. 14564–14572. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.

  @article{Vaiana2003a, abstract = {Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25\% of the R6G dye is fluorescent in comparison with 10\% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Vaiana, AC and Neuweiler, H and Schulz, A and Wolfrum, J and Sauer, M and Smith, JC}, journal = {JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, keywords = {hannes}, month = 11, number = 47, pages = {14564-14572}, publisher = {AMER CHEMICAL SOC}, title = {Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation}, type = {Article}, volume = 125, year = 2003 }

  %0 Journal Article %1 Vaiana2003a %A Vaiana, AC %A Neuweiler, H %A Schulz, A %A Wolfrum, J %A Sauer, M %A Smith, JC %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J JOURNAL OF THE AMERICAN CHEMICAL SOCIETY %N 47 %P 14564-14572 %T Fluorescence quenching of dyes by tryptophan: Interactions at atomic detail from combination of experiment and computer simulation %U http://dx.doi.org/10.1021/ja036082j %V 125 %X Fluorescence spectroscopy and molecular dynamics (MD) simulation are combined to characterize the interaction of two organic fluorescent dyes, rhodamine 6G (R6G) and an oxazine derivative (MR121), with the amino acid tryptophan in aqueous solution. Steady-state and time-resolved fluorescence quenching experiments reveal the formation of essentially nonfluorescent ground-state dye/Trp complexes. The MD simulations are used to elucidate the molecular interaction geometries involved. The MD-derived probability distribution of the distance r between the centers of geometry of the dye and quencher ring systems, P(r), extends to higher distances for R6G than for MR121 due to population in the R6G/Trp system of fluorescent interaction geometries between Trp and the phenyl ring and ester group of the dye. The consequence of this is the experimental finding that under the conditions used in the simulations about 25% of the R6G dye is fluorescent in comparison with 10% of the MR121. Combining the above findings allows determination of the ``quenching distance{''}, r{*}, above which no quenching occurs. r{*} is found to be very similar (similar to5.5 Angstrom) for both dye/Trp systems, corresponding to close to van der Waals contact. Both experimental dynamic Stern-Volmer analysis and the MD trajectories demonstrate that the main determinant of the fluorescence intensity is static quenching. The approach presented is likely to be useful in the structural interpretation of data obtained from fluorescent conjugates commonly used for monitoring the binding and dynamics of biomolecular systems.
• Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. Marme, N; Knemeyer, JP; Sauer, M; Wolfrum, J. In BIOCONJUGATE CHEMISTRY, 14(6), S. 1133–1139. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2003.
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  Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.

  @article{Marme2003, abstract = {Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Marme, N and Knemeyer, JP and Sauer, M and Wolfrum, J}, journal = {BIOCONJUGATE CHEMISTRY}, keywords = {sauer}, month = 11, number = 6, pages = {1133-1139}, publisher = {AMER CHEMICAL SOC}, title = {Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan}, type = {Article}, volume = 14, year = 2003 }

  %0 Journal Article %1 Marme2003 %A Marme, N %A Knemeyer, JP %A Sauer, M %A Wolfrum, J %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J BIOCONJUGATE CHEMISTRY %N 6 %P 1133-1139 %T Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan %U http://dx.doi.org/10.1021/bc0341324 %V 14 %X Steady-state and time-resolved fluorescence measurements were performed to elucidate the fluorescence quenching of oxazine, rhodamine, carbocyanine, and bora-diaza-indacene dyes by amino acids. Among the natural amino acids, tryptophan exhibits the most pronounced quenching efficiency. Especially, the red-absorbing dyes ATTO 655, ATTO 680, and the oxazine derivative MR 121 are strongly quenched almost exclusively by tryptophan due to the formation of weak or nonfluorescent ground-state complexes with association constants, K-ass., ranging from 96 to 206 M-1. Rhodamine, fluorescein, and bora-diaza-indacene derivatives that absorb at shorter wavelengths are also quenched substantially by tyrosine residues. The quenching of carbocyanine dyes, such as Cy5, and Alexa 647 by amino acids can be almost neglected. While quenching of ATTO 655, ATTO 680, and the oxazine derivative MR121 by tryptophan is dominated by static quenching, dynamic quenching is more efficient for the two bora-diaza-indacene dyes Bodipy-FL and Bodipy630/650. Labeling of the dyes to tryptophan, tryptophan-containing peptides, and proteins (streptavidin) demonstrates that knowledge of these fluorescence quenching processes is crucial for the development of fluorescence-based diagnostic assays. Changes in the fluorescence quantum yield of dye-labeled peptides and proteins might be used advantageously for the quantification of proteases and specific binding partners.

• Cellular Uptake Studies with beta-Peptides. Rueping, Magnus; Mahajan, Yogesh; Sauer, Markus; Seebach, Dieter. In ChemBioChem, 3(2-3), S. 257–259. WILEY-VCH Verlag GmbH, 2002.
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  Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed

  @article{CBIC:CBIC257, abstract = {Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed}, author = {Rueping, Magnus and Mahajan, Yogesh and Sauer, Markus and Seebach, Dieter}, journal = {ChemBioChem}, keywords = {sauer}, number = {2-3}, pages = {257–259}, publisher = {WILEY-VCH Verlag GmbH}, title = {Cellular Uptake Studies with beta-Peptides}, volume = 3, year = 2002 }

  %0 Journal Article %1 CBIC:CBIC257 %A Rueping, Magnus %A Mahajan, Yogesh %A Sauer, Markus %A Seebach, Dieter %D 2002 %I WILEY-VCH Verlag GmbH %J ChemBioChem %N 2-3 %P 257--259 %R 10.1002/1439-7633(20020301)3:2/3<257::AID-CBIC257>3.0.CO;2-S %T Cellular Uptake Studies with beta-Peptides %U http://dx.doi.org/10.1002/1439-7633(20020301)3:2/3<257::AID-CBIC257>3.0.CO;2-S %V 3 %X Translocation across the membrane into cells is possible for certain peptides and proteins. To investigate the structural requirements of β-peptides for this process, a series of fluorescein-labeled β-peptides has been synthesized and their cellular uptake into 3T3 mouse fibroblast cells determined by fluorescence microscopy (see picture). Polycationic β-peptides have been shown to internalize into cells, and accumulation in the cytosol and nucleus is observed
• Antibunching in the Emission of a Single Tetrachromophoric Dendritic System. Tinnefeld, Philip; Weston, Kenneth D.; Vosch, Tom; Cotlet, Mircea; Weil, Tanja; Hofkens, Johan; Müllen, Klaus; Schryver, Frans C. De; Sauer, Markus. In Journal of the American Chemical Society, 124(48), S. 14310–14311. 2002.
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  The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

  @article{doi:10.1021/ja027343c, abstract = {The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.}, author = {Tinnefeld, Philip and Weston, Kenneth D. and Vosch, Tom and Cotlet, Mircea and Weil, Tanja and Hofkens, Johan and Müllen, Klaus and Schryver, Frans C. De and Sauer, Markus}, journal = {Journal of the American Chemical Society}, keywords = {sauer}, number = 48, pages = {14310-14311}, title = {Antibunching in the Emission of a Single Tetrachromophoric Dendritic System}, volume = 124, year = 2002 }

  %0 Journal Article %1 doi:10.1021/ja027343c %A Tinnefeld, Philip %A Weston, Kenneth D. %A Vosch, Tom %A Cotlet, Mircea %A Weil, Tanja %A Hofkens, Johan %A Müllen, Klaus %A Schryver, Frans C. De %A Sauer, Markus %D 2002 %J Journal of the American Chemical Society %N 48 %P 14310-14311 %R 10.1021/ja027343c %T Antibunching in the Emission of a Single Tetrachromophoric Dendritic System %U http://pubs.acs.org/doi/abs/10.1021/ja027343c %V 124 %X The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.
• Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes. Neuweiler, Hannes; Schulz, Andreas; Vaiana, Andrea C.; Smith, Jeremy C.; Kaul, Sepp; Wolfrum, Jürgen; Sauer, Markus. In Angewandte Chemie International Edition, 41(24), S. 4769–4773. WILEY-VCH Verlag, 2002.
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  Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.

  @article{ANIE:ANIE200290044, abstract = {Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.}, author = {Neuweiler, Hannes and Schulz, Andreas and Vaiana, Andrea C. and Smith, Jeremy C. and Kaul, Sepp and Wolfrum, Jürgen and Sauer, Markus}, journal = {Angewandte Chemie International Edition}, keywords = {hannes}, number = 24, pages = {4769–4773}, publisher = {WILEY-VCH Verlag}, title = {Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes}, volume = 41, year = 2002 }

  %0 Journal Article %1 ANIE:ANIE200290044 %A Neuweiler, Hannes %A Schulz, Andreas %A Vaiana, Andrea C. %A Smith, Jeremy C. %A Kaul, Sepp %A Wolfrum, Jürgen %A Sauer, Markus %D 2002 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %N 24 %P 4769--4773 %R 10.1002/anie.200290044 %T Detection of Individual p53-Autoantibodies by Using Quenched Peptide-Based Molecular Probes %U http://dx.doi.org/10.1002/anie.200290044 %V 41 %X Single-molecule spectroscopy for cancer diagnostics: Short dye-labeled peptide epitopes derived from human p53 show strong fluorescence quenching owing to efficient electron transfer from a tryptophan residue to the fluorophore (1, see picture). The specific recognition of the peptide epitope by the antibody induces a change in the conformation which is accompanied by a strong increase in fluorescence intensity. The fluorescing antibody–peptide complex can be detected at the single-molecule level even in slightly diluted serum samples by using confocal fluorescence microscopy.
• Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons. Weston, KD; Dyck, M; Tinnefeld, P; Muller, C; Herten, DP; Sauer, M. In ANALYTICAL CHEMISTRY, 74(20), S. 5342–5349. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2002.
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  A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

  @article{Weston2002, abstract = {A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Weston, KD and Dyck, M and Tinnefeld, P and Muller, C and Herten, DP and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {sauer}, month = 10, number = 20, pages = {5342-5349}, publisher = {AMER CHEMICAL SOC}, title = {Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons}, type = {Article}, volume = 74, year = 2002 }

  %0 Journal Article %1 Weston2002 %A Weston, KD %A Dyck, M %A Tinnefeld, P %A Muller, C %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2002 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 20 %P 5342-5349 %T Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons %U http://dx.doi.org/10.1021/ac025730z %V 74 %X A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.
• Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data. Dietrich, Anja; Buschmann, Volker; Müller, Christian; Sauer, Markus. In Reviews in Molecular Biotechnology, 82(3), S. 211–231. 2002.
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  We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.

  @article{Dietrich2002211, abstract = {We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.}, author = {Dietrich, Anja and Buschmann, Volker and Müller, Christian and Sauer, Markus}, journal = {Reviews in Molecular Biotechnology}, keywords = {sauer}, number = 3, pages = {211 - 231}, title = {Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data}, volume = 82, year = 2002 }

  %0 Journal Article %1 Dietrich2002211 %A Dietrich, Anja %A Buschmann, Volker %A Müller, Christian %A Sauer, Markus %D 2002 %J Reviews in Molecular Biotechnology %N 3 %P 211 - 231 %R DOI: 10.1016/S1389-0352(01)00039-3 %T Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data %U http://www.sciencedirect.com/science/article/B6VR0-44VVW8G-4/2/5fe48e1df0969426c1254afbc5bb192b %V 82 %X We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the [pi]-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.
• Intracellular delivery of trehalose into mammalian cells by electropermeabilization. Shirakashi, R; Kostner, CM; Muller, KJ; Kurschner, M; Zimmermann, U; Sukhorukov, VL. In JOURNAL OF MEMBRANE BIOLOGY, 189(1), S. 45–54. SPRINGER-VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010 USA, 2002.
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  The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mm trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mm. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

  @article{Shirakashi2002, abstract = {The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mm trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60\% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mm. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.}, address = {175 FIFTH AVE, NEW YORK, NY 10010 USA}, author = {Shirakashi, R and Kostner, CM and Muller, KJ and Kurschner, M and Zimmermann, U and Sukhorukov, VL}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {09}, number = 1, pages = {45-54}, publisher = {SPRINGER-VERLAG}, title = {Intracellular delivery of trehalose into mammalian cells by electropermeabilization}, type = {Article}, volume = 189, year = 2002 }

  %0 Journal Article %1 Shirakashi2002 %A Shirakashi, R %A Kostner, CM %A Muller, KJ %A Kurschner, M %A Zimmermann, U %A Sukhorukov, VL %C 175 FIFTH AVE, NEW YORK, NY 10010 USA %D 2002 %I SPRINGER-VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 1 %P 45-54 %T Intracellular delivery of trehalose into mammalian cells by electropermeabilization %U http://dx.doi.org/10.1007/s00232-1003-y %V 189 %X The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mm trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mm. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.
• Interaction of fluorinated lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation and dielectrophoresis. Reuss, OR; Kurschner, M; Dilsky, S; Horbaschek, M; Schenk, WA; Zimmermann, U; Sukhorukov, VL. In JOURNAL OF ELECTROSTATICS, 56(4), S. 419–434. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2002.
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  Two complementary AC electrokinetic techniques electrorotation (ROT) and dielectrophoresis (DEP) were employed for studying the interaction of the fluorinated tungsten anion {[}W(CO)(5)(SC6H4CF3)](-) and a number of its derivatives with the plasma membrane of mouse myeloma cells. The use of microstructured electrodes allowed the measurements to be performed over wide ranges of field frequency and medium conductivity. At micromolar concentrations, most of the fluorinated anions gave rise to dramatic changes in the low-frequency part of both ROT and DEP spectra of cells, indicating that these compounds acted as lipophilic anions capable of introducing large amounts of mobile charges into the plasma membrane. Application of the theoretical models linking the ROT and DEP responses yielded not only the passive electrical properties of the plasma membrane and cytosol but also the ion transport parameters, such as the surface concentration and translocation rate of the lipophilic ions dissolved in the plasma membrane. This study demonstrated that combined ROT and DEP measurements can be employed as a powerful experimental tool for the analysis of the complex relationships between the molecular structure and membrane permeability of charged lipophilic compounds. Due to their strong lipophilicity and fairly low cytotoxicity, the fluorinated tungsten compounds presented here appear to provide a promising new class of field-sensitive molecular probes for membrane structure and transport studies. (C) 2002 Elsevier Science B.V. All rights reserved.

  @article{Reuss2002, abstract = {Two complementary AC electrokinetic techniques electrorotation (ROT) and dielectrophoresis (DEP) were employed for studying the interaction of the fluorinated tungsten anion {[}W(CO)(5)(SC6H4CF3)](-) and a number of its derivatives with the plasma membrane of mouse myeloma cells. The use of microstructured electrodes allowed the measurements to be performed over wide ranges of field frequency and medium conductivity. At micromolar concentrations, most of the fluorinated anions gave rise to dramatic changes in the low-frequency part of both ROT and DEP spectra of cells, indicating that these compounds acted as lipophilic anions capable of introducing large amounts of mobile charges into the plasma membrane. Application of the theoretical models linking the ROT and DEP responses yielded not only the passive electrical properties of the plasma membrane and cytosol but also the ion transport parameters, such as the surface concentration and translocation rate of the lipophilic ions dissolved in the plasma membrane. This study demonstrated that combined ROT and DEP measurements can be employed as a powerful experimental tool for the analysis of the complex relationships between the molecular structure and membrane permeability of charged lipophilic compounds. Due to their strong lipophilicity and fairly low cytotoxicity, the fluorinated tungsten compounds presented here appear to provide a promising new class of field-sensitive molecular probes for membrane structure and transport studies. (C) 2002 Elsevier Science B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Reuss, OR and Kurschner, M and Dilsky, S and Horbaschek, M and Schenk, WA and Zimmermann, U and Sukhorukov, VL}, journal = {JOURNAL OF ELECTROSTATICS}, keywords = {vladimir}, month = 11, number = 4, pages = {419-434}, publisher = {ELSEVIER SCIENCE BV}, title = {Interaction of fluorinated lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation and dielectrophoresis}, type = {Proceedings Paper}, volume = 56, year = 2002 }

  %0 Journal Article %1 Reuss2002 %A Reuss, OR %A Kurschner, M %A Dilsky, S %A Horbaschek, M %A Schenk, WA %A Zimmermann, U %A Sukhorukov, VL %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2002 %I ELSEVIER SCIENCE BV %J JOURNAL OF ELECTROSTATICS %N 4 %P 419-434 %T Interaction of fluorinated lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation and dielectrophoresis %U http://dx.doi.org/10.1016/S0304-3886(02)00107-9 %V 56 %X Two complementary AC electrokinetic techniques electrorotation (ROT) and dielectrophoresis (DEP) were employed for studying the interaction of the fluorinated tungsten anion {[}W(CO)(5)(SC6H4CF3)](-) and a number of its derivatives with the plasma membrane of mouse myeloma cells. The use of microstructured electrodes allowed the measurements to be performed over wide ranges of field frequency and medium conductivity. At micromolar concentrations, most of the fluorinated anions gave rise to dramatic changes in the low-frequency part of both ROT and DEP spectra of cells, indicating that these compounds acted as lipophilic anions capable of introducing large amounts of mobile charges into the plasma membrane. Application of the theoretical models linking the ROT and DEP responses yielded not only the passive electrical properties of the plasma membrane and cytosol but also the ion transport parameters, such as the surface concentration and translocation rate of the lipophilic ions dissolved in the plasma membrane. This study demonstrated that combined ROT and DEP measurements can be employed as a powerful experimental tool for the analysis of the complex relationships between the molecular structure and membrane permeability of charged lipophilic compounds. Due to their strong lipophilicity and fairly low cytotoxicity, the fluorinated tungsten compounds presented here appear to provide a promising new class of field-sensitive molecular probes for membrane structure and transport studies. (C) 2002 Elsevier Science B.V. All rights reserved.
• High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Heilemann, M; Herten, DP; Heintzmann, R; Cremer, C; Muller, C; Tinnefeld, P; Weston, KD; Wolfrum, J; Sauer, M. In ANALYTICAL CHEMISTRY, 74(14), S. 3511–3517. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2002.
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  Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.

  @article{Heilemann2002, abstract = {Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Heilemann, M and Herten, DP and Heintzmann, R and Cremer, C and Muller, C and Tinnefeld, P and Weston, KD and Wolfrum, J and Sauer, M}, journal = {ANALYTICAL CHEMISTRY}, keywords = {mike}, month = {07}, number = 14, pages = {3511-3517}, publisher = {AMER CHEMICAL SOC}, title = {High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy}, type = {Article}, volume = 74, year = 2002 }

  %0 Journal Article %1 Heilemann2002 %A Heilemann, M %A Herten, DP %A Heintzmann, R %A Cremer, C %A Muller, C %A Tinnefeld, P %A Weston, KD %A Wolfrum, J %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2002 %I AMER CHEMICAL SOC %J ANALYTICAL CHEMISTRY %N 14 %P 3511-3517 %T High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1021/ac025576g %V 74 %X Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below similar to10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 us, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.

• Time-gated biological imaging by use of colloidal quantum dots. Dahan, M.; Laurence, T.; Pinaud, F.; Chemla, D. S.; Alivisatos, A. P.; Sauer, M.; Weiss, S. In Opt. Lett., 26(11), S. 825–827. OSA, 2001.
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  The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.

