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• 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 (2022). 14 5794.
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  (1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.

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

• 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 (2021). 81(5) 905–921.e5.
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  Summary 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 = {Summary 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 Summary 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.
• 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 (2021). 35(1) 201–214.
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  Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.

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

  %0 Journal Article %1 garciaguerrero2021upregulation %A García-Guerrero, Estefanía %A Götz, Ralph %A Doose, Sören %A Sauer, Markus %A Rodríguez-Gil, Alfonso %A Nerreter, Thomas %A Kortüm, K. Martin %A Pérez-Simón, José A. %A Einsele, Hermann %A Hudecek, Michael %A Danhof, Sophia %D 2021 %J Leukemia %N 1 %P 201--214 %R 10.1038/s41375-020-0840-y %T Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab %U https://doi.org/10.1038/s41375-020-0840-y %V 35 %X Multiple myeloma (MM) is incurable, so there is a significant unmet need for effective therapy for patients with relapsed or refractory disease. This situation has not changed despite the recent approval of the anti-CD38 antibody daratumumab, one of the most potent agents in MM treatment. The efficiency of daratumumab might be improved by combining it with synergistic anti-MM agents. We therefore investigated the potential of the histone deacetylase (HDAC) inhibitor ricolinostat to up-regulate CD38 on MM cells, thereby enhancing the performance of CD38-specific therapies. Using quantitative reverse transcription polymerase chain reaction and flow cytometry, we observed that ricolinostat significantly increases CD38 RNA levels and CD38 surface expression on MM cells. Super-resolution microscopy imaging of MM cells by direct stochastic optical reconstruction microscopy confirmed this rise with molecular resolution and revealed homogeneous distribution of CD38 molecules on the cell membrane. Particularly important is that combining ricolinostat with daratumumab induced enhanced lysis of MM cells. We also evaluated next-generation HDAC6 inhibitors (ACY-241, WT-161) and observed similar increase of CD38 levels suggesting that the upregulation of CD38 expression on MM cells by HDAC6 inhibitors is a class effect. This proof-of-concept illustrates the potential benefit of combining HDAC6 inhibitors and CD38-directed immunotherapy for MM treatment.
• 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 (2021). 51(2) 342–353.
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  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.

  @article{fernandezorth2021task2, abstract = {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 = {vladimir}, month = {feb}, 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 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.
• 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 (2021). 4(1) 1151-.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.

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

  %0 Journal Article %1 trinks2021subdiffractionresolution %A Trinks, Nora %A Reinhard, Sebastian %A Drobny, Matthias %A Heilig, Linda %A Löffler, Jürgen %A Sauer, Markus %A Terpitz, Ulrich %D 2021 %J Communications Biology %N 1 %P 1151-- %R 10.1038/s42003-021-02669-y %T Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy %U https://doi.org/10.1038/s42003-021-02669-y %V 4 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells’ lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution.
• Superresolution Microscopy of Sphingolipids Schlegel, Jan; Sauer, Markus in Lipid Rafts: Methods and Protocols (2021). 303–311.
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  This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment.

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

  %0 Book Section %1 schlegel2021superresolution %A Schlegel, Jan %A Sauer, Markus %B Lipid Rafts: Methods and Protocols %C New York, NY %D 2021 %I Springer US %P 303--311 %R 10.1007/978-1-0716-0814-2_17 %T Superresolution Microscopy of Sphingolipids %U https://doi.org/10.1007/978-1-0716-0814-2_17 %X This chapter provides a step-by-step protocol to label and visualize sphingolipids by superresolution microscopy with a special focus on single-molecule localization microscopy by dSTORM. We provide information on custom fluorophore conjugation to raft-associated toxins and antibodies, and a labeling protocol for appropriate sample treatment. %@ 9781071608142
• 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 (2021). 18(3) 253–257.
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  Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.

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

  %0 Journal Article %1 helmerich2021photoblueing %A Helmerich, Dominic A. %A Beliu, Gerti %A Matikonda, Siddharth S. %A Schnermann, Martin J. %A Sauer, Markus %D 2021 %J Nature Methods %N 3 %P 253--257 %R 10.1038/s41592-021-01061-2 %T Photoblueing of organic dyes can cause artifacts in super-resolution microscopy %U https://doi.org/10.1038/s41592-021-01061-2 %V 18 %X Illumination of fluorophores can induce a loss of the ability to fluoresce, known as photobleaching. Interestingly, some fluorophores photoconvert to a blue-shifted fluorescent molecule as an intermediate on the photobleaching pathway, which can complicate multicolor fluorescence imaging, especially under the intense laser irradiation used in super-resolution fluorescence imaging. Here, we discuss the mechanisms of photoblueing of fluorophores and its impact on fluorescence imaging, and show how it can be prevented.
• 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 (2021). 51(3) 738–741.
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  Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.

