• [ 2025 ]
• [ 2024 ]
• [ 2023 ]
• [ 2022 ]
• [ 2021 ]
• [ 2020 ]
• [ 2019 ]
• [ 2018 ]
• [ 2017 ]
• [ 2016 ]
• [ 2015 ]
• [ 2014 ]
• [ 2013 ]
• [ 2012 ]
• [ 2011 ]
• [ 2010 ]
• [ 2009 ]
• [ 2008 ]
• [ 2007 ]
• [ 2006 ]
• [ 2005 ]
• [ 2004 ]
• [ 2003 ]
• [ 2002 ]
• [ 2001 ]
• [ 2000 ]
• [ 1999 ]
• [ 1998 ]
• [ 1997 ]
• [ 1996 ]
• [ 1995 ]
• [ 1994 ]
• [ 1993 ]

• 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 ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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 ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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 ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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).
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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 &amp; Neuroinflammation, 11(5), S. e200284-. Wolters Kluwer, 2024.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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 ]
  • [ BibTeX ]
  • [ EndNote ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.

• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  @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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.

• 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 ]
  • [ BibTeX ]
  • [ EndNote ]
  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 ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  (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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  (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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  @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
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.

• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• Super-resolving Microscopy in Neuroscience. Werner, Christian; Sauer, Markus; Geis, Christian. In Chem. Rev., 121(19), S. 11971–12015. American Chemical Society, 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• Superresolution Microscopy of Sphingolipids. Schlegel, Jan; Sauer, Markus. In Lipid Rafts: Methods and Protocols, S. 303–311. Springer US, New York, NY, 2021.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.

• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• A Trojan Horse for live-cell super-resolution microscopy. Beliu, Gerti; Sauer, Markus. In Light: Science & Applications, 9(1), S. 2-. 2020.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• ‘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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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).
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer. Helmerich, Dominic A.; Beliu, Gerti; Sauer, Markus. In ACS Nano. American Chemical Society, 2020.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  @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

• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.

• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
• 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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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.
  • [ Abstract ]
  • [ BibTeX ]
  • [ EndNote ]
  • [ URL ]
  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 cultu