  @article{Dahan:01, abstract = {The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.}, author = {Dahan, M. and Laurence, T. and Pinaud, F. and Chemla, D. S. and Alivisatos, A. P. and Sauer, M. and Weiss, S.}, journal = {Opt. Lett.}, keywords = {sauer}, month = {06}, number = 11, pages = {825–827}, publisher = {OSA}, title = {Time-gated biological imaging by use of colloidal quantum dots}, volume = 26, year = 2001 }

  %0 Journal Article %1 Dahan:01 %A Dahan, M. %A Laurence, T. %A Pinaud, F. %A Chemla, D. S. %A Alivisatos, A. P. %A Sauer, M. %A Weiss, S. %D 2001 %I OSA %J Opt. Lett. %N 11 %P 825--827 %R 10.1364/OL.26.000825 %T Time-gated biological imaging by use of colloidal quantum dots %U http://ol.osa.org/abstract.cfm?URI=ol-26-11-825 %V 26 %X The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.
• Electron induced chemical nanolithography with self-assembled monolayers. Geyer, W.; Stadler, V.; Eck, W.; Golzhauser, A.; Grunze, M.; Sauer, M.; Weimann, T.; Hinze, P. In The 45th international conference on electron, ion, and photon beam technology and nanofabrication, Bd. 19, S. 2732–2735. AVS, Washington, DC (USA), 2001.
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  We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society

  @conference{geyer:2732, abstract = {We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society}, author = {Geyer, W. and Stadler, V. and Eck, W. and Golzhauser, A. and Grunze, M. and Sauer, M. and Weimann, T. and Hinze, P.}, journal = {The 45th international conference on electron, ion, and photon beam technology and nanofabrication}, keywords = {sauer}, number = 6, pages = {2732-2735}, publisher = {AVS}, title = {Electron induced chemical nanolithography with self-assembled monolayers}, volume = 19, year = 2001 }

  %0 Generic %1 geyer:2732 %A Geyer, W. %A Stadler, V. %A Eck, W. %A Golzhauser, A. %A Grunze, M. %A Sauer, M. %A Weimann, T. %A Hinze, P. %D 2001 %I AVS %J The 45th international conference on electron, ion, and photon beam technology and nanofabrication %N 6 %P 2732-2735 %R 10.1116/1.1421560 %T Electron induced chemical nanolithography with self-assembled monolayers %U http://link.aip.org/link/?JVB/19/2732/1 %V 19 %X We demonstrate a simple scheme to generate chemical surface nanostructures. Electron-beam writing is used to locally modify the terminal nitro functionality in self-assembled monolayers of 4′-nitro-1,1′-biphenyl-4-thiol to amino groups, while the underlying aromatic layer is dehydrogenated and cross linked. Using low energy electron proximity printing and conventional electron-beam lithography with a beam energy of 2.5 keV and doses from 2500 to 50 000 μC/cm2, templates of reactive amino sites with lateral dimensions down to ∼20 nm could be fabricated. The templates were used for the surface immobilization of fluorinated carboxylic acid anhydrides and rhodamine dyes. The molecular structures were then imaged and analyzed by atomic force and scanning confocal fluorescence microscopy. © 2001 American Vacuum Society
• Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting. Enderlein, Jörg; Sauer, Markus. In The Journal of Physical Chemistry A, 105(1), S. 48–53. 2001.
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  A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data

  @article{doi:10.1021/jp002358n, abstract = {A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data}, author = {Enderlein, Jörg and Sauer, Markus}, journal = {The Journal of Physical Chemistry A}, keywords = {sauer}, number = 1, pages = {48-53}, title = {Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting}, volume = 105, year = 2001 }

  %0 Journal Article %1 doi:10.1021/jp002358n %A Enderlein, Jörg %A Sauer, Markus %D 2001 %J The Journal of Physical Chemistry A %N 1 %P 48-53 %R 10.1021/jp002358n %T Optimal Algorithm for Single-Molecule Identification with Time-Correlated Single-Photon Counting %U http://pubs.acs.org/doi/abs/10.1021/jp002358n %V 105 %X A pattern-matching procedure for identifying single molecules according to the temporal decay characteristics of the fluorescence is presented and applied to measured single-molecule data of three different dyes. Based on the maximum likelihood principle, this procedure is the best possible identification algorithm if the fluorescence time decays of the different molecular species are exactly known. The presented algorithm is numerically simple and fast and, thus, especially suitable to possible online applications. Exact theoretical results for the misidentification errors of the algorithm are derived and compared with the errors determined from the experimental data
• Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects. Sauer, M.; Angerer, B.; Ankenbauer, W.; Földes-Papp, Z.; Göbel, F.; Han, K.-T.; Rigler, R.; Schulz, A.; Wolfrum, J.; Zander, C. In Journal of Biotechnology, 86(3), S. 181–201. 2001.
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  We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed ~50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

  @article{Sauer2001181, abstract = {We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed  50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.}, author = {Sauer, M. and Angerer, B. and Ankenbauer, W. and Földes-Papp, Z. and Göbel, F. and Han, K.-T. and Rigler, R. and Schulz, A. and Wolfrum, J. and Zander, C.}, journal = {Journal of Biotechnology}, keywords = {sauer}, note = {DNA-Sequencing at the Single Molecule Level}, number = 3, pages = {181 - 201}, title = {Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects}, volume = 86, year = 2001 }

  %0 Journal Article %1 Sauer2001181 %A Sauer, M. %A Angerer, B. %A Ankenbauer, W. %A Földes-Papp, Z. %A Göbel, F. %A Han, K.-T. %A Rigler, R. %A Schulz, A. %A Wolfrum, J. %A Zander, C. %D 2001 %J Journal of Biotechnology %N 3 %P 181 - 201 %R DOI: 10.1016/S0168-1656(00)00413-2 %T Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects %U http://www.sciencedirect.com/science/article/B6T3C-42JHD6H-3/2/8ed6101d2ec2e07a4cf2411397f9a399 %V 86 %X We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 [mu]m. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed ~50 [mu]m in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43±0.19 ns (Cy5-dCMP), and 2.35±0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.
• A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane. Sukhorukov, VL; Meedt, G; Kurschner, M; Zimmermann, U. In JOURNAL OF ELECTROSTATICS, 50(3), S. 191–204. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2001.
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  For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity sigma (mt). On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the held component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz-MHz range) with a marked increase of the radial membrane permittivity epsilon (mr) at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells. (C) 2001 Elsevier Science B.V. All rights reserved.

  @article{Sukhorukov2001, abstract = {For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity sigma (mt). On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the held component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz-MHz range) with a marked increase of the radial membrane permittivity epsilon (mr) at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells. (C) 2001 Elsevier Science B.V. All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Sukhorukov, VL and Meedt, G and Kurschner, M and Zimmermann, U}, journal = {JOURNAL OF ELECTROSTATICS}, keywords = {vladimir}, month = {02}, number = 3, pages = {191-204}, publisher = {ELSEVIER SCIENCE BV}, title = {A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane}, type = {Article}, volume = 50, year = 2001 }

  %0 Journal Article %1 Sukhorukov2001 %A Sukhorukov, VL %A Meedt, G %A Kurschner, M %A Zimmermann, U %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2001 %I ELSEVIER SCIENCE BV %J JOURNAL OF ELECTROSTATICS %N 3 %P 191-204 %T A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane %V 50 %X For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity sigma (mt). On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the held component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz-MHz range) with a marked increase of the radial membrane permittivity epsilon (mr) at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells. (C) 2001 Elsevier Science B.V. All rights reserved.
• Time-varying photon probability distribution of individual molecules at room temperature. Tinnefeld, P.; Müller, C.; Sauer, M. In Chemical Physics Letters, 345(3-4), S. 252–258. 2001.
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  The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time

  @article{Tinnefeld2001252, abstract = {The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time}, author = {Tinnefeld, P. and Müller, C. and Sauer, M.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {252 - 258}, title = {Time-varying photon probability distribution of individual molecules at room temperature}, volume = 345, year = 2001 }

  %0 Journal Article %1 Tinnefeld2001252 %A Tinnefeld, P. %A Müller, C. %A Sauer, M. %D 2001 %J Chemical Physics Letters %N 3-4 %P 252 - 258 %R DOI: 10.1016/S0009-2614(01)00883-1 %T Time-varying photon probability distribution of individual molecules at room temperature %U http://www.sciencedirect.com/science/article/B6TFN-43XFWP4-7/2/9500b916e6f748d7c43dbff85a2e3e4f %V 345 %X The radiation field emitted from rhodamine molecules adsorbed on a glass surface is investigated at room temperature. Transitions between Poissonian and sub-Poissonian statistics are monitored by tracking the probability of detecting photon pairs after pulsed optical excitation. Fluctuations are analyzed by recording simultaneously the inter-photon time distribution, the emission maximum, and the fluorescence lifetime with a time-resolution in the ms–s time range. The presented technique enables us to determine unequivocally whether an observed chromophoric system behaves as a single quantum emitter at any time
• Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM). Tinnefeld, P; Herten, DP; Sauer, M. In JOURNAL OF PHYSICAL CHEMISTRY A, 105(34), S. 7989–8003. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2001.
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  A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.

  @article{Tinnefeld2001, abstract = {A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15\% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Tinnefeld, P and Herten, DP and Sauer, M}, journal = {JOURNAL OF PHYSICAL CHEMISTRY A}, keywords = {sauer}, month = {08}, number = 34, pages = {7989-8003}, publisher = {AMER CHEMICAL SOC}, title = {Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM)}, type = {Article}, volume = 105, year = 2001 }

  %0 Journal Article %1 Tinnefeld2001 %A Tinnefeld, P %A Herten, DP %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2001 %I AMER CHEMICAL SOC %J JOURNAL OF PHYSICAL CHEMISTRY A %N 34 %P 7989-8003 %T Photophysical dynamics of single molecules studied by spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) %U http://dx.doi.org/10.1021/jp010365l %V 105 %X A new scanning technique for simultaneous recording of intensity, fluorescence lifetime, and spectral information with single molecule sensitivity is presented. We investigated and compared the photophysical parameters of single oxazine (JA242), rhodamine (JF9), and carbocyanine (Cy5) derivatives adsorbed on glass surfaces under air-equilibrated conditions. The obtained results demonstrate that spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) is ideally suited to reveal subpopulations in inhomogeneous samples and mixtures. To obtain a more detailed insight into the underlying fluorescence dynamics of single molecules, the fluorescence characteristics of the three different chromophores were studied positioning isolated molecules in the laser focus. Two detectors with two PC plug-in cards for time-correlated single-photon counting (TCSPC) were utilized to monitor fluorescence intensity, lifetime, and spectral information simultaneously with single-molecule sensitivity and microseconds to milliseconds time resolution. Discrete jumps in fluorescence intensity from single molecules which lacked spectral diffusion and changes in radiative lifetime have been observed with correlation times (triplet lifetimes) spanning several orders of magnitude (from 2 mus for the rhodamine derivative up to several seconds for the oxazine dye) and amplitude. For the carbocyanine derivative Cy5, fast spectral fluctuations to red-shifted dim-states which appear partly as off-states with a lifetime in the millisecond range were determined. In addition, these dim-states exhibit the same radiative decay rate of similar to2 ns as the normal on-state. Our results imply that a direct correlation between the radiative decay time and spectral fluctuations is not necessarily given in each of the three chromophores. Both parameters seem to be independent characteristic of each individual molecule. About 5-15% of all molecules independent of the dye structure, respectively, exhibited a constant emission spectrum but strong fluctuations in fluorescence lifetime directly correlated to the intensity. The results indicate that a combined analysis of emission spectrum, intensity and radiative decay rate is a valuable approach for classification and quantification of the underlying photophysical dynamics.
• Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells. Sukhorukov, VL; Kurschner, M; Dilsky, S; Lisec, T; Wagner, B; Schenk, WA; Benz, R; Zimmermann, U. In BIOPHYSICAL JOURNAL, 81(2), S. 1006–1013. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2001.
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  The adsorption of the hydrophobic anion {[}W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of {[}W(CO)SCN]- across the plasma membrane of mammalian cells. The adsorption of {[}W(CO)5CN]- was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC50 for the effect of phloretin on the transport parameters of the lipophilic ion was similar to 10 muM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.

  @article{Sukhorukov2001a, abstract = {The adsorption of the hydrophobic anion {[}W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of {[}W(CO)SCN]- across the plasma membrane of mammalian cells. The adsorption of {[}W(CO)5CN]- was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC50 for the effect of phloretin on the transport parameters of the lipophilic ion was similar to 10 muM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Sukhorukov, VL and Kurschner, M and Dilsky, S and Lisec, T and Wagner, B and Schenk, WA and Benz, R and Zimmermann, U}, journal = {BIOPHYSICAL JOURNAL}, keywords = {vladimir}, month = {08}, number = 2, pages = {1006-1013}, publisher = {BIOPHYSICAL SOCIETY}, title = {Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells}, type = {Article}, volume = 81, year = 2001 }

  %0 Journal Article %1 Sukhorukov2001a %A Sukhorukov, VL %A Kurschner, M %A Dilsky, S %A Lisec, T %A Wagner, B %A Schenk, WA %A Benz, R %A Zimmermann, U %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2001 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 2 %P 1006-1013 %T Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells %V 81 %X The adsorption of the hydrophobic anion {[}W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of {[}W(CO)SCN]- across the plasma membrane of mammalian cells. The adsorption of {[}W(CO)5CN]- was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC50 for the effect of phloretin on the transport parameters of the lipophilic ion was similar to 10 muM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.
• On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives. Rueping, M; Dietrich, A; Buschmann, V; Fritz, MG; Sauer, M; Seebach, D. In MACROMOLECULES, 34(20), S. 7042–7048. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 USA, 2001.
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  Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.

  @article{Rueping2001, abstract = {Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, author = {Rueping, M and Dietrich, A and Buschmann, V and Fritz, MG and Sauer, M and Seebach, D}, journal = {MACROMOLECULES}, keywords = {sauer}, month = {09}, number = 20, pages = {7042-7048}, publisher = {AMER CHEMICAL SOC}, title = {On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives}, type = {Article}, volume = 34, year = 2001 }

  %0 Journal Article %1 Rueping2001 %A Rueping, M %A Dietrich, A %A Buschmann, V %A Fritz, MG %A Sauer, M %A Seebach, D %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2001 %I AMER CHEMICAL SOC %J MACROMOLECULES %N 20 %P 7042-7048 %T On the structure of poly(3-hydroxybutanoic acid) in solution and in phospholipid bilayers. Circular dichroism and fluorescence spectroscopy with oligo(3-hydroxybutanoic acid) derivatives %U http://dx.doi.org/10.1021/ma010520u %V 34 %X Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 I-IB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Theta /n vs (n - 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain-on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OI-IB chain. With a calculated Forster distance R-0 of 60 Angstrom the distance (in 10(-6) M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Angstrom, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not: The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 2(1)- or a 3(1)-helical arrangement in solution.
• Trehalose improves survival of electrotransfected mammalian cells. Mussauer, H; Sukhorukov, VL; Zimmermann, U. In CYTOMETRY, 45(3), S. 161–169. WILEY-LISS, DIV JOHN WILEY & SONS INC, 605 THIRD AVE, NEW YORK, NY 10158-0012 USA, 2001.
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  Background: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. Materials and Methods: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric Pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. Results: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40 -50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. Conclusions: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency. Cytometry 45:161-169, 2001. (C) 2001 Wiley-Liss, Inc.

  @article{Mussauer2001, abstract = {Background: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. Materials and Methods: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric Pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. Results: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40 -50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. Conclusions: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency. Cytometry 45:161-169, 2001. (C) 2001 Wiley-Liss, Inc.}, address = {DIV JOHN WILEY \& SONS INC, 605 THIRD AVE, NEW YORK, NY 10158-0012 USA}, author = {Mussauer, H and Sukhorukov, VL and Zimmermann, U}, journal = {CYTOMETRY}, keywords = {vladimir}, month = 11, number = 3, pages = {161-169}, publisher = {WILEY-LISS}, title = {Trehalose improves survival of electrotransfected mammalian cells}, type = {Article}, volume = 45, year = 2001 }

  %0 Journal Article %1 Mussauer2001 %A Mussauer, H %A Sukhorukov, VL %A Zimmermann, U %C DIV JOHN WILEY & SONS INC, 605 THIRD AVE, NEW YORK, NY 10158-0012 USA %D 2001 %I WILEY-LISS %J CYTOMETRY %N 3 %P 161-169 %T Trehalose improves survival of electrotransfected mammalian cells %V 45 %X Background: Electropermeabilization is widely used for introduction of DNA and other foreign molecules into eukaryotic cells. However, conditions yielding the greatest molecule uptake and gene expression can result in low cell survival. In this study, we assessed the efficiency of trehalose for enhancing cell viability after excessive electropermeabilization. This disaccharide was chosen because of its capability of stabilizing cell membranes under various stressful conditions, such as dehydration and freezing. Materials and Methods: Various mammalian cell lines were electropermeabilized by single exponentially decaying electric Pulses of few kV/cm strength and of several-microsecond duration. Propidium iodide (PI) and a plasmid encoding green fluorescent protein (GFP), respectively, served as reporter molecules. The effects of trehalose on PI-uptake, GFP gene expression, transfection yield, and short- and long-term viability were analyzed by flow cytometry and electronic cell counting. Results: The substitution of inositol by trehalose in pulse media protected cells against field-induced cell lysis. The protection effect saturated at about 40 -50 mM trehalose. Transfection yield and gene expression were not significantly affected by trehalose. But the transfection efficiency was generally higher in the presence of trehalose, mainly because of the increased cell survival. Conclusions: We demonstrated that trehalose-substituted media are superior to standard trehalose-free pulse media for improving cell survival and achieving higher electrotransfection efficiency. Cytometry 45:161-169, 2001. (C) 2001 Wiley-Liss, Inc.
• Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration. Muller, KJ; Sukhorukov, VL; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 184(2), S. 161–170. SPRINGER-VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010 USA, 2001.
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  Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to similar to 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrode-formation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigma (e) is smaller than that of the cytosol sigma (i). The stretching force vanishes when sigma (e) is equal to or larger than sigma (i). Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).

  @article{Muller2001, abstract = {Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to similar to 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrode-formation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigma (e) is smaller than that of the cytosol sigma (i). The stretching force vanishes when sigma (e) is equal to or larger than sigma (i). Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).}, address = {175 FIFTH AVE, NEW YORK, NY 10010 USA}, author = {Muller, KJ and Sukhorukov, VL and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = 11, number = 2, pages = {161-170}, publisher = {SPRINGER-VERLAG}, title = {Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration}, type = {Article}, volume = 184, year = 2001 }

  %0 Journal Article %1 Muller2001 %A Muller, KJ %A Sukhorukov, VL %A Zimmermann, U %C 175 FIFTH AVE, NEW YORK, NY 10010 USA %D 2001 %I SPRINGER-VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 161-170 %T Reversible electropermeabilization of mammalian cells by high-intensity, ultra-short pulses of submicrosecond duration %V 184 %X Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to similar to 150 kV/cm and durations of 10-100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrode-formation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity sigma (e) is smaller than that of the cytosol sigma (i). The stretching force vanishes when sigma (e) is equal to or larger than sigma (i). Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).
• Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation. Kriegmaier, M; Zimmermann, M; Wolf, K; Zimmermann, U; Sukhorukov, VL. In BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1568(2), S. 135–146. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 2001.
  • [ Abstract ]
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  Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear Field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions. (C) 2001 Elsevier Science B.V, All rights reserved.

  @article{Kriegmaier2001, abstract = {Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear Field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions. (C) 2001 Elsevier Science B.V, All rights reserved.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Kriegmaier, M and Zimmermann, M and Wolf, K and Zimmermann, U and Sukhorukov, VL}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS}, keywords = {vladimir}, month = 12, number = 2, pages = {135-146}, publisher = {ELSEVIER SCIENCE BV}, title = {Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation}, type = {Article}, volume = 1568, year = 2001 }

  %0 Journal Article %1 Kriegmaier2001 %A Kriegmaier, M %A Zimmermann, M %A Wolf, K %A Zimmermann, U %A Sukhorukov, VL %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 2001 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS %N 2 %P 135-146 %T Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation %V 1568 %X Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear Field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions. (C) 2001 Elsevier Science B.V, All rights reserved.

• Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells. Kurschner, M; Nielsen, K; von Langen, JRG; Schenk, WA; Zimmermann, U; Sukhorukov, VL. In BIOPHYSICAL JOURNAL, 79(3), S. 1490–1497. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 2000.
  • [ Abstract ]
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  The effects of the anionic tungsten carbonyl complex {[}W(CO)(5)SC6H5](-) and its fluorinated analog {[}W(CO)(5)SC6F5](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion.

  @article{Kurschner2000, abstract = {The effects of the anionic tungsten carbonyl complex {[}W(CO)(5)SC6H5](-) and its fluorinated analog {[}W(CO)(5)SC6F5](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Kurschner, M and Nielsen, K and von Langen, JRG and Schenk, WA and Zimmermann, U and Sukhorukov, VL}, journal = {BIOPHYSICAL JOURNAL}, keywords = {vladimir}, month = {09}, number = 3, pages = {1490-1497}, publisher = {BIOPHYSICAL SOCIETY}, title = {Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells}, type = {Article}, volume = 79, year = 2000 }

  %0 Journal Article %1 Kurschner2000 %A Kurschner, M %A Nielsen, K %A von Langen, JRG %A Schenk, WA %A Zimmermann, U %A Sukhorukov, VL %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 2000 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 3 %P 1490-1497 %T Effect of fluorine substitution on the interaction of lipophilic ions with the plasma membrane of mammalian cells %V 79 %X The effects of the anionic tungsten carbonyl complex {[}W(CO)(5)SC6H5](-) and its fluorinated analog {[}W(CO)(5)SC6F5](-) on the electrical properties of the plasma membrane of mouse myeloma cells were studied by the single-cell electrorotation technique. At micromolar concentrations, both compounds gave rise to an additional antifield peak in the rotational spectra of cells, indicating that the plasma membrane displayed a strong dielectric dispersion. This means that both tungsten derivatives act as lipophilic ions that are able to introduce large amounts of mobile charges into the plasma membrane. The analysis of the rotational spectra allowed the evaluation not only of the passive electric properties of the plasma membrane and cytoplasm, but also of the ion transport parameters, such as the surface concentration, partition coefficient, and translocation rate constant of the lipophilic anions dissolved in the plasma membrane. Comparison of the membrane transport parameters for the two anions showed that the fluorine-substituted analog was more lipophilic, but its translocation across the plasma membrane was slower by at least one order of magnitude than that of the parent hydrogenated anion.
• Electromanipulation of mammalian cells: Fundamentals and application. Zimmermann, U; Friedrich, U; Mussauer, H; Gessner, P; Hamel, K; Sukhoruhov, V. In IEEE TRANSACTIONS ON PLASMA SCIENCE, 28(1), S. 72–82. IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC, 345 E 47TH ST, NEW YORK, NY 10017-2394 USA, 2000.
  • [ Abstract ]
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  Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian tells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of tells, Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permeabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductivity pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed about 100 mu s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivity. Such media are also beneficial because field-induced apoptosis does not occur under these conditions tin contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: I) to high incorporation rates of plasmids CDNA) or artificial chromosomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising agents for cancer treatment, as shown by first clinical trials.