  @article{haack2021superagonistic, abstract = {Like human Th1 cells, mouse Th1 cells also secrete IFN-? upon stimulation with a superagonistic anti-CD28 monoclonal antibody (CD28-SA). Crosslinking of the CD28-SA via FcR and CD40-CD40L interactions greatly increased IFN-? release. Our data stress the utility of the mouse as a model organism for immune responses in humans.}, author = {Haack, Stephanie and Baiker, Sarah and Schlegel, Jan and Sauer, Markus and Sparwasser, Tim and Langenhorst, Daniela and Beyersdorf, Niklas}, booktitle = {European Journal of Immunology}, journal = {European Journal of Immunology}, keywords = {sauer}, month = {mar}, 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.
• 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, P. Guichard, V. Hamel (Hrsg.) (2021). (Bd. 161) 317–340.
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  This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.

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

  %0 Book Section %1 zwettler2021chapter %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Sauer, Markus %B Expansion Microscopy for Cell Biology %D 2021 %E Guichard, Paul %E Hamel, Virginie %I Academic Press %P 317--340 %R https://doi.org/10.1016/bs.mcb.2020.05.004 %T Chapter 15 - Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures %U https://www.sciencedirect.com/science/article/pii/S0091679X20301126 %V 161 %X This chapter provides a step-by-step protocol how to prepare expansion microcoscopy (ExM) treated biological samples for imaging with single-molecule localization microscopy (SMLM). For this purpose, the protocol describes the stabilization of expanded hydrogels that enables addition of photoswitching buffer without shrinkage of the sample. In addition, a guide for automated image analysis and expansion factor determination of expanded fiber-like structures is provided at the end of the chapter.
• 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 (2021). 4(1) 407-.
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  Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

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

  %0 Journal Article %1 pauli2021targeted %A Pauli, Martin %A Paul, Mila M. %A Proppert, Sven %A Mrestani, Achmed %A Sharifi, Marzieh %A Repp, Felix %A Kürzinger, Lydia %A Kollmannsberger, Philip %A Sauer, Markus %A Heckmann, Manfred %A Sirén, Anna-Leena %D 2021 %J Communications Biology %N 1 %P 407-- %R 10.1038/s42003-021-01939-z %T Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections %U https://doi.org/10.1038/s42003-021-01939-z %V 4 %X Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.
• 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 (2021). 128(2) 225–241.
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  Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.

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

  %0 Journal Article %1 jansch2021serotoninspecific %A Jansch, Charline %A Ziegler, Georg C. %A Forero, Andrea %A Gredy, Sina %A Wäldchen, Sina %A Vitale, Maria Rosaria %A Svirin, Evgeniy %A Zöller, Johanna E. M. %A Waider, Jonas %A Günther, Katharina %A Edenhofer, Frank %A Sauer, Markus %A Wischmeyer, Erhard %A Lesch, Klaus-Peter %D 2021 %J Journal of Neural Transmission %N 2 %P 225--241 %R 10.1007/s00702-021-02303-5 %T Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly %U https://doi.org/10.1007/s00702-021-02303-5 %V 128 %X Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
• 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. (2021). 19(10) 2203–2212.
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  Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

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

  %0 Journal Article %1 fink2021azidosphinganine %A Fink, Julian %A Schumacher, Fabian %A Schlegel, Jan %A Stenzel, Philipp %A Wigger, Dominik %A Sauer, Markus %A Kleuser, Burkhard %A Seibel, Jürgen %D 2021 %I The Royal Society of Chemistry %J Org. Biomol. Chem. %N 10 %P 2203--2212 %R 10.1039/D0OB02592E %T Azidosphinganine enables metabolic labeling and detection of sphingolipid de novo synthesis %U http://dx.doi.org/10.1039/D0OB02592E %V 19 %X Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.
• 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 (2021). 11(1) 4300-.
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  Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.

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

  %0 Journal Article %1 peters2021clickcorrelative %A Peters, Simon %A Kaiser, Lena %A Fink, Julian %A Schumacher, Fabian %A Perschin, Veronika %A Schlegel, Jan %A Sauer, Markus %A Stigloher, Christian %A Kleuser, Burkhard %A Seibel, Jürgen %A Schubert-Unkmeir, Alexandra %D 2021 %J Scientific Reports %N 1 %P 4300-- %R 10.1038/s41598-021-83813-w %T Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria %U https://doi.org/10.1038/s41598-021-83813-w %V 11 %X Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
• 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 (2021).
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  Summary 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 = {Summary 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}, title = {Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis}, 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 %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 %X Summary 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.