  @article{Zimmermann2000, abstract = {Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian tells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of tells, Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permeabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductivity pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed about 100 mu s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivity. Such media are also beneficial because field-induced apoptosis does not occur under these conditions tin contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: I) to high incorporation rates of plasmids CDNA) or artificial chromosomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising agents for cancer treatment, as shown by first clinical trials.}, address = {345 E 47TH ST, NEW YORK, NY 10017-2394 USA}, author = {Zimmermann, U and Friedrich, U and Mussauer, H and Gessner, P and Hamel, K and Sukhoruhov, V}, journal = {IEEE TRANSACTIONS ON PLASMA SCIENCE}, keywords = {vladimir}, month = {02}, number = 1, pages = {72-82}, publisher = {IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC}, title = {Electromanipulation of mammalian cells: Fundamentals and application}, type = {Proceedings Paper}, volume = 28, year = 2000 }

  %0 Journal Article %1 Zimmermann2000 %A Zimmermann, U %A Friedrich, U %A Mussauer, H %A Gessner, P %A Hamel, K %A Sukhoruhov, V %C 345 E 47TH ST, NEW YORK, NY 10017-2394 USA %D 2000 %I IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC %J IEEE TRANSACTIONS ON PLASMA SCIENCE %N 1 %P 72-82 %T Electromanipulation of mammalian cells: Fundamentals and application %V 28 %X Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian tells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of tells, Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permeabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermeabilization in response to a breakdown pulse, Transient stretching force assumes a maximum value in low-conductivity pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible yields of(genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed about 100 mu s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivity. Such media are also beneficial because field-induced apoptosis does not occur under these conditions tin contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: I) to high incorporation rates of plasmids CDNA) or artificial chromosomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising agents for cancer treatment, as shown by first clinical trials.
• Probes for Detection of Specific DNA Sequences at the Single-Molecule Level. Knemeyer, Jens-Peter; Marmé, Nicole; Sauer, Markus. In Analytical Chemistry, 72(16), S. 3717–3724. 2000.
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  A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules

  @article{doi:10.1021/ac000024o, abstract = {A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules}, author = {Knemeyer, Jens-Peter and Marmé, Nicole and Sauer, Markus}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 16, pages = {3717-3724}, title = {Probes for Detection of Specific DNA Sequences at the Single-Molecule Level}, volume = 72, year = 2000 }

  %0 Journal Article %1 doi:10.1021/ac000024o %A Knemeyer, Jens-Peter %A Marmé, Nicole %A Sauer, Markus %D 2000 %J Analytical Chemistry %N 16 %P 3717-3724 %R 10.1021/ac000024o %T Probes for Detection of Specific DNA Sequences at the Single-Molecule Level %U http://pubs.acs.org/doi/abs/10.1021/ac000024o %V 72 %X A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe−target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules
• Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting. Herten, D.P.; Tinnefeld, P.; Sauer, M. In Applied Physics B: Lasers and Optics, 71(5), S. 765–771. Springer Berlin / Heidelberg, 2000.
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  We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.

  @article{springerlink:10.1007/s003400000405, abstract = {We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.}, author = {Herten, D.P. and Tinnefeld, P. and Sauer, M.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400000405}, pages = {765-771}, publisher = {Springer Berlin / Heidelberg}, title = {Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting}, volume = 71, year = 2000 }

  %0 Journal Article %1 springerlink:10.1007/s003400000405 %A Herten, D.P. %A Tinnefeld, P. %A Sauer, M. %D 2000 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 765-771 %T Identification of single fluorescently labelled mononucleotide molecules in solution by spectrally resolved time-correlated single-photon counting %U http://dx.doi.org/10.1007/s003400000405 %V 71 %X We describe a method to identify single dye-labelled mononucleotide molecules in solution with high classification probability based on confocal microscopy in combination with spectrally and time-resolved fluorescence detection with two detectors. For efficient excitation of the labelled mononucleotide molecules JA133-dUTP, JA169-dUTP, Cy5-dCTP, and JA242-dUTP a short-pulse diode laser emitting at 634 nm with a repetition rate of 64 MHz was applied. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). Scatter plots of spectrally and time-resolved fluorescence data demonstrated the existence of four distinct populations with symmetrical shape. The distributions of each of the mononucleotide conjugates were determined by fitting a superposition of two independent Gaussians. Taking only those single-molecule bursts which contain more than 50 photon counts, three labelled mononucleotide molecules were identified in solution by spectrally and time-resolved fluorescence spectroscopy with a probability of correct classification of ≈99%.
• 7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain. Seela, Frank; Zulauf, Matthias; Sauer, Markus; Deimel, Michael. In Helvetica Chimica Acta, 83(5), S. 910–927. Verlag Helvetica Chimica Acta, 2000.
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  7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).

  @article{HLCA:HLCA910, abstract = {7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).}, author = {Seela, Frank and Zulauf, Matthias and Sauer, Markus and Deimel, Michael}, journal = {Helvetica Chimica Acta}, keywords = {sauer}, number = 5, pages = {910–927}, publisher = {Verlag Helvetica Chimica Acta}, title = {7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain}, volume = 83, year = 2000 }

  %0 Journal Article %1 HLCA:HLCA910 %A Seela, Frank %A Zulauf, Matthias %A Sauer, Markus %A Deimel, Michael %D 2000 %I Verlag Helvetica Chimica Acta %J Helvetica Chimica Acta %N 5 %P 910--927 %R 10.1002/(SICI)1522-2675(20000510)83:5<910::AID-HLCA910>3.0.CO;2-4 %T 7-Substituted 7-Deaza-2′-deoxyadenosines and 8-Aza-7-deaza-2′-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain %U http://dx.doi.org/10.1002/(SICI)1522-2675(20000510)83:5<910::AID-HLCA910>3.0.CO;2-4 %V 83 %X 7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).
• Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments. Neumann, M.; Herten, D.-P.; Dietrich, A.; Wolfrum, J.; Sauer, M. In Journal of Chromatography A, 871(1-2), S. 299–310. 2000.
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  The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

  @article{Neumann2000299, abstract = {The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.}, author = {Neumann, M. and Herten, D.-P. and Dietrich, A. and Wolfrum, J. and Sauer, M.}, journal = {Journal of Chromatography A}, keywords = {sauer}, number = {1-2}, pages = {299 - 310}, title = {Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments}, volume = 871, year = 2000 }

  %0 Journal Article %1 Neumann2000299 %A Neumann, M. %A Herten, D.-P. %A Dietrich, A. %A Wolfrum, J. %A Sauer, M. %D 2000 %J Journal of Chromatography A %N 1-2 %P 299 - 310 %R DOI: 10.1016/S0021-9673(99)00909-7 %T Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments %U http://www.sciencedirect.com/science/article/B6TG8-3YMF6PM-14/2/5e863581c92280fc7d00c7225fc50baa %V 871 %X The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.
• Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes. Irngartinger, Hermann; Fettel, Peter Walter; Escher, Thomas; Tinnefeld, Philip; Nord, Simon; Sauer, Markus. In European Journal of Organic Chemistry, 2000(3), S. 455–465. WILEY-VCH Verlag GmbH, 2000.
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  The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.

  @article{EJOC:EJOC455, abstract = {The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.}, author = {Irngartinger, Hermann and Fettel, Peter Walter and Escher, Thomas and Tinnefeld, Philip and Nord, Simon and Sauer, Markus}, journal = {European Journal of Organic Chemistry}, keywords = {sauer}, number = 3, pages = {455–465}, publisher = {WILEY-VCH Verlag GmbH}, title = {Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes}, volume = 2000, year = 2000 }

  %0 Journal Article %1 EJOC:EJOC455 %A Irngartinger, Hermann %A Fettel, Peter Walter %A Escher, Thomas %A Tinnefeld, Philip %A Nord, Simon %A Sauer, Markus %D 2000 %I WILEY-VCH Verlag GmbH %J European Journal of Organic Chemistry %N 3 %P 455--465 %R 10.1002/(SICI)1099-0690(200002)2000:3<455::AID-EJOC455>3.0.CO;2-N %T Substituent Effects on Redox Properties and Photoinduced Electron Transfer in Isoxazolo-Fullerenes %U http://dx.doi.org/10.1002/(SICI)1099-0690(200002)2000:3<455::AID-EJOC455>3.0.CO;2-N %V 2000 %X The new C60 and C70 adducts 1b, 1d-1k, 1m, 6d, 7d, and 8d have been synthesized by [2+3] cycloadditions of the appropriate nitrile oxides. Variations in the distance and geometry of the donor and acceptor substituents are seen to have an influence on the redox behavior of the fullerene adducts in cyclic voltammetry experiments. The isoxazolo-fullerenes 1c, 1d, and 1i show shifts of about 30 mV or 40 mV to more negative values compared with the reference compound 1a. On the other hand, strong acceptor properties are detected in the case of compound 1e, which shows a positive shift of 30 mV relative to 1a. Moreover, time-resolved fluorescence spectroscopy has shown that upon excitation of the fullerene moiety in the polar solvent benzonitrile, an electron is transferred from the donor substituent to the first excited singlet state of the fullerene, thereby reducing the excited-state lifetime. Our data demonstrate that the electron-transfer rate in donor-substituted fullerenes can be controlled by the electron-donating property of the substituent as well as the electronic structure and/or length of the spacer used. The C70 regioisomers 6d, 7d, and 8d exhibit differences in their spectroscopic characteristics.
• Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level. Tinnefeld, Philip; Buschmann, Volker; Herten, Dirk-Peter; Han, Kyung-Tae; Sauer, Markus. In Single Molecules, 1(3), S. 215–223. WILEY-VCH Verlag Berlin GmbH, 2000.
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  We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

  @article{SIMO:SIMO215, abstract = {We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of  0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.}, author = {Tinnefeld, Philip and Buschmann, Volker and Herten, Dirk-Peter and Han, Kyung-Tae and Sauer, Markus}, journal = {Single Molecules}, keywords = {sauer}, number = 3, pages = {215–223}, publisher = {WILEY-VCH Verlag Berlin GmbH}, title = {Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level}, volume = 1, year = 2000 }

  %0 Journal Article %1 SIMO:SIMO215 %A Tinnefeld, Philip %A Buschmann, Volker %A Herten, Dirk-Peter %A Han, Kyung-Tae %A Sauer, Markus %D 2000 %I WILEY-VCH Verlag Berlin GmbH %J Single Molecules %N 3 %P 215--223 %R 10.1002/1438-5171(200009)1:3<215::AID-SIMO215>3.0.CO;2-S %T Confocal Fluorescence Lifetime Imaging Microscopy (FLIM) at the Single Molecule Level %U http://dx.doi.org/10.1002/1438-5171(200009)1:3<215::AID-SIMO215>3.0.CO;2-S %V 1 %X We report on confocal fluorescence lifetime imaging microscopy (CFLIM) of single dye molecules adsorbed on glass surface. Applying a short-pulse diode laser emitting at 635 nm with a repetition rate of 64 MHz we studied the time-resolved identification of individual carbocyanine and oxazine dyes via their characteristic fluorescence lifetimes of 2.06±0.37 ns (Cy5) and 3.89±0.91 ns (JA242). Fluctuations in fluorescence intensity and lifetime of individual adsorbed molecules were investigated with millisecond time resolution. These jumps exhibit short off-states toff of 0.5 ms which can be ascribed to the triplet state lifetime under dry conditions with an intersystem crossing yield YISC of ~0.2 %. Besides triplet states, other quantum jumps into longer lived states (several milliseconds) with lower transition probability were observed. The correlation of rotational and spectral jumps of single and coupled fluorophores with changes in the observed fluorescence lifetime are discussed.

• Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures. Neumann, M.; Ehrfeld, W.; Han, K.-T.; Jacob, P.; Konrad, R.; Siebert, S.; Wolfrum, J.; Sauer, M. In Biomedical Chromatography, 13(2), S. 109–110. John Wiley & Sons, Ltd., 1999.
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  @article{BMC:BMC854, author = {Neumann, M. and Ehrfeld, W. and Han, K.-T. and Jacob, P. and Konrad, R. and Siebert, S. and Wolfrum, J. and Sauer, M.}, journal = {Biomedical Chromatography}, keywords = {sauer}, number = 2, pages = {109–110}, publisher = {John Wiley & Sons, Ltd.}, title = {Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures}, volume = 13, year = 1999 }

  %0 Journal Article %1 BMC:BMC854 %A Neumann, M. %A Ehrfeld, W. %A Han, K.-T. %A Jacob, P. %A Konrad, R. %A Siebert, S. %A Wolfrum, J. %A Sauer, M. %D 1999 %I John Wiley & Sons, Ltd. %J Biomedical Chromatography %N 2 %P 109--110 %R 10.1002/(SICI)1099-0801(199904)13:2<109::AID-BMC854>3.0.CO;2-R %T Electrophoretic sizing of DNA fragments and detection of single mononucleotide molecules in microstructures %U http://dx.doi.org/10.1002/(SICI)1099-0801(199904)13:2<109::AID-BMC854>3.0.CO;2-R %V 13
• Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique. Sauer, M; Angerer, B; Han, K-T; Zander, C. In Phys. Chem. Chem. Phys., 1(10), S. 2471–2477. The Royal Society of Chemistry, 1999.
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  We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.

  @article{sauer1999detection, abstract = {We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.}, author = {Sauer, M and Angerer, B and Han, K-T and Zander, C}, journal = {Phys. Chem. Chem. Phys.}, keywords = {sauer}, number = 10, pages = {2471–2477}, publisher = {The Royal Society of Chemistry}, title = {Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique}, volume = 1, year = 1999 }

  %0 Journal Article %1 sauer1999detection %A Sauer, M %A Angerer, B %A Han, K-T %A Zander, C %D 1999 %I The Royal Society of Chemistry %J Phys. Chem. Chem. Phys. %N 10 %P 2471--2477 %R 10.1039/A901411J %T Detection and identification of single dye labeled mononucleotide molecules released from an optical fiber in a microcapillary: First steps towards a new single molecule DNA sequencing technique %U http://dx.doi.org/10.1039/A901411J %V 1 %X We present efficient detection of single dye labeled mononucleotide molecules released from an optical fiber in a cone shaped microcapillary with an inner diameter of 0.5[small micro]m at the small end. A 3[small micro]m diameter streptavidin coated optical fiber was doped with dye labeled mononucleotide molecules and positioned about 400[small micro]m in front of the detection area. The flow of the negatively charged analyte molecules towards the detection area was established by electrokinetic forces. Adsorption of the dye labeled mononucleotide molecules on the capillary walls as well as the electroosmotic (EOF) flow could be drastically reduced using a 3% polyvinylpyrrolidone (PVP) matrix. We studied bursts of fluorescence photons from single fluorescently labeled mononucleotide molecules Cy5-dCTP and MR121-dUTP. The conjugates were excited by a short-pulse diode laser emitting at a wavelength of 638 nm with a repetition rate of 57 MHz. The setup consists essentially of a confocal microscope for time-correlated single-photon counting (TCSPC). Detection was performed by a single-photon avalanche-photodiode in combination with a PC plug-in card for TCSPC. The time-resolved fluorescence signals of individual molecules were analyzed and identified by a maximum likelihood estimator (MLE). We obtain an overall classification probability of about 96% for the time-resolved identification of Cy5-dCTP and MR121-dUTP their characteristic fluorescence decay times of 1.42+/-0.21 ns (Cy5-dCTP) and 2.41+/-0.32 ns (MR121-dUTP). In addition we report on detection and identification of single Cy5-dCMP and MR121-dUMP molecules degraded enzymatically with the 3[prime or minute][rightward arrow]5[prime or minute] proofreading activity of T4-DNA-polymerase from statistically labeled DNA strands bound to the optical fiber. The technique presented permits counting and identification of fluorescently labeled mononucleotide molecules cleaved from DNA strands with counting rates>200 nucleotides s and represents a suitable alternative for single molecule sequencing.
• Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC). Becker, W.; Hickl, H.; Zander, C.; Drexhage, K. H.; Sauer, M.; Siebert, S.; Wolfrum, J. In Review of Scientific Instruments, 70(3), S. 1835–1841. American Institute of Physics, 1999.
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  A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.

  @article{becker1999timeresolved, abstract = {A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.}, author = {Becker, W. and Hickl, H. and Zander, C. and Drexhage, K. H. and Sauer, M. and Siebert, S. and Wolfrum, J.}, booktitle = {Review of Scientific Instruments}, journal = {Review of Scientific Instruments}, keywords = {sauer}, month = {03}, number = 3, pages = {1835–1841}, publisher = {American Institute of Physics}, title = {Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)}, volume = 70, year = 1999 }

  %0 Journal Article %1 becker1999timeresolved %A Becker, W. %A Hickl, H. %A Zander, C. %A Drexhage, K. H. %A Sauer, M. %A Siebert, S. %A Wolfrum, J. %B Review of Scientific Instruments %D 1999 %I American Institute of Physics %J Review of Scientific Instruments %N 3 %P 1835--1841 %R 10.1063/1.1149677 %T Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC) %U https://doi.org/10.1063/1.1149677 %V 70 %X A PC plug-in card for on-line time resolved fluorescence detection of single dye molecules based on a new time-correlated single photon counting (TCSPC) module is described. The module contains all electronic components constant fraction discriminators (CFDs), time-to-amplitude converter (TAC), analog-to-digital converter (ADC), multichannel analyzer (MCA timers) on board required for TCSPC. A fast TAC design in combination with a fast flash ADC and an error-correcting ADC/MCA principle results in a maximum count rate of 8 MHz (dead time 125 ns). A dual memory architecture allows for unlimited recording of decay curves with collection times down to 150 μs without time gaps between subsequent recordings. Applying a short-pulse diode laser emitting at 640 nm with a repetition rate of 60 MHz in combination with a confocal microscope, we studied bursts of fluorescence photons from individual dye labeled mononucleotide molecules (Cy5-dCTP) in a cone shaped microcapillary with an inner diameter of 0.5 μm at the end of the tip. The flow of the conjugates was controlled by electrokinetic forces. The presented technique permits the counting and identification of all labeled analyte molecules present in a given sample due to their characteristic velocities, burst sizes, and fluorescence decay times.
• Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents. Mussauer, H; Sukhorukov, VL; Haase, A; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 170(2), S. 121–133. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010 USA, 1999.
  • [ Abstract ]
  • [ BibTeX ]
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  The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca2+ sequestration) in the Vicinity of the cell membrane.

  @article{Mussauer1999, abstract = {The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca2+ sequestration) in the Vicinity of the cell membrane.}, address = {175 FIFTH AVE, NEW YORK, NY 10010 USA}, author = {Mussauer, H and Sukhorukov, VL and Haase, A and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {07}, number = 2, pages = {121-133}, publisher = {SPRINGER VERLAG}, title = {Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents}, type = {Article}, volume = 170, year = 1999 }

  %0 Journal Article %1 Mussauer1999 %A Mussauer, H %A Sukhorukov, VL %A Haase, A %A Zimmermann, U %C 175 FIFTH AVE, NEW YORK, NY 10010 USA %D 1999 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 121-133 %T Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents %V 170 %X The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca2+ sequestration) in the Vicinity of the cell membrane.
• Dielectric properties of alginate beads and bound water relaxation studied by electrorotation. Esch, M; Sukhorukov, VL; Kurschner, M; Zimmermann, U. In BIOPOLYMERS, 50(3), S. 227–237. JOHN WILEY & SONS INC, 605 THIRD AVE, NEW YORK, NY 10158-0012 USA, 1999.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  The electrical and dielectric properties of Ba2+ and Ca2+ cross-linked alginate hydrogel beads were studied by means of single-particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well-resolved co- and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. (C) 1999 John Wiley & Sons, Inc.

  @article{Esch1999, abstract = {The electrical and dielectric properties of Ba2+ and Ca2+ cross-linked alginate hydrogel beads were studied by means of single-particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well-resolved co- and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. (C) 1999 John Wiley \& Sons, Inc.}, address = {605 THIRD AVE, NEW YORK, NY 10158-0012 USA}, author = {Esch, M and Sukhorukov, VL and Kurschner, M and Zimmermann, U}, journal = {BIOPOLYMERS}, keywords = {vladimir}, month = {09}, number = 3, pages = {227-237}, publisher = {JOHN WILEY \& SONS INC}, title = {Dielectric properties of alginate beads and bound water relaxation studied by electrorotation}, type = {Article}, volume = 50, year = 1999 }

  %0 Journal Article %1 Esch1999 %A Esch, M %A Sukhorukov, VL %A Kurschner, M %A Zimmermann, U %C 605 THIRD AVE, NEW YORK, NY 10158-0012 USA %D 1999 %I JOHN WILEY & SONS INC %J BIOPOLYMERS %N 3 %P 227-237 %T Dielectric properties of alginate beads and bound water relaxation studied by electrorotation %V 50 %X The electrical and dielectric properties of Ba2+ and Ca2+ cross-linked alginate hydrogel beads were studied by means of single-particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well-resolved co- and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. (C) 1999 John Wiley & Sons, Inc.

• The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low- and high-conductivity media. Sukhorukov, VL; Mussauer, H; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 163(3), S. 235–245. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010 USA, 1998.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks{''} in the plasma membrane generated by electric breakdown.

  @article{Sukhorukov1998, abstract = {Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks{''} in the plasma membrane generated by electric breakdown.}, address = {175 FIFTH AVE, NEW YORK, NY 10010 USA}, author = {Sukhorukov, VL and Mussauer, H and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {06}, number = 3, pages = {235-245}, publisher = {SPRINGER VERLAG}, title = {The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low- and high-conductivity media}, type = {Article}, volume = 163, year = 1998 }

  %0 Journal Article %1 Sukhorukov1998 %A Sukhorukov, VL %A Mussauer, H %A Zimmermann, U %C 175 FIFTH AVE, NEW YORK, NY 10010 USA %D 1998 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 3 %P 235-245 %T The effect of electrical deformation forces on the electropermeabilization of erythrocyte membranes in low- and high-conductivity media %V 163 %X Electrical breakdown of erythrocytes induces hemoglobin release which increases markedly with decreasing conductivity of the pulse medium. This effect presumably results from the transient, conductivity-dependent deformation forces (elongation or compression) on the cell caused by Maxwell stress. The deformation force is exerted on the plasma membrane of the cell, which can be viewed as a transient dipole induced by an applied DC electric field pulse. The induced dipole arises from the free charges that accumulate at the cell interfaces via the Maxwell-Wagner polarization mechanism. The polarization response of erythrocytes to a DC field pulse was estimated from the experimental data obtained by using two complementary frequency-domain techniques. The response is very rapid, due to the highly conductive cytosol. Measurements of the electrorotation and electrodeformation spectra over a wide conductivity range yielded the information and data required for the calculation of the deformation force as a function of frequency and external conductivity and for the calculation of the transient development of the deformation forces during the application of a DC-field pulse. These calculations showed that (i) electric force precedes and accompanies membrane charging (up to the breakdown voltage) and (ii) that under low-conductivity conditions, the electric stretching force contributes significantly to the enlargement of ``electroleaks{''} in the plasma membrane generated by electric breakdown.
• Interaction of lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation. Kurschner, M; Nielsen, K; Andersen, C; Sukhorukov, VL; Schenk, WA; Benz, R; Zimmermann, U. In BIOPHYSICAL JOURNAL, 74(6), S. 3031–3043. BIOPHYSICAL SOCIETY, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA, 1998.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as {[}W(CO)(5)(CN)](-), {[}W(CO)(5)(NCS)](-), {[}W-2(CO)(10)(CN)](-), and {[}W(CO)(5)(SCH2C6H5)](-), using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.

  @article{Kurschner1998, abstract = {The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as {[}W(CO)(5)(CN)](-), {[}W(CO)(5)(NCS)](-), {[}W-2(CO)(10)(CN)](-), and {[}W(CO)(5)(SCH2C6H5)](-), using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA}, author = {Kurschner, M and Nielsen, K and Andersen, C and Sukhorukov, VL and Schenk, WA and Benz, R and Zimmermann, U}, journal = {BIOPHYSICAL JOURNAL}, keywords = {vladimir}, month = {06}, number = 6, pages = {3031-3043}, publisher = {BIOPHYSICAL SOCIETY}, title = {Interaction of lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation}, type = {Article}, volume = 74, year = 1998 }

  %0 Journal Article %1 Kurschner1998 %A Kurschner, M %A Nielsen, K %A Andersen, C %A Sukhorukov, VL %A Schenk, WA %A Benz, R %A Zimmermann, U %C 9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3998 USA %D 1998 %I BIOPHYSICAL SOCIETY %J BIOPHYSICAL JOURNAL %N 6 %P 3031-3043 %T Interaction of lipophilic ions with the plasma membrane of mammalian cells studied by electrorotation %V 74 %X The electrical properties of biological and artificial membranes were studied in the presence of a number of negatively charged tungsten carbonyl complexes, such as {[}W(CO)(5)(CN)](-), {[}W(CO)(5)(NCS)](-), {[}W-2(CO)(10)(CN)](-), and {[}W(CO)(5)(SCH2C6H5)](-), using the single-cell electrorotation and the charge-pulse relaxation techniques. Most of the negatively charged tungsten complexes were able to introduce mobile charges into the membranes, as judged from electrorotation spectra and relaxation experiments. This means that the tungsten derivatives act as lipophilic anions. They greatly contributed to the polarizability of the membranes and led to a marked dielectric dispersion (frequency dependence of the membrane capacitance and conductance). The increment and characteristic frequency of the dispersion reflect the structure, environment, and mobility of the charged probe molecule in electrorotation experiments with biological membranes. The partition coefficients and the translocation rate constants derived from the electrorotation spectra of cells agreed well with the corresponding data obtained from charge-pulse experiments on artificial lipid bilayers.
• Electrorotation of isolated generative and vegetative cells, and of intact pollen grains of Lilium longiflorum. Sukhorukov, VL; Benkert, R; Obermeyer, G; Bentrup, FW; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 161(1), S. 21–32. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010 USA, 1998.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m(-1). The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.

  @article{Sukhorukov1998a, abstract = {The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m(-1). The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.}, address = {175 FIFTH AVE, NEW YORK, NY 10010 USA}, author = {Sukhorukov, VL and Benkert, R and Obermeyer, G and Bentrup, FW and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {01}, number = 1, pages = {21-32}, publisher = {SPRINGER VERLAG}, title = {Electrorotation of isolated generative and vegetative cells, and of intact pollen grains of Lilium longiflorum}, type = {Article}, volume = 161, year = 1998 }

  %0 Journal Article %1 Sukhorukov1998a %A Sukhorukov, VL %A Benkert, R %A Obermeyer, G %A Bentrup, FW %A Zimmermann, U %C 175 FIFTH AVE, NEW YORK, NY 10010 USA %D 1998 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 1 %P 21-32 %T Electrorotation of isolated generative and vegetative cells, and of intact pollen grains of Lilium longiflorum %V 161 %X The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m(-1). The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.
• Single-molecule counting and identification in a microcapillary. Zander, C.; Drexhage, K.H.; Han, K.-T.; Wolfrum, J.; Sauer, M. In Chemical Physics Letters, 286(5-6), S. 457–465. 1998.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.

  @article{Zander1998457, abstract = {Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.}, author = {Zander, C. and Drexhage, K.H. and Han, K.-T. and Wolfrum, J. and Sauer, M.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {5-6}, pages = {457 - 465}, title = {Single-molecule counting and identification in a microcapillary}, volume = 286, year = 1998 }

  %0 Journal Article %1 Zander1998457 %A Zander, C. %A Drexhage, K.H. %A Han, K.-T. %A Wolfrum, J. %A Sauer, M. %D 1998 %J Chemical Physics Letters %N 5-6 %P 457 - 465 %R DOI: 10.1016/S0009-2614(98)00096-7 %T Single-molecule counting and identification in a microcapillary %U http://www.sciencedirect.com/science/article/B6TFN-3VN2MVG-3B/2/6f1d57b094658a541d630753d82f717f %V 286 %X Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 [mu]m at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser ([lambda]=640 nm, average power 800 [mu]W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.
• Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection. Lieberwirth, Ulrike; Arden-Jacob, Jutta; Drexhage, Karl H.; Herten, Dirk P.; Müller, Ralph; Neumann, Michael; Schulz, Andreas; Siebert, Stefan; Sagner, Gregor; Klingel, Sven; Sauer, Markus; Wolfrum, Jürgen. In Analytical Chemistry, 70(22), S. 4771–4779. 1998.
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  A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods

  @article{doi:10.1021/ac980230k, abstract = {A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods}, author = {Lieberwirth, Ulrike and Arden-Jacob, Jutta and Drexhage, Karl H. and Herten, Dirk P. and Müller, Ralph and Neumann, Michael and Schulz, Andreas and Siebert, Stefan and Sagner, Gregor and Klingel, Sven and Sauer, Markus and Wolfrum, Jürgen}, journal = {Analytical Chemistry}, keywords = {sauer}, number = 22, pages = {4771-4779}, title = {Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection}, volume = 70, year = 1998 }

  %0 Journal Article %1 doi:10.1021/ac980230k %A Lieberwirth, Ulrike %A Arden-Jacob, Jutta %A Drexhage, Karl H. %A Herten, Dirk P. %A Müller, Ralph %A Neumann, Michael %A Schulz, Andreas %A Siebert, Stefan %A Sagner, Gregor %A Klingel, Sven %A Sauer, Markus %A Wolfrum, Jürgen %D 1998 %J Analytical Chemistry %N 22 %P 4771-4779 %R 10.1021/ac980230k %T Multiplex Dye DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection %U http://pubs.acs.org/doi/abs/10.1021/ac980230k %V 70 %X A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of >90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods
• Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase. SEELA, F.; CHEN, Y.; SAUER, M. In ChemInform, 29(28), S. no-no. WILEY-VCH Verlag, 1998.
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  Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

  @article{CHIN:CHIN199828268, abstract = {Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.}, author = {SEELA, F. and CHEN, Y. and SAUER, M.}, journal = {ChemInform}, keywords = {sauer}, number = 28, pages = {no–no}, publisher = {WILEY-VCH Verlag}, title = {Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase.}, volume = 29, year = 1998 }

  %0 Journal Article %1 CHIN:CHIN199828268 %A SEELA, F. %A CHEN, Y. %A SAUER, M. %D 1998 %I WILEY-VCH Verlag %J ChemInform %N 28 %P no--no %R 10.1002/chin.199828268 %T Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase. %U http://dx.doi.org/10.1002/chin.199828268 %V 29 %X Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
• Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level. Sauer, M; Drexhage, K.H; Lieberwirth, U; Müller, R; Nord, S; Zander, C. In Chemical Physics Letters, 284(3-4), S. 153–163. 1998.
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  Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.

  @article{Sauer1998153, abstract = {Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.}, author = {Sauer, M and Drexhage, K.H and Lieberwirth, U and Müller, R and Nord, S and Zander, C}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {153 - 163}, title = {Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level}, volume = 284, year = 1998 }

  %0 Journal Article %1 Sauer1998153 %A Sauer, M %A Drexhage, K.H %A Lieberwirth, U %A Müller, R %A Nord, S %A Zander, C %D 1998 %J Chemical Physics Letters %N 3-4 %P 153 - 163 %R DOI: 10.1016/S0009-2614(97)01377-8 %T Dynamics of the electron transfer reaction between an oxazine dye and DNA oligonucleotides monitored on the single-molecule level %U http://www.sciencedirect.com/science/article/B6TFN-3VN2FFT-29/2/0d5cca7d9662f7bc3754f4f96d675166 %V 284 %X Single-molecule spectroscopy in water has been investigated by monitoring the dynamical behaviour of the electron transfer reaction between guanosine-containing oligonucleotides and the covalently attached oxazine dye MR121, using diode laser based far-field fluorescence microscopy. In this system, each oligonucleotide molecule exhibits multiexponential electron transfer kinetics. The influence of the guanosine position on the degree of quenching is shown using different oligonucleotide sequences. The dynamical behaviour of conformational transitions between various states with different electron transfer efficiency is monitored by time-resolved fluorescence spectroscopy on the [mu]s- and ms-time scales. In addition, guanosine specific on/off blinking of individual labeled oligonucleotide molecules in aqueous solution is shown.
• Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers. Sauer, Markus; Arden-Jacob, Jutta; Drexhage, Karl H; Göbel, Florian; Lieberwirth, Ulrike; Mühlegger, Klaus; Müller, Ralph; Wolfrum, Jürgen; Zander, Christoph. In Bioimaging, 6(1), S. 14–24. IOP Publishing Ltd, 1998.
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  We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)

  @article{BIO:BIO3, abstract = {We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)}, author = {Sauer, Markus and Arden-Jacob, Jutta and Drexhage, Karl H and Göbel, Florian and Lieberwirth, Ulrike and Mühlegger, Klaus and Müller, Ralph and Wolfrum, Jürgen and Zander, Christoph}, journal = {Bioimaging}, keywords = {sauer}, number = 1, pages = {14–24}, publisher = {IOP Publishing Ltd}, title = {Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers}, volume = 6, year = 1998 }

  %0 Journal Article %1 BIO:BIO3 %A Sauer, Markus %A Arden-Jacob, Jutta %A Drexhage, Karl H %A Göbel, Florian %A Lieberwirth, Ulrike %A Mühlegger, Klaus %A Müller, Ralph %A Wolfrum, Jürgen %A Zander, Christoph %D 1998 %I IOP Publishing Ltd %J Bioimaging %N 1 %P 14--24 %R 10.1002/1361-6374(199803)6:1<14::AID-BIO3>3.0.CO;2-O %T Time-resolved identification of individual mononucleotide molecules in aqueous solution with pulsed semiconductor lasers %U http://dx.doi.org/10.1002/1361-6374(199803)6:1<14::AID-BIO3>3.0.CO;2-O %V 6 %X We applied a short-pulse diode laser emitting at 640 nm with a repetition rate of 56 MHz in combination with a confocal microscope to study bursts of fluorescence photons from individual differently labeled mononucleotide molecules in water. Two newly synthesized dyes, an oxazine dye (MR121) and a rhodamine dye (JA53), and two commercially available dyes, a carbocyanine dye (Cy5) and a bora-diaza-indacene dye (Bodipy630/650), were used as fluorescent labels. The time-resolved fluorescence signals of individual mononucleotiode molecules in water were analyzed and identified by a maximum likelihood estimator (MLE). Taking only those single molecule transits which contain more than 30 collected photoelectrons, the two labeled mononucleotide molecules, Cy5-dCTP and Bodipy-dUTP, can be identified by time-resolved fluorescence spectroscopy with a probability of correct classification of greater than 99%. Our results show that at least three differently labeled mononucleotide molecules can be identified in a common aqueous solution. We obtain an overall classification probability of 90% for the time-resolved identification of Cy5-dCTP, MR121-dUTP and Bodipy-dUTP molecules via their characteristic fluorescence lifetimes of 1.05 ± 0.33 ns (Cy5-dCTP), 2.07 ± 0.59 ns (MR121-dUTP) and 3.88 ± 1.71 ns (Bodipy-dUTP)

• Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine. Nord, S.; Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Lieberwirth, U.; Seeger, S.; Wolfrum, J. In J. Fluoresc., 7, S. 15–18. 1997.
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  @article{nord1997ground, author = {Nord, S. and Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Lieberwirth, U. and Seeger, S. and Wolfrum, J.}, journal = {J. Fluoresc.}, keywords = {sauer}, pages = {15-18}, title = {Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine}, volume = 7, year = 1997 }

  %0 Journal Article %1 nord1997ground %A Nord, S. %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Lieberwirth, U. %A Seeger, S. %A Wolfrum, J. %D 1997 %J J. Fluoresc. %P 15-18 %T Ground and excited state reactions of new red fluorescent dyes with the DNA base guanosine %V 7
• Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes. Lieberwirth, U.; Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Nord, S.; Seeger, S.; Schulz, A; Wolfrum, J. In J. Fluoresc., 7, S. 55–58. 1997.
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  @article{lieberwirth1997development, author = {Lieberwirth, U. and Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Nord, S. and Seeger, S. and Schulz, A and Wolfrum, J.}, journal = {J. Fluoresc.}, keywords = {sauer}, pages = {55-58}, title = {Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes}, volume = 7, year = 1997 }

  %0 Journal Article %1 lieberwirth1997development %A Lieberwirth, U. %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Nord, S. %A Seeger, S. %A Schulz, A %A Wolfrum, J. %D 1997 %J J. Fluoresc. %P 55-58 %T Development of Multiplex Dyes: Intramolecular fluorescence quenching of rhodamine dyes %V 7
• Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set. Müller, Ralph; Herten, Dirk P.; Lieberwirth, Ulrike; Neumann, Michael; Sauer, Markus; Schulz, Andreas; Siebert, Stefan; Drexhage, Karl H.; Wolfrum, Jürgen. In Chemical Physics Letters, 279(5-6), S. 282–288. 1997.
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  A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.

  @article{Müller1997282, abstract = {A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.}, author = {Müller, Ralph and Herten, Dirk P. and Lieberwirth, Ulrike and Neumann, Michael and Sauer, Markus and Schulz, Andreas and Siebert, Stefan and Drexhage, Karl H. and Wolfrum, Jürgen}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {5-6}, pages = {282 - 288}, title = {Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set}, volume = 279, year = 1997 }

  %0 Journal Article %1 Müller1997282 %A Müller, Ralph %A Herten, Dirk P. %A Lieberwirth, Ulrike %A Neumann, Michael %A Sauer, Markus %A Schulz, Andreas %A Siebert, Stefan %A Drexhage, Karl H. %A Wolfrum, Jürgen %D 1997 %J Chemical Physics Letters %N 5-6 %P 282 - 288 %R DOI: 10.1016/S0009-2614(97)01041-5 %T Efficient DNA sequencing with a pulsed semiconductor laser and a new fluorescent dye set %U http://www.sciencedirect.com/science/article/B6TFN-3SFVBFC-47/2/985d40ed8deaa5d0ca07432857da5550 %V 279 %X A new method is presented for automated one-lane four-dye DNA sequencing in capillary gel electrophoresis based on semiconductor technology and a special set of multiplex fluorescent dyes which exhibit similar absorption and emission spectra but different fluorescent lifetimes. The primer sequencing reaction was applied in a confocal optical system. Detection and identification of the differently 5'-labeled primers was done by time-correlated single-photon counting and a specially developed pattern-recognition technique based on the characteristic fluorescence lifetimes of the fluorescent dyes used as labels. Efficient excitation was performed at 630 nm by a short-pulsed semiconductor laser with a repetition rate of 22 MHz and pulsewidth of about 500 ps (FWHM). With the new dye set, no mobility shift correction is required for a separation up to 350 base pairs during separation in a linear 5% PAA gel. This technique of multiplex-dye DNA sequencing shows potential for high-throughput DNA sequencing in parallel capillaries or microfabricated DNA quenching chips.
• On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis. Sauer, M.; Arden-Jacob, J.; Drexhage, K. H.; Marx, N. J.; Karger, A. E.; Lieberwirth, U.; Müller, R.; Neumann, M.; Nord, S.; Paulus, A.; Schulz, A.; Seeger, S.; Zander, C.; Wolfrum, J. In Biomedical Chromatography, 11(2), S. 81–82. John Wiley & Sons, Ltd., 1997.
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  @article{BMC:BMC645, author = {Sauer, M. and Arden-Jacob, J. and Drexhage, K. H. and Marx, N. J. and Karger, A. E. and Lieberwirth, U. and Müller, R. and Neumann, M. and Nord, S. and Paulus, A. and Schulz, A. and Seeger, S. and Zander, C. and Wolfrum, J.}, journal = {Biomedical Chromatography}, keywords = {sauer}, number = 2, pages = {81–82}, publisher = {John Wiley & Sons, Ltd.}, title = {On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis}, volume = 11, year = 1997 }

  %0 Journal Article %1 BMC:BMC645 %A Sauer, M. %A Arden-Jacob, J. %A Drexhage, K. H. %A Marx, N. J. %A Karger, A. E. %A Lieberwirth, U. %A Müller, R. %A Neumann, M. %A Nord, S. %A Paulus, A. %A Schulz, A. %A Seeger, S. %A Zander, C. %A Wolfrum, J. %D 1997 %I John Wiley & Sons, Ltd. %J Biomedical Chromatography %N 2 %P 81--82 %R 10.1002/(SICI)1099-0801(199703)11:2<81::AID-BMC645>3.0.CO;2-U %T On-Line Diode Laser Based Time-Resolved Fluorescence Detection of Labelled Oligonucleotides in Capillary Gel Electrophoresis %U http://dx.doi.org/10.1002/(SICI)1099-0801(199703)11:2<81::AID-BMC645>3.0.CO;2-U %V 11
• Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers. Sauer, M.; Zander, C.; Müller, R.; Ullrich, B.; Drexhage, K.H.; Kaul, S.; Wolfrum, J. In Applied Physics B: Lasers and Optics, 65(3), S. 427–431. Springer Berlin / Heidelberg, 1997.
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   M without separation steps.