• 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 (2020). 11(1) 3388-.
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  Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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

  %0 Journal Article %1 zwettler2020molecular %A Zwettler, Fabian U. %A Reinhard, Sebastian %A Gambarotto, Davide %A Bell, Toby D. M. %A Hamel, Virginie %A Guichard, Paul %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3388-- %R 10.1038/s41467-020-17086-8 %T Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM) %U https://doi.org/10.1038/s41467-020-17086-8 %V 11 %X Expansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining ExM with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.
• Multiple-Labeled Antibodies Behave Like Single Emitters in Photoswitching Buffer Helmerich, Dominic A.; Beliu, Gerti; Sauer, Markus in ACS Nano (2020).
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  The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.

  @article{helmerich2020multiplelabeled, abstract = {The degree of labeling (DOL) of antibodies has so far been optimized for high brightness and specific and efficient binding. The influence of the DOL on the blinking performance of antibodies used in direct stochastic optical reconstruction microscopy (dSTORM) has so far attained limited attention. Here, we investigated the spectroscopic characteristics of IgG antibodies labeled at DOLs of 1.1–8.3 with Alexa Fluor 647 (Al647) at the ensemble and single-molecule level. Multiple-Al647-labeled antibodies showed weak and strong quenching interactions in aqueous buffer but could all be used for dSTORM imaging with spatial resolutions of ∼20 nm independent of the DOL. Single-molecule fluorescence trajectories and photon antibunching experiments revealed that individual multiple-Al647-labeled antibodies show complex photophysics in aqueous buffer but behave as single emitters in photoswitching buffer independent of the DOL. We developed a model that explains the observed blinking of multiple-labeled antibodies and can be used for the development of improved fluorescent probes for dSTORM experiments.}, author = {Helmerich, Dominic A. and Beliu, Gerti and Sauer, Markus}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = {aug}, 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.
• 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 (2020). 14(10) 14190–14200.
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  Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

  @article{thiele2020confocal, abstract = {Fluorescence lifetime imaging microscopy is an important technique that adds another dimension to intensity and color acquired by conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is stimulated emission depletion microscopy. In contrast, all single-molecule localization microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine fluorescence-lifetime confocal laser-scanning microscopy with SMLM for realizing single-molecule localization-based fluorescence-lifetime super-resolution imaging. Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localized molecules. We validate our technique by applying it to direct stochastic optical reconstruction microscopy and points accumulation for imaging in nanoscale topography imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.}, author = {Thiele, Jan Christoph and Helmerich, Dominic A. and Oleksiievets, Nazar and Tsukanov, Roman and Butkevich, Eugenia and Sauer, Markus and Nevskyi, Oleksii and Enderlein, Jörg}, booktitle = {ACS Nano}, journal = {ACS Nano}, keywords = {sauer}, month = {oct}, 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.
• 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, D. M. Chenoweth (Hrsg.) (2020). (Bd. 641) 225–244.
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  Pentamethine cyanines are a class of far-red fluorophores that find extensive use in single-molecule localization microscopy (SMLM), as well as a broad range of other techniques. A drawback of this scaffold is its relatively low quantum yields, which is due to excited state deactivation via trans-to-cis chromophore isomerization. Here we describe a synthetic strategy to improve the photon output of these molecules. In the key synthetic transformation, a protected dialdehyde precursor undergoes a cascade reaction that forms a tetracyclic ring system. The resulting conformationally restrained analogs exhibit improved fluorescence quantum yield and extended fluorescence lifetimes. These properties, together with their ability to efficiently recover from hydride reduction, enable a uniquely simple form of single-molecule localization microscopy (SMLM).

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

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

  %0 Journal Article %1 kouhestani2020variant %A Kouhestani, Dina %A Geis, Maria %A Alsouri, Saed %A Bumm, Thomas G. P. %A Einsele, Hermann %A Sauer, Markus %A Stuhler, Gernot %D 2020 %J Cellular & Molecular Immunology %R 10.1038/s41423-020-0507-7 %T Variant signaling topology at the cancer cell–T-cell interface induced by a two-component T-cell engager %U https://doi.org/10.1038/s41423-020-0507-7
• 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 (2020). 130(11) 6064–6079.
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  Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

  @article{volz2020orchestrates, abstract = {Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.}, author = {Volz, Julia and Kusch, Charly and Beck, Sarah and Popp, Michael and Vögtle, Timo and Meub, Mara and Scheller, Inga and Heil, Hannah S. and Preu, Julia and Schuhmann, Michael K. and Hemmen, Katherina and Premsler, Thomas and Sickmann, Albert and Heinze, Katrin G. and Stegner, David and Stoll, Guido and Braun, Attila and Sauer, Markus and Nieswandt, Bernhard}, journal = {The Journal of Clinical Investigation}, keywords = {sauer}, month = {nov}, 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.
• 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. (2020). 40(33) 6289-.
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  Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.