  @article{springerlink:10.1007/s003400050292, abstract = { M without separation steps.}, author = {Sauer, M. and Zander, C. and Müller, R. and Ullrich, B. and Drexhage, K.H. and Kaul, S. and Wolfrum, J.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400050292}, pages = {427-431}, publisher = {Springer Berlin / Heidelberg}, title = {Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers}, volume = 65, year = 1997 }

  %0 Journal Article %1 springerlink:10.1007/s003400050292 %A Sauer, M. %A Zander, C. %A Müller, R. %A Ullrich, B. %A Drexhage, K.H. %A Kaul, S. %A Wolfrum, J. %D 1997 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 427-431 %T Detection and identification of individual antigen molecules in human serum with pulsed semiconductor lasers %U http://dx.doi.org/10.1007/s003400050292 %V 65 %X  M without separation steps.
• Electrorotational spectra of protoplasts generated from the giant marine alga Valonia utricularis. Wang, J; Sukhorukov, VL; Djuzenova, CS; Zimmermann, U; Muller, T; Fuhr, G. In PROTOPLASMA, 196(3-4), S. 123–134. SPRINGER-VERLAG WIEN, SACHSENPLATZ 4-6, PO BOX 89, A-1201 VIENNA, AUSTRIA, 1997.
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  Protoplasts of Valonia utricularis is lacking the large central vacuole can be generated by cutting multi-nucleated, giant `'mother'' cells into small pieces after short exposure to air. When the protoplasmic content was squeezed out into sea water, irregularly shaped, green coloured aggregates were formed which changed into spherical protoplasts (radius of 20-60 mu m) after about 2 h. In these protoplasts the dense internal material (consisting mainly of organelles) was separated from the plasmalemma by a thin transparent layer containing a large number of small lipid vesicles. Cell wall regeneration occurred rapidly after protoplast formation. A central vacuole developed after about 10 h. The regenerated cells continued to grow and were viable for several months. Electrorotation studies on 2-3 h old protoplasts at pH 7 in low- and fairly high-conductivity solutions showed one or two anti-field rotation peaks (depending on medium conductivity) between 10 kHz to 1 MHz as well as one co-field rotation peak between 10 MHz to 100 MHz. The rotation spec tra could not be fitted on the basis of the single- (or multi-) shell model (i.e., by modelling the cells as a homogeneous sphere surrounded by one or more layers). However, fairly good agreement between the experimental data and theory could be obtained by assuming that the rotational behaviour of the protoplasts depends not only on passive electrical properties of the plasmalemma but is influenced by `'mobile charges'' of carrier transport systems and/or the dielectric behaviour of the aggregated chloroplasts and vesicles.

  @article{Wang1997, abstract = {Protoplasts of Valonia utricularis is lacking the large central vacuole can be generated by cutting multi-nucleated, giant `'mother'' cells into small pieces after short exposure to air. When the protoplasmic content was squeezed out into sea water, irregularly shaped, green coloured aggregates were formed which changed into spherical protoplasts (radius of 20-60 mu m) after about 2 h. In these protoplasts the dense internal material (consisting mainly of organelles) was separated from the plasmalemma by a thin transparent layer containing a large number of small lipid vesicles. Cell wall regeneration occurred rapidly after protoplast formation. A central vacuole developed after about 10 h. The regenerated cells continued to grow and were viable for several months. Electrorotation studies on 2-3 h old protoplasts at pH 7 in low- and fairly high-conductivity solutions showed one or two anti-field rotation peaks (depending on medium conductivity) between 10 kHz to 1 MHz as well as one co-field rotation peak between 10 MHz to 100 MHz. The rotation spec tra could not be fitted on the basis of the single- (or multi-) shell model (i.e., by modelling the cells as a homogeneous sphere surrounded by one or more layers). However, fairly good agreement between the experimental data and theory could be obtained by assuming that the rotational behaviour of the protoplasts depends not only on passive electrical properties of the plasmalemma but is influenced by `'mobile charges'' of carrier transport systems and/or the dielectric behaviour of the aggregated chloroplasts and vesicles.}, address = {SACHSENPLATZ 4-6, PO BOX 89, A-1201 VIENNA, AUSTRIA}, author = {Wang, J and Sukhorukov, VL and Djuzenova, CS and Zimmermann, U and Muller, T and Fuhr, G}, journal = {PROTOPLASMA}, keywords = {vladimir}, number = {3-4}, pages = {123-134}, publisher = {SPRINGER-VERLAG WIEN}, title = {Electrorotational spectra of protoplasts generated from the giant marine alga Valonia utricularis}, type = {Article}, volume = 196, year = 1997 }

  %0 Journal Article %1 Wang1997 %A Wang, J %A Sukhorukov, VL %A Djuzenova, CS %A Zimmermann, U %A Muller, T %A Fuhr, G %C SACHSENPLATZ 4-6, PO BOX 89, A-1201 VIENNA, AUSTRIA %D 1997 %I SPRINGER-VERLAG WIEN %J PROTOPLASMA %N 3-4 %P 123-134 %T Electrorotational spectra of protoplasts generated from the giant marine alga Valonia utricularis %V 196 %X Protoplasts of Valonia utricularis is lacking the large central vacuole can be generated by cutting multi-nucleated, giant `'mother'' cells into small pieces after short exposure to air. When the protoplasmic content was squeezed out into sea water, irregularly shaped, green coloured aggregates were formed which changed into spherical protoplasts (radius of 20-60 mu m) after about 2 h. In these protoplasts the dense internal material (consisting mainly of organelles) was separated from the plasmalemma by a thin transparent layer containing a large number of small lipid vesicles. Cell wall regeneration occurred rapidly after protoplast formation. A central vacuole developed after about 10 h. The regenerated cells continued to grow and were viable for several months. Electrorotation studies on 2-3 h old protoplasts at pH 7 in low- and fairly high-conductivity solutions showed one or two anti-field rotation peaks (depending on medium conductivity) between 10 kHz to 1 MHz as well as one co-field rotation peak between 10 MHz to 100 MHz. The rotation spec tra could not be fitted on the basis of the single- (or multi-) shell model (i.e., by modelling the cells as a homogeneous sphere surrounded by one or more layers). However, fairly good agreement between the experimental data and theory could be obtained by assuming that the rotational behaviour of the protoplasts depends not only on passive electrical properties of the plasmalemma but is influenced by `'mobile charges'' of carrier transport systems and/or the dielectric behaviour of the aggregated chloroplasts and vesicles.
• Determination of the diffusion coefficient of dye in solution at single molecule level. Ko, D. S.; Sauer, M.; Nord, S.; Müller, R.; Wolfrum, J. In Chemical Physics Letters, 269(1-2), S. 54–58. 1997.
  • [ Abstract ]
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  We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.

  @article{Ko199754, abstract = {We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.}, author = {Ko, D. S. and Sauer, M. and Nord, S. and Müller, R. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {1-2}, pages = {54 - 58}, title = {Determination of the diffusion coefficient of dye in solution at single molecule level}, volume = 269, year = 1997 }

  %0 Journal Article %1 Ko199754 %A Ko, D. S. %A Sauer, M. %A Nord, S. %A Müller, R. %A Wolfrum, J. %D 1997 %J Chemical Physics Letters %N 1-2 %P 54 - 58 %R DOI: 10.1016/S0009-2614(97)00246-7 %T Determination of the diffusion coefficient of dye in solution at single molecule level %U http://www.sciencedirect.com/science/article/B6TFN-3S9MRPW-4G/2/74f26e268928991c260b84a3f926321a %V 269 %X We have analyzed the width distributions of fluorescence bursts emitted from single dye in solution. Here we used the confocal microscope combined with a short-pulsed diode laser providing an excitation wavelength of 638 nm and a repetition rate of 17 MHz. Under the experimental condition that the transit times of dye molecules through a probe space were shorter than the average photodestruction time, the data obtained using a multichannel scaler showed a linear trend between the photon counts and the width of fluorescence bursts. From the width distributions of burst, the translational diffusion coefficient of JA22 in ethylene glycol was determined.

• Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells. Djuzenova, CS; Zimmermann, U; Frank, H; Sukhorukov, VL; Richter, E; Fuhr, G. In BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1284(2), S. 143–152. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 1996.
  • [ Abstract ]
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  The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 mu g ml(-1) PI at different conductivities (0.8-14 mS cm(-1)) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO42-. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the `electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm(-1) in K+-free, NaCl-containing medium and about 0.9 kV cm(-1) in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K+-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.

  @article{Djuzenova1996, abstract = {The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 mu g ml(-1) PI at different conductivities (0.8-14 mS cm(-1)) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO42-. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the `electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm(-1) in K+-free, NaCl-containing medium and about 0.9 kV cm(-1) in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K+-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.}, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {Djuzenova, CS and Zimmermann, U and Frank, H and Sukhorukov, VL and Richter, E and Fuhr, G}, journal = {BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES}, keywords = {vladimir}, month = 10, number = 2, pages = {143-152}, publisher = {ELSEVIER SCIENCE BV}, title = {Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells}, type = {Article}, volume = 1284, year = 1996 }

  %0 Journal Article %1 Djuzenova1996 %A Djuzenova, CS %A Zimmermann, U %A Frank, H %A Sukhorukov, VL %A Richter, E %A Fuhr, G %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 1996 %I ELSEVIER SCIENCE BV %J BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES %N 2 %P 143-152 %T Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells %V 1284 %X The effects of ionic composition and conductivity of the medium on electropermeabilization of the plasma membrane of mammalian cells were studied. Temporal and spatial uptake of propidium iodide (PI) into field-treated cells was measured by means of flow cytometry, spectrofluorimetry and confocal laser scanning microscopy. Murine myeloma cells were electropulsed in iso-osmolar solutions. These contained 10-100 mu g ml(-1) PI at different conductivities (0.8-14 mS cm(-1)) and ionic strengths, adjusted by varying concentrations of K+, Na+, Cl- and SO42-. Field-induced incorporation of PI into reversibly permeabilized cells was (almost) independent of ionic composition and strength (at a fixed medium conductivity), but increased dramatically with decreasing medium conductivity at a fixed field strength. The time-course of PI uptake (which apparently reflected the resealing process of the membrane) could be fitted by a single-exponential curve (relaxation time about 2 min in the absence of Ca2+) and was independent of medium conductivity and composition. These and other data suggested that the expansion of the `electroleaks' during the breakdown process is field-controlled and depends, therefore, on the (conductivity-dependent) discharging process of the permeabilized membrane. The threshold field strength for dye uptake increased with increasing K+ concentration (about 0.6 kV cm(-1) in K+-free, NaCl-containing medium and about 0.9 kV cm(-1) in 30 mM KCl-containing medium). Also, the spatial uptake pattern of PI shifted from an asymmetric permeation through the cell hemisphere facing the anode to a more symmetric uptake through both hemispheres. These results suggested that the generated potential is superimposed on the (K+-dependent) resting membrane potential. Taking this into account, the breakdown voltage of the membrane was estimated to be about 1 V.
• Absorption of tungsten carbonyl anions into the lipid bilayer membrane of mouse myeloma cells. Nielsen, K; Schenk, WA; Kriegmeier, M; Sukhorukov, VL; Zimmermann, U. In INORGANIC CHEMISTRY, 35(20), S. 5762-&. AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036, 1996.
  • [ Abstract ]
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  The anions of the tungsten carbonyl salts Na{[}W(CO)(5)(CN)]. 3H2(O) (1), K{[}W(CO)(5)(NCS)] (2), and K{[}W-2(CO)(10)(mu-SCN)] (3) are readily taken up by the lipid membrane of living cells. Cell rotation experiments show that at solution concentrations in the micromolar range the lipid bilayer absorbs up to 1.0 pmol cm(-2) of these anions, which leads to a pronounced increase of both the conductance, G(m), and the capacitance, C-m, of the membrane. 1-3 and similar salts are thus seen as ideal instruments to further develop models of mass transport through lipid membranes.

  @article{Nielsen1996, abstract = {The anions of the tungsten carbonyl salts Na{[}W(CO)(5)(CN)]. 3H2(O) (1), K{[}W(CO)(5)(NCS)] (2), and K{[}W-2(CO)(10)(mu-SCN)] (3) are readily taken up by the lipid membrane of living cells. Cell rotation experiments show that at solution concentrations in the micromolar range the lipid bilayer absorbs up to 1.0 pmol cm(-2) of these anions, which leads to a pronounced increase of both the conductance, G(m), and the capacitance, C-m, of the membrane. 1-3 and similar salts are thus seen as ideal instruments to further develop models of mass transport through lipid membranes.}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036}, author = {Nielsen, K and Schenk, WA and Kriegmeier, M and Sukhorukov, VL and Zimmermann, U}, journal = {INORGANIC CHEMISTRY}, keywords = {vladimir}, month = {09}, number = 20, pages = {5762-\&}, publisher = {AMER CHEMICAL SOC}, title = {Absorption of tungsten carbonyl anions into the lipid bilayer membrane of mouse myeloma cells}, type = {Article}, volume = 35, year = 1996 }

  %0 Journal Article %1 Nielsen1996 %A Nielsen, K %A Schenk, WA %A Kriegmeier, M %A Sukhorukov, VL %A Zimmermann, U %C 1155 16TH ST, NW, WASHINGTON, DC 20036 %D 1996 %I AMER CHEMICAL SOC %J INORGANIC CHEMISTRY %N 20 %P 5762-& %T Absorption of tungsten carbonyl anions into the lipid bilayer membrane of mouse myeloma cells %V 35 %X The anions of the tungsten carbonyl salts Na{[}W(CO)(5)(CN)]. 3H2(O) (1), K{[}W(CO)(5)(NCS)] (2), and K{[}W-2(CO)(10)(mu-SCN)] (3) are readily taken up by the lipid membrane of living cells. Cell rotation experiments show that at solution concentrations in the micromolar range the lipid bilayer absorbs up to 1.0 pmol cm(-2) of these anions, which leads to a pronounced increase of both the conductance, G(m), and the capacitance, C-m, of the membrane. 1-3 and similar salts are thus seen as ideal instruments to further develop models of mass transport through lipid membranes.
• Electrorotation of erythrocytes treated with dipicrylamine: Mobile charges within the membrane show their `’signature’’ in rotational spectra. Sukhorukov, VL; Zimmermann, U. In JOURNAL OF MEMBRANE BIOLOGY, 153(2), S. 161–169. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010, 1996.
  • [ Abstract ]
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  In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2-5 mS m(-1)). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values.

  @article{Sukhorukov1996, abstract = {In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2-5 mS m(-1)). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values.}, address = {175 FIFTH AVE, NEW YORK, NY 10010}, author = {Sukhorukov, VL and Zimmermann, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {09}, number = 2, pages = {161-169}, publisher = {SPRINGER VERLAG}, title = {Electrorotation of erythrocytes treated with dipicrylamine: Mobile charges within the membrane show their `'signature'' in rotational spectra}, type = {Article}, volume = 153, year = 1996 }

  %0 Journal Article %1 Sukhorukov1996 %A Sukhorukov, VL %A Zimmermann, U %C 175 FIFTH AVE, NEW YORK, NY 10010 %D 1996 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 2 %P 161-169 %T Electrorotation of erythrocytes treated with dipicrylamine: Mobile charges within the membrane show their `'signature'' in rotational spectra %V 153 %X In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2-5 mS m(-1)). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values.
• Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies. Seidel, Claus A. M.; Schulz, Andreas; Sauer, Markus H. M. In The Journal of Physical Chemistry, 100(13), S. 5541–5553. 1996.
  • [ Abstract ]
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  Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.

  @article{doi:10.1021/jp951507c, abstract = {Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.}, author = {Seidel, Claus A. M. and Schulz, Andreas and Sauer, Markus H. M.}, journal = {The Journal of Physical Chemistry}, keywords = {sauer}, number = 13, pages = {5541-5553}, title = {Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies}, volume = 100, year = 1996 }

  %0 Journal Article %1 doi:10.1021/jp951507c %A Seidel, Claus A. M. %A Schulz, Andreas %A Sauer, Markus H. M. %D 1996 %J The Journal of Physical Chemistry %N 13 %P 5541-5553 %R 10.1021/jp951507c %T Nucleobase-Specific Quenching of Fluorescent Dyes. 1. Nucleobase One-Electron Redox Potentials and Their Correlation with Static and Dynamic Quenching Efficiencies %U http://pubs.acs.org/doi/abs/10.1021/jp951507c %V 100 %X Intermolecular static and dynamic fluorescence quenching constants of eight coumarin derivatives by nucleobase derivatives have been determined in aqueous media. One common sequence of the quenching efficiency has been found for the nucleobases. The feasibility of a photoinduced electron transfer reaction for the nucleobase-specific quenching of fluorescent dyes is investigated by the calculation of the standard free energy changes with the Rehm−Weller equation. A complete set of one-electron redox potential data for the nucleobases are determined electrochemically in aprotic solvents for the first time, which are compared with values obtained by various other methods. Depending on the redox properties of the fluorescent dyes, the sequences of the quenching efficiencies can be rationalized by the orders of electrochemical oxidation potentials (vs NHE) of nucleosides (dG (+1.47 V) < dA < dC ≈ dT < U (≥ +2.39 V)) and reduction potentials (dG (< −2.76 V) < dA < dC < dT < U (−2.07 V)). The correlation between the intermolecular dynamic quenching constants and the standard free energy of photoinduced electron transfer according to the classical Marcus equation indicates that photoinduced electron transfer is the rate-limiting step. However, an additional, water-specific gain of free energy between −0.5 and −0.9 eV shows that additional effects, like a coupled proton transfer and a hydrophobic effect, have to be considered, too. Furthermore, the capability of the nucleobases to form ground state complexes with fluorescent dyes is influenced by their redox potentials. The relevance of these observations to current efforts for DNA sequencing with a detection by laser-induced fluorescence and their application to other dyes are discussed.
• Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser. Müller, R.; Zander, C.; Sauer, M.; Deimel, M.; Ko, D. S.; Siebert, S.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H.; Wolfrum, J. In Chemical Physics Letters, 262(6), S. 716–722. 1996.
  • [ Abstract ]
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  We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).

  @article{Müller1996716, abstract = {We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).}, author = {Müller, R. and Zander, C. and Sauer, M. and Deimel, M. and Ko, D. S. and Siebert, S. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Drexhage, K. H. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = 6, pages = {716 - 722}, title = {Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser}, volume = 262, year = 1996 }

  %0 Journal Article %1 Müller1996716 %A Müller, R. %A Zander, C. %A Sauer, M. %A Deimel, M. %A Ko, D. S. %A Siebert, S. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Drexhage, K. H. %A Wolfrum, J. %D 1996 %J Chemical Physics Letters %N 6 %P 716 - 722 %R DOI: 10.1016/S0009-2614(96)01147-5 %T Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser %U http://www.sciencedirect.com/science/article/B6TFN-3VV60GR-2X/2/d00a127d7acc02c711f22e8392d20f98 %V 262 %X We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).
• Diode laser based detection of single molecules in solutions. Sauer, M.; Drexhage, K. H.; Zander, C.; Wolfrum, J. In Chemical Physics Letters, 254(3-4), S. 223–228. 1996.
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  Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.

  @article{Sauer1996223, abstract = {Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.}, author = {Sauer, M. and Drexhage, K. H. and Zander, C. and Wolfrum, J.}, journal = {Chemical Physics Letters}, keywords = {sauer}, number = {3-4}, pages = {223 - 228}, title = {Diode laser based detection of single molecules in solutions}, volume = 254, year = 1996 }

  %0 Journal Article %1 Sauer1996223 %A Sauer, M. %A Drexhage, K. H. %A Zander, C. %A Wolfrum, J. %D 1996 %J Chemical Physics Letters %N 3-4 %P 223 - 228 %R DOI: 10.1016/0009-2614(96)00303-X %T Diode laser based detection of single molecules in solutions %U http://www.sciencedirect.com/science/article/B6TFN-3V8YMT0-3K/2/b453bf51d51141737d98f04f0a321859 %V 254 %X Using a confocal microscope we studied bursts of fluorescence photons from single dye molecules that were excited at 632 nm with a CW diode laser or helium neon laser. The measurements were performed with new rhodamine derivatives in ethylene glycol solution. The potential of this method for future construction of a low-cost instrument for single-molecule detection is pointed out. To demonstrate the utility of our new dyes in bioanalytical applications we showed the single-molecule detection of labeled oligonucleotides in water.
• Detection and characterization of single molecules in aqueous solution. Zander, C.; Sauer, M.; Drexhage, K. H.; Ko, D.-S.; Schulz, A.; Wolfrum, J.; Brand, L.; Eggeling, C.; Seidel, C. A. M. In Applied Physics B: Lasers and Optics, 63(5), S. 517–523. Springer Berlin / Heidelberg, 1996.
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  Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100&#114ps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79&#450.33&#114ns (RB) and 3.79&#450.38&#114ns (R6G) in aqueous solution.

  @article{zander1996detection, abstract = {Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100rps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79ǂ.33rns (RB) and 3.79ǂ.38rns (R6G) in aqueous solution.}, author = {Zander, C. and Sauer, M. and Drexhage, K. H. and Ko, D.-S. and Schulz, A. and Wolfrum, J. and Brand, L. and Eggeling, C. and Seidel, C. A. M.}, journal = {Applied Physics B: Lasers and Optics}, keywords = {sauer}, note = {10.1007/s003400050118}, pages = {517-523}, publisher = {Springer Berlin / Heidelberg}, title = {Detection and characterization of single molecules in aqueous solution}, volume = 63, year = 1996 }

  %0 Journal Article %1 zander1996detection %A Zander, C. %A Sauer, M. %A Drexhage, K. H. %A Ko, D.-S. %A Schulz, A. %A Wolfrum, J. %A Brand, L. %A Eggeling, C. %A Seidel, C. A. M. %D 1996 %I Springer Berlin / Heidelberg %J Applied Physics B: Lasers and Optics %P 517-523 %T Detection and characterization of single molecules in aqueous solution %U http://dx.doi.org/10.1007/s003400050118 %V 63 %X Using a confocal microscope with a single-photon avalanche photodiode as detector, we studied photon bursts of single Rhodamine 6G (R6G) and Rhodamin B-zwitterion (RB) molecules in aqueous solution by excitation of the lowest excited singlet state S1 with a frequency-doubled titanium:sapphire laser. Multichannel scaler traces, the fluorescence autocorrelation function and fluorescence decay times determined by time-correlated single-photon counting have been measured simultaneously. The time-resolved fluorescence signals were analyzed with a maximum likelihood estimator. Fluorescence lifetime patterns in steps of 100&#114ps were generated by convolution with the excitation pulse. The lifetime of the S1 state was derived from the Kullback-Leibler minimum discrimination information. We are able to demonstrate for the first time identification of two different single dye molecules via their characteristic fluorescence lifetimes of 1.79&#450.33&#114ns (RB) and 3.79&#450.38&#114ns (R6G) in aqueous solution.

• ELECTROPERMEABILIZATION AND FLUORESCENT TRACER EXCHANGE - THE ROLE OF WHOLE-CELL CAPACITANCE. SUKHORUKOV, VL; DJUZENOVA, CS; FRANK, H; ARNOLD, WM; ZIMMERMANN, U. In CYTOMETRY, 21(3), S. 230–240. WILEY-LISS, DIV JOHN WILEY & SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012, 1995.
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  Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxy-fluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 mu s) in medium containing 25-50 mu g/ml PI at 21-23 degrees C. Cytograms of PI vs, CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95\% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm, This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecoles (and/or organelles), In isotonic `'pulse medium,'' the membranes resealed after electropulsing with a time constant (tau(R)) of about 2 min, La 150 mOsm medium, resealing was faster (typically tau(R) similar to 0.5 min). The population distribution of PI uptake {[}coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius similar to 10\%), The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C-c), was taken into account, Evaluation of the C-c values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. (C) 1995 Wiley-Liss, Inc.

  @article{SUKHORUKOV1995, abstract = {Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxy-fluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 mu s) in medium containing 25-50 mu g/ml PI at 21-23 degrees C. Cytograms of PI vs, CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95\% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm, This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecoles (and/or organelles), In isotonic `'pulse medium,'' the membranes resealed after electropulsing with a time constant (tau(R)) of about 2 min, La 150 mOsm medium, resealing was faster (typically tau(R) similar to 0.5 min). The population distribution of PI uptake {[}coefficient of variation (CV) > 40\%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius similar to 10\%), The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C-c), was taken into account, Evaluation of the C-c values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. (C) 1995 Wiley-Liss, Inc.}, address = {DIV JOHN WILEY \& SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012}, author = {SUKHORUKOV, VL and DJUZENOVA, CS and FRANK, H and ARNOLD, WM and ZIMMERMANN, U}, journal = {CYTOMETRY}, keywords = {vladimir}, month = 11, number = 3, pages = {230-240}, publisher = {WILEY-LISS}, title = {ELECTROPERMEABILIZATION AND FLUORESCENT TRACER EXCHANGE - THE ROLE OF WHOLE-CELL CAPACITANCE}, type = {Article}, volume = 21, year = 1995 }

  %0 Journal Article %1 SUKHORUKOV1995 %A SUKHORUKOV, VL %A DJUZENOVA, CS %A FRANK, H %A ARNOLD, WM %A ZIMMERMANN, U %C DIV JOHN WILEY & SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012 %D 1995 %I WILEY-LISS %J CYTOMETRY %N 3 %P 230-240 %T ELECTROPERMEABILIZATION AND FLUORESCENT TRACER EXCHANGE - THE ROLE OF WHOLE-CELL CAPACITANCE %V 21 %X Transmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxy-fluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0-Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5-3.0 kV/cm, 40 mu s) in medium containing 25-50 mu g/ml PI at 21-23 degrees C. Cytograms of PI vs, CF fluorescence showed three readily distinguishable subpopulations: 1) intact living cells with CF but without PI (these form > 95\% of the prepulsed population), 2) transiently electropermeabilized but resealed cells showing both CF and low-level PI fluorescence, and 3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm, This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecoles (and/or organelles), In isotonic `'pulse medium,'' the membranes resealed after electropulsing with a time constant (tau(R)) of about 2 min, La 150 mOsm medium, resealing was faster (typically tau(R) similar to 0.5 min). The population distribution of PI uptake {[}coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse-induced voltage (CVradius similar to 10\%), The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole-cell capacitance (C-c), was taken into account, Evaluation of the C-c values with single-cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. (C) 1995 Wiley-Liss, Inc.
• New fluorescent dyes in the red region for biodiagnostics. Sauer, M.; Han, K. T.; Müller, R.; Nord, S.; Schulz, A.; Seeger, S.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Zander, C.; Drexhage, K. H. In Journal of Fluorescence, 5(3), S. 247–261. Springer Netherlands, 1995.
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  The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

  @article{springerlink:10.1007/BF00723896, abstract = {The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.}, author = {Sauer, M. and Han, K. T. and Müller, R. and Nord, S. and Schulz, A. and Seeger, S. and Wolfrum, J. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Zander, C. and Drexhage, K. H.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF00723896}, pages = {247-261}, publisher = {Springer Netherlands}, title = {New fluorescent dyes in the red region for biodiagnostics}, volume = 5, year = 1995 }

  %0 Journal Article %1 springerlink:10.1007/BF00723896 %A Sauer, M. %A Han, K. T. %A Müller, R. %A Nord, S. %A Schulz, A. %A Seeger, S. %A Wolfrum, J. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Zander, C. %A Drexhage, K. H. %D 1995 %I Springer Netherlands %J Journal of Fluorescence %P 247-261 %T New fluorescent dyes in the red region for biodiagnostics %U http://dx.doi.org/10.1007/BF00723896 %V 5 %X The increased sensitivity together with the advent of low-cost optical sources and detectors in the visible-near IR region has led us to current efforts to develop new efficient fluorescent labels for biodiagnostics with absorption and emission beyond 600 nm. In view of the general fluorescence decrease with increasing emission wavelength, we investigated the possibility to shift the absorption of rhodamine dyes toward the region 620–670 nm. The hydrophobic nature of all known long-wavelength dyes results in the tendency to form intra- and intermolecular aggregates in hydrophilic solvents, especially in aqueous environment. Due to the aggregation with biological materials, fluorescence quenching of the dyes is often observed. New strategies for prevention of these processes are considered.

• Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes. Bachteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Han, K. T.; Köllner, M.; Müller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. In Journal of Luminescence, 62(3-4), S. 101–108. 1994.
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  A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.

  @article{Bachteler1994101, abstract = {A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.}, author = {Bachteler, G. and Drexhage, K. H. and Arden-Jacob, J. and Han, K. T. and Köllner, M. and Müller, R. and Sauer, M. and Seeger, S. and Wolfrum, J.}, journal = {Journal of Luminescence}, keywords = {sauer}, number = {3-4}, pages = {101 - 108}, title = {Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes}, volume = 62, year = 1994 }

  %0 Journal Article %1 Bachteler1994101 %A Bachteler, G. %A Drexhage, K. H. %A Arden-Jacob, J. %A Han, K. T. %A Köllner, M. %A Müller, R. %A Sauer, M. %A Seeger, S. %A Wolfrum, J. %D 1994 %J Journal of Luminescence %N 3-4 %P 101 - 108 %R DOI: 10.1016/0022-2313(94)90336-0 %T Sensitive fluorescence detection in capillary electrophoresis using laser diodes and multiplex dyes %U http://www.sciencedirect.com/science/article/B6TJH-46FHCFY-2/2/86c15278719c7f6788703c0fe8cd6023 %V 62 %X A detector for laser induced fluorescence based on laser diole technology was designed and sensitivity measurements with rhodamine 800 were performed. The detection limit is around 100 molecules in the detection volume. Using the laser diode in combination with a commercial capillary electrophoresis system, about 1200 molecules were detectable. Using time-resolved fluorescence measurements multiplex dyes were recognized with a pulsed laser diode and a pattern recognition technique collecting only a few hundred photons.
• Simultaneous antigen detection using multiplex dyes. Galla, K.; Arden-Jacob, J.; Deltau, G.; Drexhage, K. H.; Martin, M.; Sauer, M.; Wolfrum, J.; Seeger, S. In Journal of Fluorescence, 4(1), S. 111–115. Springer Netherlands, 1994.
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  To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.

  @article{galla1994simultaneous, abstract = {To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.}, author = {Galla, K. and Arden-Jacob, J. and Deltau, G. and Drexhage, K. H. and Martin, M. and Sauer, M. and Wolfrum, J. and Seeger, S.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF01876665}, pages = {111-115}, publisher = {Springer Netherlands}, title = {Simultaneous antigen detection using multiplex dyes}, volume = 4, year = 1994 }

  %0 Journal Article %1 galla1994simultaneous %A Galla, K. %A Arden-Jacob, J. %A Deltau, G. %A Drexhage, K. H. %A Martin, M. %A Sauer, M. %A Wolfrum, J. %A Seeger, S. %D 1994 %I Springer Netherlands %J Journal of Fluorescence %P 111-115 %T Simultaneous antigen detection using multiplex dyes %U http://dx.doi.org/10.1007/BF01876665 %V 4 %X To detect several antigens simultaneously, antibodies directed against different antigens were immobilized on a quartz surface. The antigens were tagged with multiplex, dyes, which show different fluorescence lifetimes but similar excitation and emission spectra. The antigens were detected by recognizing the characteristic fluorescence lifetime. Furthermore, the effect of labeling of the dye on the antigen molecules was examined.
• Sensitive fluorescence detection using laser diodes and multiplex dyes. Bachteler, G.; Drexhage, K.-H.; Arden-Jacob, J.; Han, K.-T.; Ko¨llner, M.; Mu¨ller, R.; Sauer, M.; Seeger, S.; Wolfrum, J. In Journal of Luminescence, 60-61, S. 511–514. 1994.
  • [ Abstract ]
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  A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.

  @article{Bachteler1994511, abstract = {A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.}, author = {Bachteler, G. and Drexhage, K.-H. and Arden-Jacob, J. and Han, K.-T. and Ko¨llner, M. and Mu¨ller, R. and Sauer, M. and Seeger, S. and Wolfrum, J.}, journal = {Journal of Luminescence}, keywords = {sauer}, note = {International Conference on Luminescence}, pages = {511 - 514}, title = {Sensitive fluorescence detection using laser diodes and multiplex dyes}, volume = {60-61}, year = 1994 }

  %0 Journal Article %1 Bachteler1994511 %A Bachteler, G. %A Drexhage, K.-H. %A Arden-Jacob, J. %A Han, K.-T. %A Ko¨llner, M. %A Mu¨ller, R. %A Sauer, M. %A Seeger, S. %A Wolfrum, J. %D 1994 %J Journal of Luminescence %P 511 - 514 %R DOI: 10.1016/0022-2313(94)90204-6 %T Sensitive fluorescence detection using laser diodes and multiplex dyes %U http://www.sciencedirect.com/science/article/B6TJH-46G3CGF-7R/2/13b4a8aed95422d6c11ea658258477ec %V 60-61 %X A laser diode based detector for laser-induced fluorescence was designed. Sensitivity measurements with rhodamine 800 showed a detection limit of 100 molecules. Using time-resolved fluorescence measurements new fluorescent dyes, called multiplex dyes, were recognized with a pulsed laser diode by their characteristic fluorescence lifetime and a pattern recognition technique collecting only a few hundred photons.
• EFFECT OF ELECTRIC-FIELD PULSES ON THE VIABILITY AND ON THE MEMBRANE-BOUND IMMUNOGLOBULINS OF LPS-ACTIVATED MURINE B-LYMPHOCYTES - CORRELATION WITH THE CELL-CYCLE. DJUZENOVA, CS; SUKHORUKOV, VL; KLOCK, G; ARNOLD, WM; ZIMMERMANN, U. In CYTOMETRY, 15(1), S. 35–45. WILEY-LISS, DIV JOHN WILEY & SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012, 1994.
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  The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 mu s) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37 degrees C for 30 min (''resealing'') significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing. The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, tau = 5 mu s, three pulses, 4 degrees C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. (C) 1994 Wiley-Liss, Inc.

  @article{DJUZENOVA1994, abstract = {The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 mu s) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20\%, 45\%, or 70\% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37 degrees C for 30 min (''resealing'') significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing. The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, tau = 5 mu s, three pulses, 4 degrees C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. (C) 1994 Wiley-Liss, Inc.}, address = {DIV JOHN WILEY \& SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012}, author = {DJUZENOVA, CS and SUKHORUKOV, VL and KLOCK, G and ARNOLD, WM and ZIMMERMANN, U}, journal = {CYTOMETRY}, keywords = {vladimir}, month = {01}, number = 1, pages = {35-45}, publisher = {WILEY-LISS}, title = {EFFECT OF ELECTRIC-FIELD PULSES ON THE VIABILITY AND ON THE MEMBRANE-BOUND IMMUNOGLOBULINS OF LPS-ACTIVATED MURINE B-LYMPHOCYTES - CORRELATION WITH THE CELL-CYCLE}, type = {Article}, volume = 15, year = 1994 }

  %0 Journal Article %1 DJUZENOVA1994 %A DJUZENOVA, CS %A SUKHORUKOV, VL %A KLOCK, G %A ARNOLD, WM %A ZIMMERMANN, U %C DIV JOHN WILEY & SONS INC 605 THIRD AVE, NEW YORK, NY 10158-0012 %D 1994 %I WILEY-LISS %J CYTOMETRY %N 1 %P 35-45 %T EFFECT OF ELECTRIC-FIELD PULSES ON THE VIABILITY AND ON THE MEMBRANE-BOUND IMMUNOGLOBULINS OF LPS-ACTIVATED MURINE B-LYMPHOCYTES - CORRELATION WITH THE CELL-CYCLE %V 15 %X The effects of microsecond electropulses (1-5 kV/cm) on the viability of murine B lymphocytes and on their binding of antibodies by surface immunoglobulin (Ig) were studied in relation to the cell cycle. Before electropulsing, cultures given 48 h mitogenic stimulation showed at least two cell subpopulations, which were distinguishable by their levels of surface-Ig expression as assessed with FITC-labelled antibodies against mouse Ig. The immunofluorescence intensity of cells in S and G2/M phases was higher than that of G0/G1 cells. After exposure of the mitogen-stimulated lymphocytes to three exponentially decaying (time constant tau = 5-40 mu s) electric field pulses, dye exclusion assay showed that pulsing at 1 or 2 kV/cm (at 4 degrees C or 20 degrees C) did not cause permeabilization. Field strengths of 3, 4, or 5 kV/cm resulted in 20%, 45%, or 70% of dye-permeable cells, respectively, if the pulsed cells were transferred to phosphate-buffered saline on ice for 30 min. Incubation in full medium at 37 degrees C for 30 min (''resealing'') significantly decreased the percentage of permeabilized cells. Electropulsed G0/G1 cells were not only more resistant to direct electric exposure (tolerated higher field strengths) than S + G2/M cells but also responded better to resealing. The surface Ig of lymphocytes pulsed at higher fields and low temperature (4 or 5 kV/cm, tau = 5 mu s, three pulses, 4 degrees C) was less easily immunostained than in controls or in cells pulsed at 2 kV/cm or less. At 5 kV/cm those cells that were not permeabilized showed a greater reduction in immunostaining, especially if resealed. (C) 1994 Wiley-Liss, Inc.
• DNA, PROTEIN, AND PLASMA-MEMBRANE INCORPORATION BY ARRESTED MAMMALIAN-CELLS. SUKHORUKOV, VL; DJUZENOVA, CS; ARNOLD, WM; ZIMMERMANN, U. In JOURNAL OF MEMBRANE BIOLOGY, 142(1), S. 77–92. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010, 1994.
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  Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrororation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated eel populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 X 5kV/cm, 40 mu sec decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.

  @article{SUKHORUKOV1994, abstract = {Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrororation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated eel populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 X 5kV/cm, 40 mu sec decay time) of aphidicolin-synchronized L cells in G2/M led to a 22\% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.}, address = {175 FIFTH AVE, NEW YORK, NY 10010}, author = {SUKHORUKOV, VL and DJUZENOVA, CS and ARNOLD, WM and ZIMMERMANN, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = 10, number = 1, pages = {77-92}, publisher = {SPRINGER VERLAG}, title = {DNA, PROTEIN, AND PLASMA-MEMBRANE INCORPORATION BY ARRESTED MAMMALIAN-CELLS}, type = {Article}, volume = 142, year = 1994 }

  %0 Journal Article %1 SUKHORUKOV1994 %A SUKHORUKOV, VL %A DJUZENOVA, CS %A ARNOLD, WM %A ZIMMERMANN, U %C 175 FIFTH AVE, NEW YORK, NY 10010 %D 1994 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 1 %P 77-92 %T DNA, PROTEIN, AND PLASMA-MEMBRANE INCORPORATION BY ARRESTED MAMMALIAN-CELLS %V 142 %X Incorporation of DNA, protein, and plasma membrane during blockage by aphidicolin or by doxorubicin was studied by flow cytometry and electrororation of three cell lines (mouse-myeloma Sp2/0-Ag14, hybridoma H73C11, and fibroblast-like L929 cells). Drug-mediated arrest at the G1-S boundary (aphidicolin) or in G2/M (doxorubicin) did not arrest synthesis of either protein or total membrane area, the increases in which outstripped growth in cell volume and apparent cell area, respectively. Measurements of membrane capacity in normal and hypo-osmotic media showed that the drugs had not changed the fundamental bilayer, but that an increase in the number or size of microvilli must have occurred. Aphidicolin-arrested cells withstood hypo-osmotic stress better than untreated cells could, indicating that the membrane excess can be utilized as a reserve during rapid cell expansion. Hypo-osmotically treated cell populations exhibited only about half the coefficient of variance (CV) in membrane properties of cells at physiological osmolality. Populations of arrested cells exhibited the same high CV as asynchronous cells, indicating that chemical arrest does not give uniformly villated eel populations. However, the lowest CV values were given by some synchronized (aphidicolin-blocked, then released) populations. Removal of aphidicolin allowed most cells to progress through S and G2, and then divide. During these processes, the membrane excess was reduced. After removal of doxorubicin, the cells did not divide: some continued protein synthesis, grew abnormally large, and further increased their membrane excess. Membrane breakdown by electric pulsing (3 X 5kV/cm, 40 mu sec decay time) of aphidicolin-synchronized L cells in G2/M led to a 22% loss of plasma membrane (both the area-specific and the whole-cell capacitance were reduced), presumably via endocytosis-like vesiculation.

• PRE-IRRADIATED PHOTOOXIDIZED PSORALEN INDUCES HEMOLYSIS AND CHANGES IN ELECTRICAL PARAMETERS OF ERYTHROCYTE-MEMBRANES. WUNDERLICH, S; PLIQUETT, F; MORICH, E; KYAGOVA, AA; SUKHORUKOV, VL; POTAPENKO, AY. In BIOELECTROCHEMISTRY AND BIOENERGETICS, 31(2), S. 193–202. ELSEVIER SCIENCE SA LAUSANNE, PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND, 1993.
  • [ Abstract ]
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  The effects of pre-irradiated photo-oxidized psoralen (POP) on human erythrocytes (RBCs) were investigated. Psoralen in ethanol solutions was photo-oxidized and then added to a suspension of erythrocytes. The hemolysis and the electric impedance of the suspension were measured in the frequency range 5-20 MHz. The membrane capacity C(M), and the extracellular and intracellular conductivitieS kappa(a) and kappa(1) were calculated from these data. There appeared to be a threshold concentration of POP for hemolysis. When the concentration of POP slightly exceeded this threshold, the hemolytic effect rapidly increased to a maximum, but a further increase in POP concentration slowed down the rate of hemolysis. POP induced a decrease in extracellular conductivity and an increase in membrane capacity, but did not affect intracellular conductivity. The changes in extracellular conductivity correlated well with hemolysis.