  @article{andreska2020induction, abstract = {Motor learning depends on synaptic plasticity between corticostriatal projections and striatal medium spiny neurons. Retrograde tracing from the dorsolateral striatum reveals that both layer II/III and V neurons in the motor cortex express BDNF as a potential regulator of plasticity in corticostriatal projections in male and female mice. The number of these BDNF-expressing cortical neurons and levels of BDNF protein are highest in juvenile mice when adult motor patterns are shaped, while BDNF levels in the adult are low. When mice are trained by physical exercise in the adult, BDNF expression in motor cortex is reinduced, especially in layer II/III projection neurons. Reduced expression of cortical BDNF in 3-month-old mice results in impaired motor learning while space memory is preserved. These findings suggest that activity regulates BDNF expression differentially in layers II/III and V striatal afferents from motor cortex and that cortical BDNF is essential for motor learning.SIGNIFICANCE STATEMENT Motor learning in mice depends on corticostriatal BDNF supply, and regulation of BDNF expression during motor learning is highest in corticostriatal projection neurons in cortical layer II/III.}, author = {Andreska, Thomas and Rauskolb, Stefanie and Schukraft, Nina and Lüningschrör, Patrick and Sasi, Manju and Signoret-Genest, Jeremy and Behringer, Marcus and Blum, Robert and Sauer, Markus and Tovote, Philip and Sendtner, Michael}, journal = {J. Neurosci.}, keywords = {sauer}, month = {aug}, 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.
• 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 (2020). 11
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  Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

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

  %0 Journal Article %1 weiss2020reconstituting %A Weiss, Esther %A Schlegel, Jan %A Terpitz, Ulrich %A Weber, Michael %A Linde, Jörg %A Schmitt, Anna-Lena %A Hünniger, Kerstin %A Marischen, Lothar %A Gamon, Florian %A Bauer, Joachim %A Löffler, Claudia %A Kurzai, Oliver %A Morton, Charles Oliver %A Sauer, Markus %A Einsele, Hermann %A Loeffler, Juergen %B 2117 %D 2020 %J Frontiers in Immunology %T Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus %U https://www.frontiersin.org/article/10.3389/fimmu.2020.02117 %V 11 %X Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56^brightCD16^dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
• 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 (2020). 8 617-.
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  Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.

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

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

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

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

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

  %0 Journal Article %1 forero2020serotonin %A Forero, Andrea %A Ku, Hsing-Ping %A Malpartida, Ana Belén %A Wäldchen, Sina %A Alhama-Riba, Judit %A Kulka, Christina %A Aboagye, Benjamin %A Norton, William H.J %A Young, Andrew M.J %A Ding, Yu-Qiang %A Blum, Robert %A Sauer, Markus %A Rivero, Olga %A Lesch, Klaus-Peter %D 2020 %J Neuropharmacology %P 108018-- %R https://doi.org/10.1016/j.neuropharm.2020.108018 %T Serotonin (5-HT) neuron-specific inactivation of Cadherin-13 impacts 5-HT system formation and cognitive function %U https://www.sciencedirect.com/science/article/pii/S0028390820300848 %V 168 %X Genome-wide screening approaches identified the cell adhesion molecule Cadherin-13 (CDH13) as a risk factor for neurodevelopmental disorders, nevertheless the contribution of CDH13 to the disease mechanism remains obscure. CDH13 is involved in neurite outgrowth and axon guidance during early brain development and we previously provided evidence that constitutive CDH13 deficiency influences the formation of the raphe serotonin (5-HT) system by modifying neuron-radial glia interaction. Here, we dissect the specific impact of CDH13 on 5-HT system development and function using a 5-HT neuron-specific Cdh13 knockout mouse model (conditional Cdh13 knockout, Cdh13 cKO). Our results show that exclusive inactivation of CDH13 in 5-HT neurons selectively increases 5-HT neuron density in the embryonic dorsal raphe, with persistence into adulthood, and serotonergic innervation of the developing prefrontal cortex. At the behavioral level, adult Cdh13 cKO mice display delayed acquisition of several learning tasks and a subtle impulsive-like phenotype, with decreased latency in a sociability paradigm alongside with deficits in visuospatial memory. Anxiety-related traits were not observed in Cdh13 cKO mice. Our findings further support the critical role of CDH13 in the development of dorsal raphe 5-HT circuitries, a mechanism that may underlie specific clinical features observed in neurodevelopmental disorders.
• ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein Rozario, Ashley M.; Zwettler, Fabian; Duwé, Sam; Hargreaves, Riley B.; Brice, Aaron; Dedecker, Peter; Sauer, Markus; Moseley, Gregory W.; Whelan, Donna R.; Bell, Toby D. M. in Aust. J. Chem. (2020).
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  The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

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

  %0 Journal Article %1 rozario2020large %A Rozario, Ashley M. %A Zwettler, Fabian %A Duwé, Sam %A Hargreaves, Riley B. %A Brice, Aaron %A Dedecker, Peter %A Sauer, Markus %A Moseley, Gregory W. %A Whelan, Donna R. %A Bell, Toby D. M. %D 2020 %J Aust. J. Chem. %T ‘Live and Large’: Super-Resolution Optical Fluctuation Imaging (SOFI) and Expansion Microscopy (ExM) of Microtubule Remodelling by Rabies Virus P Protein %U https://doi.org/10.1071/CH19571 %X The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.
• 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 (2020). 11(1) 3222-.
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  The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