  @article{WUNDERLICH1993, abstract = {The effects of pre-irradiated photo-oxidized psoralen (POP) on human erythrocytes (RBCs) were investigated. Psoralen in ethanol solutions was photo-oxidized and then added to a suspension of erythrocytes. The hemolysis and the electric impedance of the suspension were measured in the frequency range 5-20 MHz. The membrane capacity C(M), and the extracellular and intracellular conductivitieS kappa(a) and kappa(1) were calculated from these data. There appeared to be a threshold concentration of POP for hemolysis. When the concentration of POP slightly exceeded this threshold, the hemolytic effect rapidly increased to a maximum, but a further increase in POP concentration slowed down the rate of hemolysis. POP induced a decrease in extracellular conductivity and an increase in membrane capacity, but did not affect intracellular conductivity. The changes in extracellular conductivity correlated well with hemolysis.}, address = {PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND}, author = {WUNDERLICH, S and PLIQUETT, F and MORICH, E and KYAGOVA, AA and SUKHORUKOV, VL and POTAPENKO, AY}, journal = {BIOELECTROCHEMISTRY AND BIOENERGETICS}, keywords = {vladimir}, month = {07}, number = 2, pages = {193-202}, publisher = {ELSEVIER SCIENCE SA LAUSANNE}, title = {PRE-IRRADIATED PHOTOOXIDIZED PSORALEN INDUCES HEMOLYSIS AND CHANGES IN ELECTRICAL PARAMETERS OF ERYTHROCYTE-MEMBRANES}, type = {Article}, volume = 31, year = 1993 }

  %0 Journal Article %1 WUNDERLICH1993 %A WUNDERLICH, S %A PLIQUETT, F %A MORICH, E %A KYAGOVA, AA %A SUKHORUKOV, VL %A POTAPENKO, AY %C PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND %D 1993 %I ELSEVIER SCIENCE SA LAUSANNE %J BIOELECTROCHEMISTRY AND BIOENERGETICS %N 2 %P 193-202 %T PRE-IRRADIATED PHOTOOXIDIZED PSORALEN INDUCES HEMOLYSIS AND CHANGES IN ELECTRICAL PARAMETERS OF ERYTHROCYTE-MEMBRANES %V 31 %X The effects of pre-irradiated photo-oxidized psoralen (POP) on human erythrocytes (RBCs) were investigated. Psoralen in ethanol solutions was photo-oxidized and then added to a suspension of erythrocytes. The hemolysis and the electric impedance of the suspension were measured in the frequency range 5-20 MHz. The membrane capacity C(M), and the extracellular and intracellular conductivitieS kappa(a) and kappa(1) were calculated from these data. There appeared to be a threshold concentration of POP for hemolysis. When the concentration of POP slightly exceeded this threshold, the hemolytic effect rapidly increased to a maximum, but a further increase in POP concentration slowed down the rate of hemolysis. POP induced a decrease in extracellular conductivity and an increase in membrane capacity, but did not affect intracellular conductivity. The changes in extracellular conductivity correlated well with hemolysis.
• HYPOTONICALLY INDUCED CHANGES IN THE PLASMA-MEMBRANE OF CULTURED-MAMMALIAN-CELLS. SUKHORUKOV, VL; ARNOLD, WM; ZIMMERMANN, U. In JOURNAL OF MEMBRANE BIOLOGY, 132(1), S. 27–40. SPRINGER VERLAG, 175 FIFTH AVE, NEW YORK, NY 10010, 1993.
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  Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C(m)) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C(m) values of 1.01 +/- 0.04 muF/cm2 and 1.09 +/- 0.03 muF/cm2, respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C(m) = 2.18 +/- 0.10 muF/cm2. As the osmolality was reduced, the C(m) reached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C(m), after which a plateau close to 0.80 muF/cm2 was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C(m) between 280 mOsm and 150 mOsm, but below 150 mOsm the C(m) decreased rapidly. The changes in C(m) correlate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C(m), but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of `'extra'' membrane into the cell surface.

  @article{SUKHORUKOV1993, abstract = {Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C(m)) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C(m) values of 1.01 +/- 0.04 muF/cm2 and 1.09 +/- 0.03 muF/cm2, respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C(m) = 2.18 +/- 0.10 muF/cm2. As the osmolality was reduced, the C(m) reached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7\% increase in C(m), after which a plateau close to 0.80 muF/cm2 was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C(m) between 280 mOsm and 150 mOsm, but below 150 mOsm the C(m) decreased rapidly. The changes in C(m) correlate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C(m), but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of `'extra'' membrane into the cell surface.}, address = {175 FIFTH AVE, NEW YORK, NY 10010}, author = {SUKHORUKOV, VL and ARNOLD, WM and ZIMMERMANN, U}, journal = {JOURNAL OF MEMBRANE BIOLOGY}, keywords = {vladimir}, month = {02}, number = 1, pages = {27-40}, publisher = {SPRINGER VERLAG}, title = {HYPOTONICALLY INDUCED CHANGES IN THE PLASMA-MEMBRANE OF CULTURED-MAMMALIAN-CELLS}, type = {Article}, volume = 132, year = 1993 }

  %0 Journal Article %1 SUKHORUKOV1993 %A SUKHORUKOV, VL %A ARNOLD, WM %A ZIMMERMANN, U %C 175 FIFTH AVE, NEW YORK, NY 10010 %D 1993 %I SPRINGER VERLAG %J JOURNAL OF MEMBRANE BIOLOGY %N 1 %P 27-40 %T HYPOTONICALLY INDUCED CHANGES IN THE PLASMA-MEMBRANE OF CULTURED-MAMMALIAN-CELLS %V 132 %X Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C(m)) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C(m) values of 1.01 +/- 0.04 muF/cm2 and 1.09 +/- 0.03 muF/cm2, respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C(m) = 2.18 +/- 0.10 muF/cm2. As the osmolality was reduced, the C(m) reached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C(m), after which a plateau close to 0.80 muF/cm2 was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C(m) between 280 mOsm and 150 mOsm, but below 150 mOsm the C(m) decreased rapidly. The changes in C(m) correlate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C(m), but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of `'extra'' membrane into the cell surface.
• FE-2+ IONS AND REDUCED GLUTATHIONE - CHEMICAL ACTIVATORS OF PSORALEN-SENSITIZED PHOTOHEMOLYSIS. POTAPENKO, AY; SAPAROV, SM; AGAMALIEVA, MA; LYSENKO, EP; BEZDETNAYA, LN; SUKHORUKOV, VL. In JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY, 17(1), S. 69–75. ELSEVIER SCIENCE SA LAUSANNE, PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND, 1993.
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  The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe'' ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.

  @article{POTAPENKO1993, abstract = {The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe'' ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.}, address = {PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND}, author = {POTAPENKO, AY and SAPAROV, SM and AGAMALIEVA, MA and LYSENKO, EP and BEZDETNAYA, LN and SUKHORUKOV, VL}, journal = {JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY}, keywords = {vladimir}, month = {01}, number = 1, pages = {69-75}, publisher = {ELSEVIER SCIENCE SA LAUSANNE}, title = {FE-2+ IONS AND REDUCED GLUTATHIONE - CHEMICAL ACTIVATORS OF PSORALEN-SENSITIZED PHOTOHEMOLYSIS}, type = {Article}, volume = 17, year = 1993 }

  %0 Journal Article %1 POTAPENKO1993 %A POTAPENKO, AY %A SAPAROV, SM %A AGAMALIEVA, MA %A LYSENKO, EP %A BEZDETNAYA, LN %A SUKHORUKOV, VL %C PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND %D 1993 %I ELSEVIER SCIENCE SA LAUSANNE %J JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY %N 1 %P 69-75 %T FE-2+ IONS AND REDUCED GLUTATHIONE - CHEMICAL ACTIVATORS OF PSORALEN-SENSITIZED PHOTOHEMOLYSIS %V 17 %X The influence of Fe2+ ions and reduced glutathione (GSH) on the haemolysis of erythrocytes photosensitized (366 nm) by psoralen (PUVA haemolysis) was investigated. PUVA haemolysis was induced by the low fluence rate (24 W m-2) and high fluence (greater than 150 W m-2) of UV-A irradiation. It has been shown that Fe2+ ions and GSH activated PUVA haemolysis at both fluence rates of irradiation. PUVA haemolysis activation by Fe'' ions was more pronounced than that by GSH. It is supposed that activation caused by Fe2+ ions and GSH is connected with their ability to reduce lipid peroxides or psoralen peroxides with the subsequent formation of free radicals. The regeneration of endogenous Fe2+ by reduced glutathione is also possible.
• Design of Multiplex Dyes. Sauer, Markus; Schulz, Andreas; Seeger, Stefan; Wolfrum, Jürgen; Arden-Jacob, Jutta; Deltau, Gerhard; Drexhage, Karl H. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1734–1737. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
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  Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.

  @article{BBPC:BBPC19930971240, abstract = {Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.}, author = {Sauer, Markus and Schulz, Andreas and Seeger, Stefan and Wolfrum, Jürgen and Arden-Jacob, Jutta and Deltau, Gerhard and Drexhage, Karl H.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1734–1737}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Design of Multiplex Dyes}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971240 %A Sauer, Markus %A Schulz, Andreas %A Seeger, Stefan %A Wolfrum, Jürgen %A Arden-Jacob, Jutta %A Deltau, Gerhard %A Drexhage, Karl H. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1734--1737 %R 10.1002/bbpc.19930971240 %T Design of Multiplex Dyes %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971240/abstract %V 97 %X Abstract Fluorescent dyes which are almost identical in their absorption and emission spectral characteristics but differ in fluorescence lifetime (“Multiplex Dyes”) have been developed for analytical applications. First investigations to design such dyes were carried out with rhodamines. In order to influence the excited state lifetime without changing the spectral characteristics we studied rhodamines modified with non-conjugated substituents that promote non-radiative transitions. We used two different methods to control the fluorescence lifetime in rhodamine dyes with rigidized amino groups: (A) Substitution with benzyl derivatives of different electron acceptor strength at the amino position. (B) Substitution of the carboxyphenyl group by phenyl groups with reduced steric requirements and varying acceptor properties.
• New fluorescent labels for time-resolved detection of biomolecules. Sauer, M.; Han, K-T; Müller, R.; Schulz, A.; Tadday, R.; Seeger, S.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.; Marx, N. J.; Drexhage, K. H. In Journal of Fluorescence, 3(3), S. 131–139. Springer Netherlands, 1993.
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  New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.

  @article{sauer1993fluorescent, abstract = {New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.}, author = {Sauer, M. and Han, K-T and Müller, R. and Schulz, A. and Tadday, R. and Seeger, S. and Wolfrum, J. and Arden-Jacob, J. and Deltau, G. and Marx, N. J. and Drexhage, K. H.}, journal = {Journal of Fluorescence}, keywords = {sauer}, note = {10.1007/BF00862730}, pages = {131-139}, publisher = {Springer Netherlands}, title = {New fluorescent labels for time-resolved detection of biomolecules}, volume = 3, year = 1993 }

  %0 Journal Article %1 sauer1993fluorescent %A Sauer, M. %A Han, K-T %A Müller, R. %A Schulz, A. %A Tadday, R. %A Seeger, S. %A Wolfrum, J. %A Arden-Jacob, J. %A Deltau, G. %A Marx, N. J. %A Drexhage, K. H. %D 1993 %I Springer Netherlands %J Journal of Fluorescence %P 131-139 %T New fluorescent labels for time-resolved detection of biomolecules %U http://dx.doi.org/10.1007/BF00862730 %V 3 %X New dyes with characteristic fluorescence lifetimes have been developed for bioanalytical applications. Based upon the concept of multiplex dyes, we have designed rhodamine dyes with nearly identical absorption and emission spectral characteristics but different fluorescence lifetimes. Extending this principle to applications with laser diodes, new rhodamines with functional groups for covalent coupling of analytes have been developed. The new labels exhibit absortion and fluorescence beyond 600 nm and have a high quantum efficiency, even in aqueous buffer systems.
• Biodiagnostics and Polymer Identification with Multiplex Dyes. Seeger, S.; Bachteler, G.; Drexhage, K. H.; Arden-Jacob, J.; Deltau, G.; Galla, K.; Han, K. T.; Müller, R.; Köllner, M.; Rumphorst, A.; Sauer, M.; Schulz, A.; Wolfrum, J. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1542–1547. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
  • [ Abstract ]
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  Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.

  @article{BBPC:BBPC19930971208, abstract = {Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.}, author = {Seeger, S. and Bachteler, G. and Drexhage, K. H. and Arden-Jacob, J. and Deltau, G. and Galla, K. and Han, K. T. and Müller, R. and Köllner, M. and Rumphorst, A. and Sauer, M. and Schulz, A. and Wolfrum, J.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1542–1547}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Biodiagnostics and Polymer Identification with Multiplex Dyes}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971208 %A Seeger, S. %A Bachteler, G. %A Drexhage, K. H. %A Arden-Jacob, J. %A Deltau, G. %A Galla, K. %A Han, K. T. %A Müller, R. %A Köllner, M. %A Rumphorst, A. %A Sauer, M. %A Schulz, A. %A Wolfrum, J. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1542--1547 %R 10.1002/bbpc.19930971208 %T Biodiagnostics and Polymer Identification with Multiplex Dyes %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971208/abstract %V 97 %X Abstract A new analytical concept based on time-resolved fluorescence spectroscopy is presented, which uses the characteristic fluorescence lifetimes as information to identify new fluorescent dyes, called multiplex dyes. Several dyes can be distinguished at nearly fixed excitation and emission wavelength by recognition of the lifetimes. A pattern recognition technique is used for data analysis. As light sources a pulsed laser diode is used. First experiments are shown in several application fields, like DNA sequencing, immunoassays and tagging of polymer material.
• Time-Resolved Fluorescence Studies of Labelled Nucleosides. Han, K.-T.; Sauer, M.; Schulz, A.; Seeger, S.; Wolfrum, J. In Berichte der Bunsengesellschaft für physikalische Chemie, 97(12), S. 1728–1730. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1993.
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  Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.

  @article{BBPC:BBPC19930971238, abstract = {Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.}, author = {Han, K.-T. and Sauer, M. and Schulz, A. and Seeger, S. and Wolfrum, J.}, journal = {Berichte der Bunsengesellschaft für physikalische Chemie}, keywords = {sauer}, number = 12, pages = {1728–1730}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim}, title = {Time-Resolved Fluorescence Studies of Labelled Nucleosides}, volume = 97, year = 1993 }

  %0 Journal Article %1 BBPC:BBPC19930971238 %A Han, K.-T. %A Sauer, M. %A Schulz, A. %A Seeger, S. %A Wolfrum, J. %D 1993 %I Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim %J Berichte der Bunsengesellschaft für physikalische Chemie %N 12 %P 1728--1730 %R 10.1002/bbpc.19930971238 %T Time-Resolved Fluorescence Studies of Labelled Nucleosides %U http://onlinelibrary.wiley.com/doi/10.1002/bbpc.19930971238/abstract %V 97 %X Synthesis, steady-state and time-resolved fluorescence measurements of carboxycoumari labelled aminonucleosides (thymidine, cytidine, guanosine) are described. The data indicate a strong influence of the coupling position, the linker length and the structure of the coumarin dye on the quenching rates.

• PHOTOHEMOLYSIS SENSITIZED BY PSORALEN - RECIPROCITY LAW IS NOT FULFILLED. POTAPENKO, AY; AGAMALIEVA, MA; NAGIEV, AI; LYSENKO, EP; BEZDETNAYA, LN; SUKHORUKOV, VL. In PHOTOCHEMISTRY AND PHOTOBIOLOGY, 54(3), S. 375–379. AMER SOC PHOTOBIOLOGY, BIOTECH PARK, 1021 15TH ST, SUITE 9, AUGUSTA, GA 30901-3158, 1991.
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  The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100\%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.

  @article{POTAPENKO1991, abstract = {The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100\%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.}, address = {BIOTECH PARK, 1021 15TH ST, SUITE 9, AUGUSTA, GA 30901-3158}, author = {POTAPENKO, AY and AGAMALIEVA, MA and NAGIEV, AI and LYSENKO, EP and BEZDETNAYA, LN and SUKHORUKOV, VL}, journal = {PHOTOCHEMISTRY AND PHOTOBIOLOGY}, keywords = {vladimir}, month = {09}, number = 3, pages = {375-379}, publisher = {AMER SOC PHOTOBIOLOGY}, title = {PHOTOHEMOLYSIS SENSITIZED BY PSORALEN - RECIPROCITY LAW IS NOT FULFILLED}, type = {Article}, volume = 54, year = 1991 }

  %0 Journal Article %1 POTAPENKO1991 %A POTAPENKO, AY %A AGAMALIEVA, MA %A NAGIEV, AI %A LYSENKO, EP %A BEZDETNAYA, LN %A SUKHORUKOV, VL %C BIOTECH PARK, 1021 15TH ST, SUITE 9, AUGUSTA, GA 30901-3158 %D 1991 %I AMER SOC PHOTOBIOLOGY %J PHOTOCHEMISTRY AND PHOTOBIOLOGY %N 3 %P 375-379 %T PHOTOHEMOLYSIS SENSITIZED BY PSORALEN - RECIPROCITY LAW IS NOT FULFILLED %V 54 %X The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100\%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.
• EFFECT OF CALCIUM-IONS ON PSORALEN-SENSITIZED PHOTOHEMOLYSIS. SAPAROV, SM; ZHURAVEL, NN; MOLLAEV, RE; SUKHORUKOV, VL; POTAPENKO, AY. In JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY, 10(1-2), S. 159–164. ELSEVIER SCIENCE SA LAUSANNE, PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND, 1991.
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  @article{SAPAROV1991, address = {PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND}, author = {SAPAROV, SM and ZHURAVEL, NN and MOLLAEV, RE and SUKHORUKOV, VL and POTAPENKO, AY}, journal = {JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY}, keywords = {vladimir}, month = {07}, number = {1-2}, pages = {159-164}, publisher = {ELSEVIER SCIENCE SA LAUSANNE}, title = {EFFECT OF CALCIUM-IONS ON PSORALEN-SENSITIZED PHOTOHEMOLYSIS}, type = {Note}, volume = 10, year = 1991 }

  %0 Journal Article %1 SAPAROV1991 %A SAPAROV, SM %A ZHURAVEL, NN %A MOLLAEV, RE %A SUKHORUKOV, VL %A POTAPENKO, AY %C PO BOX 564, 1001 LAUSANNE 1, SWITZERLAND %D 1991 %I ELSEVIER SCIENCE SA LAUSANNE %J JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY %N 1-2 %P 159-164 %T EFFECT OF CALCIUM-IONS ON PSORALEN-SENSITIZED PHOTOHEMOLYSIS %V 10

• AGGREGATION EFFECT ON FLUORESCENT PROPERTIES OF FUROCOUMARINS IN SOLUTIONS. LYSENKO, EP; SUKHORUKOV, VL; VUNDERLIKH, Z; SHAGOVA, LI; KUZNETSOVA, GA; KUZMINA, LV; POTAPENKO, AY. In ZHURNAL FIZICHESKOI KHIMII, 63(10), S. 2768–2773. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1989.
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  @article{LYSENKO1989, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {LYSENKO, EP and SUKHORUKOV, VL and VUNDERLIKH, Z and SHAGOVA, LI and KUZNETSOVA, GA and KUZMINA, LV and POTAPENKO, AY}, journal = {ZHURNAL FIZICHESKOI KHIMII}, keywords = {vladimir}, month = 10, number = 10, pages = {2768-2773}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {AGGREGATION EFFECT ON FLUORESCENT PROPERTIES OF FUROCOUMARINS IN SOLUTIONS}, type = {Article}, volume = 63, year = 1989 }

  %0 Journal Article %1 LYSENKO1989 %A LYSENKO, EP %A SUKHORUKOV, VL %A VUNDERLIKH, Z %A SHAGOVA, LI %A KUZNETSOVA, GA %A KUZMINA, LV %A POTAPENKO, AY %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1989 %I MEZHDUNARODNAYA KNIGA %J ZHURNAL FIZICHESKOI KHIMII %N 10 %P 2768-2773 %T AGGREGATION EFFECT ON FLUORESCENT PROPERTIES OF FUROCOUMARINS IN SOLUTIONS %V 63
• ANTIOXIDANTS INHIBIT HEMOLYSIS INDUCED BY PHOTOOXIDIZED PSORALEN. POTAPENKO, AY; BEZDETNAYA, LN; AKHTYAMOV, SN; PERKHOVA, OY; SUKHORUKOV, VL. In BIOFIZIKA, 34(2), S. 195–198. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1989.
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  @article{POTAPENKO1989, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {POTAPENKO, AY and BEZDETNAYA, LN and AKHTYAMOV, SN and PERKHOVA, OY and SUKHORUKOV, VL}, journal = {BIOFIZIKA}, keywords = {vladimir}, month = {03}, number = 2, pages = {195-198}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {ANTIOXIDANTS INHIBIT HEMOLYSIS INDUCED BY PHOTOOXIDIZED PSORALEN}, type = {Article}, volume = 34, year = 1989 }

  %0 Journal Article %1 POTAPENKO1989 %A POTAPENKO, AY %A BEZDETNAYA, LN %A AKHTYAMOV, SN %A PERKHOVA, OY %A SUKHORUKOV, VL %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1989 %I MEZHDUNARODNAYA KNIGA %J BIOFIZIKA %N 2 %P 195-198 %T ANTIOXIDANTS INHIBIT HEMOLYSIS INDUCED BY PHOTOOXIDIZED PSORALEN %V 34

• PHOTOHEMOLYSIS SENSITIZED BY PSORALEN, PARADOXICAL DEPENDENCE ON THE LIGHT-INTENSITY. POTAPENKO, AJ; SUKHORUKOV, VL; AGAMALIEVA, MA; LYSENKO, EP. In DOKLADY AKADEMII NAUK SSSR, 302(5), S. 1258–1260. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1988.
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  @article{POTAPENKO1988a, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {POTAPENKO, AJ and SUKHORUKOV, VL and AGAMALIEVA, MA and LYSENKO, EP}, journal = {DOKLADY AKADEMII NAUK SSSR}, keywords = {vladimir}, number = 5, pages = {1258-1260}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {PHOTOHEMOLYSIS SENSITIZED BY PSORALEN, PARADOXICAL DEPENDENCE ON THE LIGHT-INTENSITY}, type = {Article}, volume = 302, year = 1988 }

  %0 Journal Article %1 POTAPENKO1988a %A POTAPENKO, AJ %A SUKHORUKOV, VL %A AGAMALIEVA, MA %A LYSENKO, EP %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1988 %I MEZHDUNARODNAYA KNIGA %J DOKLADY AKADEMII NAUK SSSR %N 5 %P 1258-1260 %T PHOTOHEMOLYSIS SENSITIZED BY PSORALEN, PARADOXICAL DEPENDENCE ON THE LIGHT-INTENSITY %V 302
• DEPENDENCE OF ABSORPTION AND FLUORESCENCE EXCITATION-SPECTRA ON THE CONCENTRATION OF FUROCOUMARINS AND COUMARINS. LYSENKO, EP; POTAPENKO, AY; SUKHORUKOV, VL. In BIOFIZIKA, 33(5), S. 747–750. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1988.
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  @article{LYSENKO1988, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {LYSENKO, EP and POTAPENKO, AY and SUKHORUKOV, VL}, journal = {BIOFIZIKA}, keywords = {vladimir}, month = {09}, number = 5, pages = {747-750}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {DEPENDENCE OF ABSORPTION AND FLUORESCENCE EXCITATION-SPECTRA ON THE CONCENTRATION OF FUROCOUMARINS AND COUMARINS}, type = {Article}, volume = 33, year = 1988 }

  %0 Journal Article %1 LYSENKO1988 %A LYSENKO, EP %A POTAPENKO, AY %A SUKHORUKOV, VL %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1988 %I MEZHDUNARODNAYA KNIGA %J BIOFIZIKA %N 5 %P 747-750 %T DEPENDENCE OF ABSORPTION AND FLUORESCENCE EXCITATION-SPECTRA ON THE CONCENTRATION OF FUROCOUMARINS AND COUMARINS %V 33
• HYPOTHESIS OF THE INDUCTION OF PSORALEN PHOTOTOXIC EFFECTS THROUGH THE STAGE OF PHOTOOXIDIZED PSORALEN FORMATION - MODEL STUDIES OF ERYTHROCYTES. POTAPENKO, AY; BEZDETNAYA, LN; LYSENKO, EP; AKHTYAMOV, SN; TOMASHAEVA, SK; SUKHORUKOV, VL. In STUDIA BIOPHYSICA, 124(2-3), S. 205–223. GORDON BREACH SCI PUBL LTD, C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL, 1988.
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  @article{POTAPENKO1988, address = {C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL}, author = {POTAPENKO, AY and BEZDETNAYA, LN and LYSENKO, EP and AKHTYAMOV, SN and TOMASHAEVA, SK and SUKHORUKOV, VL}, journal = {STUDIA BIOPHYSICA}, keywords = {vladimir}, number = {2-3}, pages = {205-223}, publisher = {GORDON BREACH SCI PUBL LTD}, title = {HYPOTHESIS OF THE INDUCTION OF PSORALEN PHOTOTOXIC EFFECTS THROUGH THE STAGE OF PHOTOOXIDIZED PSORALEN FORMATION - MODEL STUDIES OF ERYTHROCYTES}, type = {Article}, volume = 124, year = 1988 }

  %0 Journal Article %1 POTAPENKO1988 %A POTAPENKO, AY %A BEZDETNAYA, LN %A LYSENKO, EP %A AKHTYAMOV, SN %A TOMASHAEVA, SK %A SUKHORUKOV, VL %C C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL %D 1988 %I GORDON BREACH SCI PUBL LTD %J STUDIA BIOPHYSICA %N 2-3 %P 205-223 %T HYPOTHESIS OF THE INDUCTION OF PSORALEN PHOTOTOXIC EFFECTS THROUGH THE STAGE OF PHOTOOXIDIZED PSORALEN FORMATION - MODEL STUDIES OF ERYTHROCYTES %V 124
• ENHANCING EFFECT OF CATECHOLAMINE DIHYDROXYPHENYLALANINE ON PSORALEN PHOTOSENSITIZED HEMOLYSIS. SUKHORUKOV, VL; POTAPENKO, AY; CHUDINOVA, OI; LYSENKO, EP; BEZDETNAYA, LN. In STUDIA BIOPHYSICA, 124(2-3), S. 235–238. GORDON BREACH SCI PUBL LTD, C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL, 1988.
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  @article{SUKHORUKOV1988, address = {C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL}, author = {SUKHORUKOV, VL and POTAPENKO, AY and CHUDINOVA, OI and LYSENKO, EP and BEZDETNAYA, LN}, journal = {STUDIA BIOPHYSICA}, keywords = {vladimir}, number = {2-3}, pages = {235-238}, publisher = {GORDON BREACH SCI PUBL LTD}, title = {ENHANCING EFFECT OF CATECHOLAMINE DIHYDROXYPHENYLALANINE ON PSORALEN PHOTOSENSITIZED HEMOLYSIS}, type = {Article}, volume = 124, year = 1988 }

  %0 Journal Article %1 SUKHORUKOV1988 %A SUKHORUKOV, VL %A POTAPENKO, AY %A CHUDINOVA, OI %A LYSENKO, EP %A BEZDETNAYA, LN %C C/O STBS LTD PO BOX 90, READING, BERKS, ENGLAND RG1 8JL %D 1988 %I GORDON BREACH SCI PUBL LTD %J STUDIA BIOPHYSICA %N 2-3 %P 235-238 %T ENHANCING EFFECT OF CATECHOLAMINE DIHYDROXYPHENYLALANINE ON PSORALEN PHOTOSENSITIZED HEMOLYSIS %V 124

• PHOTOSENSITIZED LESION OF ERYTHROCYTE-MEMBRANE INDUCED BY PSORALEN - 2 MECHANISMS. BEZDETNAYA, LN; POTAPENKO, AY; PERKHOVA, OY; NAGIEV, AI; SUKHORUKOV, VL; VLADIMIROV, YA. In BIOLOGICHESKIE MEMBRANY, 4(3), S. 270–279. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1987.
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  @article{BEZDETNAYA1987, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {BEZDETNAYA, LN and POTAPENKO, AY and PERKHOVA, OY and NAGIEV, AI and SUKHORUKOV, VL and VLADIMIROV, YA}, journal = {BIOLOGICHESKIE MEMBRANY}, keywords = {vladimir}, month = {03}, number = 3, pages = {270-279}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {PHOTOSENSITIZED LESION OF ERYTHROCYTE-MEMBRANE INDUCED BY PSORALEN - 2 MECHANISMS}, type = {Article}, volume = 4, year = 1987 }

  %0 Journal Article %1 BEZDETNAYA1987 %A BEZDETNAYA, LN %A POTAPENKO, AY %A PERKHOVA, OY %A NAGIEV, AI %A SUKHORUKOV, VL %A VLADIMIROV, YA %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1987 %I MEZHDUNARODNAYA KNIGA %J BIOLOGICHESKIE MEMBRANY %N 3 %P 270-279 %T PHOTOSENSITIZED LESION OF ERYTHROCYTE-MEMBRANE INDUCED BY PSORALEN - 2 MECHANISMS %V 4

• EFFECTS OF ANTIOXIDANTS ON PHOTOOXIDATION OF PSORALEN. POTAPENKO, AY; BEZDETNAYA, LN; WUNDERLICH, Z; SUKHORUKOV, VL; PLIQUETT, F; DUBURG, GY. In BIOFIZIKA, 31(4), S. 549–554. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1986.
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  @article{POTAPENKO1986, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {POTAPENKO, AY and BEZDETNAYA, LN and WUNDERLICH, Z and SUKHORUKOV, VL and PLIQUETT, F and DUBURG, GY}, journal = {BIOFIZIKA}, keywords = {vladimir}, month = {07}, number = 4, pages = {549-554}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {EFFECTS OF ANTIOXIDANTS ON PHOTOOXIDATION OF PSORALEN}, type = {Article}, volume = 31, year = 1986 }

  %0 Journal Article %1 POTAPENKO1986 %A POTAPENKO, AY %A BEZDETNAYA, LN %A WUNDERLICH, Z %A SUKHORUKOV, VL %A PLIQUETT, F %A DUBURG, GY %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1986 %I MEZHDUNARODNAYA KNIGA %J BIOFIZIKA %N 4 %P 549-554 %T EFFECTS OF ANTIOXIDANTS ON PHOTOOXIDATION OF PSORALEN %V 31
• PHOTOSENSITIZED MODIFICATION OF ERYTHROCYTE-MEMBRANES INDUCED BY FUROCOUMARINS. POTAPENKO, AY; WUNDERLICH, S; PLIQUETT, F; BEZDETNAYA, LN; SUKHORUKOV, VL. In PHOTOBIOCHEMISTRY AND PHOTOBIOPHYSICS, 10(3), S. 175–180. ELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS, 1986.
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  @article{POTAPENKO1986b, address = {PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS}, author = {POTAPENKO, AY and WUNDERLICH, S and PLIQUETT, F and BEZDETNAYA, LN and SUKHORUKOV, VL}, journal = {PHOTOBIOCHEMISTRY AND PHOTOBIOPHYSICS}, keywords = {vladimir}, month = {01}, number = 3, pages = {175-180}, publisher = {ELSEVIER SCIENCE BV}, title = {PHOTOSENSITIZED MODIFICATION OF ERYTHROCYTE-MEMBRANES INDUCED BY FUROCOUMARINS}, type = {Article}, volume = 10, year = 1986 }

  %0 Journal Article %1 POTAPENKO1986b %A POTAPENKO, AY %A WUNDERLICH, S %A PLIQUETT, F %A BEZDETNAYA, LN %A SUKHORUKOV, VL %C PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDS %D 1986 %I ELSEVIER SCIENCE BV %J PHOTOBIOCHEMISTRY AND PHOTOBIOPHYSICS %N 3 %P 175-180 %T PHOTOSENSITIZED MODIFICATION OF ERYTHROCYTE-MEMBRANES INDUCED BY FUROCOUMARINS %V 10
• FUROCOUMARIN-PHOTOSENSITIZED FORMATION OF SINGLET MOLECULAR-OXYGEN IN AQUEOUS AND ALCOHOL SOLUTION. EGOROV, SY; KRASNOVSKY, AA; SUKHORUKOV, VL; POTAPENKO, AY. In BIOFIZIKA, 31(1), S. 154–156. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1986.
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  @article{EGOROV1986, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {EGOROV, SY and KRASNOVSKY, AA and SUKHORUKOV, VL and POTAPENKO, AY}, journal = {BIOFIZIKA}, keywords = {vladimir}, month = {01}, number = 1, pages = {154-156}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {FUROCOUMARIN-PHOTOSENSITIZED FORMATION OF SINGLET MOLECULAR-OXYGEN IN AQUEOUS AND ALCOHOL SOLUTION}, type = {Letter}, volume = 31, year = 1986 }

  %0 Journal Article %1 EGOROV1986 %A EGOROV, SY %A KRASNOVSKY, AA %A SUKHORUKOV, VL %A POTAPENKO, AY %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1986 %I MEZHDUNARODNAYA KNIGA %J BIOFIZIKA %N 1 %P 154-156 %T FUROCOUMARIN-PHOTOSENSITIZED FORMATION OF SINGLET MOLECULAR-OXYGEN IN AQUEOUS AND ALCOHOL SOLUTION %V 31
• GENERATION AND QUENCHING OF SINGLET MOLECULAR-OXYGEN BY FUROCOUMARINS - DIRECT MEASUREMENTS. KRASNOVSKY, AA; SUKHORUKOV, VL; EGOROV, SY; POTAPENKO, AY. In STUDIA BIOPHYSICA, 114(1-3), S. 149–158. GORDON BREACH SCI PUBL LTD, C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL, 1986.
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  @article{KRASNOVSKY1986, address = {C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL}, author = {KRASNOVSKY, AA and SUKHORUKOV, VL and EGOROV, SY and POTAPENKO, AY}, journal = {STUDIA BIOPHYSICA}, keywords = {vladimir}, number = {1-3}, pages = {149-158}, publisher = {GORDON BREACH SCI PUBL LTD}, title = {GENERATION AND QUENCHING OF SINGLET MOLECULAR-OXYGEN BY FUROCOUMARINS - DIRECT MEASUREMENTS}, type = {Article}, volume = 114, year = 1986 }

  %0 Journal Article %1 KRASNOVSKY1986 %A KRASNOVSKY, AA %A SUKHORUKOV, VL %A EGOROV, SY %A POTAPENKO, AY %C C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL %D 1986 %I GORDON BREACH SCI PUBL LTD %J STUDIA BIOPHYSICA %N 1-3 %P 149-158 %T GENERATION AND QUENCHING OF SINGLET MOLECULAR-OXYGEN BY FUROCOUMARINS - DIRECT MEASUREMENTS %V 114
• MECHANISMS OF FUROCOUMARIN-SENSITIZED DAMAGE TO BIOLOGICAL-MEMBRANES. POTAPENKO, AY; BEZDETNAYA, LN; LYSENKO, EP; SUKHORUKOV, VL; REMISOV, AN; VLADIMIROV, YA. In STUDIA BIOPHYSICA, 114(1-3), S. 159–170. GORDON BREACH SCI PUBL LTD, C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL, 1986.
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  @article{POTAPENKO1986a, address = {C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL}, author = {POTAPENKO, AY and BEZDETNAYA, LN and LYSENKO, EP and SUKHORUKOV, VL and REMISOV, AN and VLADIMIROV, YA}, journal = {STUDIA BIOPHYSICA}, keywords = {vladimir}, number = {1-3}, pages = {159-170}, publisher = {GORDON BREACH SCI PUBL LTD}, title = {MECHANISMS OF FUROCOUMARIN-SENSITIZED DAMAGE TO BIOLOGICAL-MEMBRANES}, type = {Article}, volume = 114, year = 1986 }

  %0 Journal Article %1 POTAPENKO1986a %A POTAPENKO, AY %A BEZDETNAYA, LN %A LYSENKO, EP %A SUKHORUKOV, VL %A REMISOV, AN %A VLADIMIROV, YA %C C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL %D 1986 %I GORDON BREACH SCI PUBL LTD %J STUDIA BIOPHYSICA %N 1-3 %P 159-170 %T MECHANISMS OF FUROCOUMARIN-SENSITIZED DAMAGE TO BIOLOGICAL-MEMBRANES %V 114

• A COMPARISON BETWEEN SKIN-PHOTOSENSITIZING (334 NM) ACTIVITIES OF 8-METHOXYPSORALEN AND ANGELICIN. POTAPENKO, AY; SUKHORUKOV, VL; DAVIDOV, BV. In EXPERIENTIA, 40(3), S. 264–265. BIRKHAUSER VERLAG AG, PO BOX 133 KLOSTERBERG 23, CH-4010 BASEL, SWITZERLAND, 1984.
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  @article{POTAPENKO1984a, address = {PO BOX 133 KLOSTERBERG 23, CH-4010 BASEL, SWITZERLAND}, author = {POTAPENKO, AY and SUKHORUKOV, VL and DAVIDOV, BV}, journal = {EXPERIENTIA}, keywords = {vladimir}, number = 3, pages = {264-265}, publisher = {BIRKHAUSER VERLAG AG}, title = {A COMPARISON BETWEEN SKIN-PHOTOSENSITIZING (334 NM) ACTIVITIES OF 8-METHOXYPSORALEN AND ANGELICIN}, type = {Note}, volume = 40, year = 1984 }

  %0 Journal Article %1 POTAPENKO1984a %A POTAPENKO, AY %A SUKHORUKOV, VL %A DAVIDOV, BV %C PO BOX 133 KLOSTERBERG 23, CH-4010 BASEL, SWITZERLAND %D 1984 %I BIRKHAUSER VERLAG AG %J EXPERIENTIA %N 3 %P 264-265 %T A COMPARISON BETWEEN SKIN-PHOTOSENSITIZING (334 NM) ACTIVITIES OF 8-METHOXYPSORALEN AND ANGELICIN %V 40
• PHOTOLYSIS OF 3,4-DIOXYPHENYLALANINE SENSITIZED BY 8-METHOXYPSORALENE - ACCELERATION BY SULFOHYDRYL GROUPS. SUKHORUKOV, VL; POTAPENKO, AY. In ZHURNAL FIZICHESKOI KHIMII, 58(2), S. 470–472. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1984.
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  @article{SUKHORUKOV1984, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {SUKHORUKOV, VL and POTAPENKO, AY}, journal = {ZHURNAL FIZICHESKOI KHIMII}, keywords = {vladimir}, number = 2, pages = {470-472}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {PHOTOLYSIS OF 3,4-DIOXYPHENYLALANINE SENSITIZED BY 8-METHOXYPSORALENE - ACCELERATION BY SULFOHYDRYL GROUPS}, type = {Note}, volume = 58, year = 1984 }

  %0 Journal Article %1 SUKHORUKOV1984 %A SUKHORUKOV, VL %A POTAPENKO, AY %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1984 %I MEZHDUNARODNAYA KNIGA %J ZHURNAL FIZICHESKOI KHIMII %N 2 %P 470-472 %T PHOTOLYSIS OF 3,4-DIOXYPHENYLALANINE SENSITIZED BY 8-METHOXYPSORALENE - ACCELERATION BY SULFOHYDRYL GROUPS %V 58
• PHOTOOXIDATIVE REACTIONS OF PSORALENS. POTAPENKO, AY; SUKHORUKOV, VL. In STUDIA BIOPHYSICA, 101, S. 89–98. GORDON BREACH SCI PUBL LTD, C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL, 1984.
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  @article{POTAPENKO1984, address = {C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL}, author = {POTAPENKO, AY and SUKHORUKOV, VL}, journal = {STUDIA BIOPHYSICA}, keywords = {vladimir}, pages = {89-98}, publisher = {GORDON BREACH SCI PUBL LTD}, title = {PHOTOOXIDATIVE REACTIONS OF PSORALENS}, type = {Article}, volume = 101, year = 1984 }

  %0 Journal Article %1 POTAPENKO1984 %A POTAPENKO, AY %A SUKHORUKOV, VL %C C/O STBS LTD, PO BOX 90, READING, BERKS, ENGLAND RG1 8JL %D 1984 %I GORDON BREACH SCI PUBL LTD %J STUDIA BIOPHYSICA %P 89-98 %T PHOTOOXIDATIVE REACTIONS OF PSORALENS %V 101

• OXIDATION OF LIPIDS BY DIMERIC PHOTOOXIDIZING 8-METHOXYPSORALENE. SUKHORUKOV, VL; POTAPENKO, AY. In ZHURNAL FIZICHESKOI KHIMII, 57(5), S. 1320–1321. MEZHDUNARODNAYA KNIGA, 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA, 1983.
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  @article{SUKHORUKOV1983a, address = {39 DIMITROVA UL., 113095 MOSCOW, RUSSIA}, author = {SUKHORUKOV, VL and POTAPENKO, AY}, journal = {ZHURNAL FIZICHESKOI KHIMII}, keywords = {vladimir}, number = 5, pages = {1320-1321}, publisher = {MEZHDUNARODNAYA KNIGA}, title = {OXIDATION OF LIPIDS BY DIMERIC PHOTOOXIDIZING 8-METHOXYPSORALENE}, type = {Letter}, volume = 57, year = 1983 }

  %0 Journal Article %1 SUKHORUKOV1983a %A SUKHORUKOV, VL %A POTAPENKO, AY %C 39 DIMITROVA UL., 113095 MOSCOW, RUSSIA %D 1983 %I MEZHDUNARODNAYA KNIGA %J ZHURNAL FIZICHESKOI KHIMII %N 5 %P 1320-1321 %T OXIDATION OF LIPIDS BY DIMERIC PHOTOOXIDIZING 8-METHOXYPSORALENE %V 57