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

  %0 Journal Article %1 zwettler2020tracking %A Zwettler, Fabian U. %A Spindler, Marie-Christin %A Reinhard, Sebastian %A Klein, Teresa %A Kurz, Andreas %A Benavente, Ricardo %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 3222-- %R 10.1038/s41467-020-17017-7 %T Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy %U https://doi.org/10.1038/s41467-020-17017-7 %V 11 %X The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different expansion microscopy (ExM) protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased comparison of structural properties pre- and post-expansion. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20–30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.
• 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 (2020). 6(16) eaay7193-.
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  G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.

  @article{siddig2020superresolution, abstract = {G protein–coupled receptors (GPCRs) play a fundamental role in the modulation of synaptic transmission. A pivotal example is provided by the metabotropic glutamate receptor type 4 (mGluR4), which inhibits glutamate release at presynaptic active zones (AZs). However, how GPCRs are organized within AZs to regulate neurotransmission remains largely unknown. Here, we applied two-color super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the nanoscale organization of mGluR4 at parallel fiber AZs in the mouse cerebellum. We find an inhomogeneous distribution, with multiple nanodomains inside AZs, each containing, on average, one to two mGluR4 subunits. Within these nanodomains, mGluR4s are often localized in close proximity to voltage-dependent CaV2.1 channels and Munc-18-1, which are both essential for neurotransmitter release. These findings provide previously unknown insights into the molecular organization of GPCRs at AZs, suggesting a likely implication of a close association between mGluR4 and the secretory machinery in modulating synaptic transmission.}, author = {Siddig, Sana and Aufmkolk, Sarah and Doose, Sören and Jobin, Marie-Lise and Werner, Christian and Sauer, Markus and Calebiro, Davide}, journal = {Sci Adv}, keywords = {werner}, month = {apr}, 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.
• 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 (2020). 11
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  Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

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

  %0 Journal Article %1 gotz2020expansion %A Götz, Ralph %A Panzer, Sabine %A Trinks, Nora %A Eilts, Janna %A Wagener, Johannes %A Turrà, David %A Di Pietro, Antonio %A Sauer, Markus %A Terpitz, Ulrich %B 574 %D 2020 %J Frontiers in Microbiology %T Expansion Microscopy for Cell Biology Analysis in Fungi %U https://www.frontiersin.org/article/10.3389/fmicb.2020.00574 %V 11 %X Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.
• A Trojan Horse for live-cell super-resolution microscopy Beliu, Gerti; Sauer, Markus in Light: Science & Applications (2020). 9(1) 2-.
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  New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.

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

  %0 Journal Article %1 beliu2020trojan %A Beliu, Gerti %A Sauer, Markus %D 2020 %J Light: Science & Applications %N 1 %P 2-- %R 10.1038/s41377-019-0238-7 %T A Trojan Horse for live-cell super-resolution microscopy %U https://doi.org/10.1038/s41377-019-0238-7 %V 9 %X New peptide vehicles enable the efficient live-cell labeling of intracellular organelles with cell-impermeable fluorescent probes by simple coincubation, paving the way for refined multicolor super-resolution fluorescence imaging.
• 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 (2020). 11(1) 887-.
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  The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.

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

  %0 Journal Article %1 waldchen2020wholecell %A Wäldchen, Felix %A Schlegel, Jan %A Götz, Ralph %A Luciano, Michael %A Schnermann, Martin %A Doose, Sören %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 887-- %R 10.1038/s41467-020-14731-0 %T Whole-cell imaging of plasma membrane receptors by 3D lattice light-sheet dSTORM %U https://doi.org/10.1038/s41467-020-14731-0 %V 11 %X The molecular organization of receptors in the plasma membrane of cells is paramount for their functionality. We combined lattice light-sheet (LLS) microscopy with three-dimensional (3D) single-molecule localization microscopy (dSTORM) and single-particle tracking to quantify the expression and distribution, and mobility of CD56 receptors on whole fixed and living cells, finding that CD56 accumulated at cell–cell interfaces. For comparison, we investigated two other receptors, CD2 and CD45, which showed different expression levels and distributions in the plasma membrane. Overall, 3D-LLS-dSTORM enabled imaging and single-particle tracking of plasma membrane receptors with single-molecule sensitivity unperturbed by surface effects. Our results demonstrate that receptor distribution and mobility are largely unaffected by contact to the coverslip but the measured localization densities are in general lower at the basal plasma membrane due to partial limited accessibility for antibodies.
• 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 (2020). 11(1) 6173-.
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  Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

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

  %0 Journal Article %1 gotz2020nanoscale %A Götz, Ralph %A Kunz, Tobias C. %A Fink, Julian %A Solger, Franziska %A Schlegel, Jan %A Seibel, Jürgen %A Kozjak-Pavlovic, Vera %A Rudel, Thomas %A Sauer, Markus %D 2020 %J Nature Communications %N 1 %P 6173-- %R 10.1038/s41467-020-19897-1 %T Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy %U https://doi.org/10.1038/s41467-020-19897-1 %V 11 %X Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

• 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 (2019). 10 1294-.
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  Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.

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

  %0 Journal Article %1 derakhshani2019measles %A Derakhshani, Shaghayegh %A Kurz, Andreas %A Japtok, Lukasz %A Schumacher, Fabian %A Pilgram, Lisa %A Steinke, Maria %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2019 %J Frontiers in Immunology %P 1294-- %T Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium %U https://www.frontiersin.org/article/10.3389/fimmu.2019.01294 %V 10 %X Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility towards the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity cell autonomously. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus towards epithelial cells during viral exit.
• Super-resolution microscopy demystified Schermelleh, Lothar; Ferrand, Alexia; Huser, Thomas; Eggeling, Christian; Sauer, Markus; Biehlmaier, Oliver; Drummen, Gregor P. C. in Nature Cell Biology (2019). 21(1) 72–84.
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  Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

  @article{schermelleh2019superresolution, abstract = {Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.}, author = {Schermelleh, Lothar and Ferrand, Alexia and Huser, Thomas and Eggeling, Christian and Sauer, Markus and Biehlmaier, Oliver and Drummen, Gregor P. C.}, journal = {Nature Cell Biology}, keywords = {sauer}, 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.
• Registration and Visualization of Correlative Super-Resolution Microscopy Data Reinhard, Sebastian; Aufmkolk, Sarah; Sauer, Markus; Doose, Sören in Biophysical Journal (2019). 116(11) 2073–2078.
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  We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.

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

  %0 Journal Article %1 reinhard2019registration %A Reinhard, Sebastian %A Aufmkolk, Sarah %A Sauer, Markus %A Doose, Sören %D 2019 %J Biophysical Journal %N 11 %P 2073--2078 %R https://doi.org/10.1016/j.bpj.2019.04.029 %T Registration and Visualization of Correlative Super-Resolution Microscopy Data %U http://www.sciencedirect.com/science/article/pii/S000634951930373X %V 116 %X We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
• 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 (2019). 116(5) 1017–1028.
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  Abstract Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the Fc?RIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased Fc?RIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

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

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

  @article{zilkowski2019nanogels, abstract = {Multiple myeloma is a common plasma-cell-derived hematologic neoplasm. While the delivery of growth-inhibiting miRNA to multiple myeloma cells would be a promising strategy to evaluate treatment options, most multiple myeloma cells are transfection-resistant with established methods. Nonviral nanoparticulate transfection systems are particularly promising in this context, but so far struggle with transfection and knockdown efficiency. Here, we present poly(glycidol)-based nanogels with covalently bound cell-penetrating peptide TAT (transactivator of transcription from HIV). TAT facilitated a varying internalization efficiency of the nanogels depending on the cell line. The positively charged peptide also served as complexation agent for miRNA and enabled covalent binding of the TAT/miR-34a complex in the nanogels. These TAT/miRNA-loaded nanogels delivered and released miR-34a with high efficiency into OPM-2 multiple myeloma cells that are known as transfection-resistant. Delivery resulted in efficient downregulation of known target genes such as Notch1, Hey1, Hes6, and Hes1. Thus, these nanogel constructs offer a new tool to enhance gene delivery into multiple myeloma cells with immediate value in cancer research.}, author = {Zilkowski, Ilona and Ziouti, Fani and Schulze, Andres and Hauck, Stefanie and Schmidt, Stefanie and Mainz, Laura and Sauer, Markus and Albrecht, Krystyna and Jundt, Franziska and Groll, Jürgen}, booktitle = {Biomacromolecules}, journal = {Biomacromolecules}, keywords = {sauer}, month = {feb}, 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.
• 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., (A. J. Bäumler, Hrsg.) (2019). 87(8) e00410–19.
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  Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

  @article{peters2019ltspan, abstract = {Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.}, author = {Peters, Simon and Schlegel, Jan and Becam, Jérôme and Avota, Elita and Sauer, Markus and Schubert-Unkmeir, Alexandra}, editor = {Bäumler, Andreas J.}, journal = {Infect. Immun.}, keywords = {sauer}, month = {aug}, 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.
• 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 (2019). 2(1) 261-.
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  Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

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

  %0 Journal Article %1 beliu2019bioorthogonal %A Beliu, Gerti %A Kurz, Andreas J. %A Kuhlemann, Alexander C. %A Behringer-Pliess, Lisa %A Meub, Mara %A Wolf, Natalia %A Seibel, Jürgen %A Shi, Zhen-Dan %A Schnermann, Martin %A Grimm, Jonathan B. %A Lavis, Luke D. %A Doose, Sören %A Sauer, Markus %D 2019 %J Communications Biology %N 1 %P 261-- %R 10.1038/s42003-019-0518-z %T Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy %U https://doi.org/10.1038/s42003-019-0518-z %V 2 %X Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
• Super-resolution microscopy 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 (2019). 10(1) 3137-.
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  Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.

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

  %0 Journal Article %1 nerreter2019superresolution %A Nerreter, Thomas %A Letschert, Sebastian %A Götz, Ralph %A Doose, Sören %A Danhof, Sophia %A Einsele, Hermann %A Sauer, Markus %A Hudecek, Michael %D 2019 %J Nature Communications %N 1 %P 3137-- %R 10.1038/s41467-019-10948-w %T Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T %U https://doi.org/10.1038/s41467-019-10948-w %V 10 %X Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
• 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 (2019). 16(1) 71–74.
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  Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

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

  %0 Journal Article %1 gambarotto2019imaging %A Gambarotto, Davide %A Zwettler, Fabian U. %A Le Guennec, Maeva %A Schmidt-Cernohorska, Marketa %A Fortun, Denis %A Borgers, Susanne %A Heine, Jörn %A Schloetel, Jan-Gero %A Reuss, Matthias %A Unser, Michael %A Boyden, Edward S. %A Sauer, Markus %A Hamel, Virginie %A Guichard, Paul %D 2019 %J Nature Methods %N 1 %P 71--74 %R 10.1038/s41592-018-0238-1 %T Imaging cellular ultrastructures using expansion microscopy (U-ExM) %U https://doi.org/10.1038/s41592-018-0238-1 %V 16 %X Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
• 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 (2019). 7 194-.
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  Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.

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

  %0 Journal Article %1 schlegel2019superresolution %A Schlegel, Jan %A Peters, Simon %A Doose, Sören %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2019 %J Frontiers in Cell and Developmental Biology %P 194-- %T Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites %U https://www.frontiersin.org/article/10.3389/fcell.2019.00194 %V 7 %X Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.
• 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 (2019). 134 2318–2329.
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  During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.

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

  %0 Journal Article %1 schurr2019platelet %A Schurr, Yvonne %A Sperr, Andreas %A Volz, Julia %A Beck, Sarah %A Reil, Lucy %A Kusch, Charly %A Eiring, Patrick %A Bryson, Sheila %A Sauer, Markus %A Nieswandt, Bernhard %A Machesky, Laura %A Bender, Markus %B Blood %D 2019 %J Blood %P 2318-2329 %T Platelet lamellipodium formation is not required for thrombus formation and stability %U https://doi.org/10.1182/blood.2019002105 %V 134 %X During platelet spreading, the actin cytoskeleton undergoes rapid rearrangement, forming filopodia and lamellipodia. Controversial data have been published on the role of lamellipodia in thrombus formation and stability. The Wiskott-Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex, which has been shown in other cells to drive lamellipodium formation by enhancing actin nucleation via the actin-related protein 2/3 (Arp2/3) complex, is activated by Ras-related C3 botulinum toxin substrate 1 (Rac1) interaction with the WAVE complex subunit cytoplasmic fragile X mental retardation 1–interacting protein 1 (Cyfip1). We analyzed Cyfip1flox/floxPf4-Cre mice to investigate the role of Cyfip1 in platelet function. These mice displayed normal platelet counts and a slight reduction in platelet volume. Activation of mutant platelets was only moderately reduced to all tested agonists as measured by αIIbβ3 integrin activation and P-selectin surface exposure. However, lamellipodium formation of mutant platelets was completely abolished on different matrices. Nevertheless, Cyfip1-deficient platelets formed stable thrombi on collagen fibers ex vivo and in 2 models of occlusive arterial thrombosis in vivo. Similarly, the hemostatic function and maintenance of vascular integrity during inflammation of the skin and lung were unaltered in the mutant mice. Investigation of platelet morphology in an induced thrombus under flow revealed that platelets rather form filopodia in the thrombus shell, and are flattened with filopodium-like structures when in direct contact to collagen fibers at the bottom of the thrombus. We provide for the first time direct evidence that platelet lamellipodium formation is not required for stable thrombus formation, and that morphological changes of platelets differ between a static spreading assay and thrombus formation under flow.
• 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 (2019). 9 276-.
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  Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

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

  %0 Journal Article %1 kunz2019detection %A Kunz, Tobias C. %A Götz, Ralph %A Sauer, Markus %A Rudel, Thomas %D 2019 %J Frontiers in Cellular and Infection Microbiology %P 276-- %T Detection of Chlamydia Developmental Forms and Secreted Effectors by Expansion Microscopy %U https://www.frontiersin.org/article/10.3389/fcimb.2019.00276 %V 9 %X Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.
• 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 (2019). 575(7784) 693–698.
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  Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

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

  %0 Journal Article %1 doll2019glutathioneindependent %A Doll, Sebastian %A Freitas, Florencio Porto %A Shah, Ron %A Aldrovandi, Maceler %A da Silva, Milene Costa %A Ingold, Irina %A Grocin, Andrea Goya %A Xavier da Silva, Thamara Nishida %A Panzilius, Elena %A Scheel, Christina H. %A Mourão, André %A Buday, Katalin %A Sato, Mami %A Wanninger, Jonas %A Vignane, Thibaut %A Mohana, Vaishnavi %A Rehberg, Markus %A Flatley, Andrew %A Schepers, Aloys %A Kurz, Andreas %A White, Daniel %A Sauer, Markus %A Sattler, Michael %A Tate, Edward William %A Schmitz, Werner %A Schulze, Almut %A O’Donnell, Valerie %A Proneth, Bettina %A Popowicz, Grzegorz M. %A Pratt, Derek A. %A Angeli, José Pedro Friedmann %A Conrad, Marcus %D 2019 %J Nature %N 7784 %P 693--698 %R 10.1038/s41586-019-1707-0 %T FSP1 is a glutathione-independent ferroptosis suppressor %U https://doi.org/10.1038/s41586-019-1707-0 %V 575 %X Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

• 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 (2018). 57(50) 16364–16369.
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  Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.

  @article{neubert2018bioorthogonal, abstract = {Abstract Super-resolution microscopy requires small fluorescent labels. We report the application of genetic code expansion in combination with bioorthogonal click chemistry to label the NR1 domain of the NMDA receptor. We generated NR1 mutants incorporating an unnatural amino acid at various positions in order to attach small organic fluorophores such as Cy5-tetrazine site-specifically to the extracellular domain of the receptor. Mutants were optimized with regard to protein expression, labeling efficiency and receptor functionality as tested by fluorescence microscopy and whole-cell patch clamp. The results show that bioorthogonal click chemistry in combination with small organic dyes is superior to available immunocytochemistry protocols for receptor labeling in live and fixed cells and enables single-molecule sensitive super-resolution microscopy experiments.}, author = {Neubert, Franziska and Beliu, Gerti and Terpitz, Ulrich and Werner, Christian and Geis, Christian and Sauer, Markus and Doose, Sören}, booktitle = {Angewandte Chemie International Edition}, journal = {Angewandte Chemie International Edition}, keywords = {werner}, month = {oct}, 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.
• 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 (2018). fj.201701435-.
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  Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.

  @article{sisario2018nanostructure, abstract = {Induction of DNA double-strand breaks (DSBs) by ionizing radiation leads to formation of micrometer-sized DNA-repair foci, whose organization on the nanometer-scale remains unknown because of the diffraction limit (?200 nm) of conventional microscopy. Here, we applied diffraction-unlimited, direct stochastic optical-reconstruction microscopy (dSTORM) with a lateral resolution of ?20 nm to analyze the focal nanostructure of the DSB marker histone ?H2AX and the DNA-repair protein kinase (DNA-PK) in irradiated glioblastoma multiforme cells. Although standard confocal microscopy revealed substantial colocalization of immunostained ?H2AX and DNA-PK, in our dSTORM images, the 2 proteins showed very little (if any) colocalization despite their close spatial proximity. We also found that ?H2AX foci consisted of distinct circular subunits (?nanofoci?) with a diameter of ?45 nm, whereas DNA-PK displayed a diffuse, intrafocal distribution. We conclude that ?H2AX nanofoci represent the elementary, structural units of DSB repair foci, that is, individual ?H2AX-containing nucleosomes. dSTORM-based ?H2AX nanofoci counting and distance measurements between nanofoci provided quantitative information on the total amount of chromatin involved in DSB repair as well as on the number and longitudinal distribution of ?H2AX-containing nucleosomes in a chromatin fiber. We thus estimate that a single focus involves between ?0.6 and ?1.1 Mbp of chromatin, depending on radiation treatment. Because of their ability to unravel the nanostructure of DSB-repair foci, dSTORM and related single-molecule localization nanoscopy methods will likely emerge as powerful tools in biology and medicine to elucidate the effects of DNA damaging agents in cells.?Sisario, D., Memmel, S., Doose, S., Neubauer, J., Zimmermann, H., Flentje, M., Djuzenova, C. S., Sauer, M., Sukhorukov, V. L. Nanostructure of DNA repair foci revealed by superresolution microscopy.}, author = {Sisario, Dmitri and Memmel, Simon and Doose, Sören and Neubauer, Julia and Zimmermann, Heiko and Flentje, Michael and Djuzenova, Cholpon S. and Sauer, Markus and Sukhorukov, Vladimir L.}, booktitle = {The FASEB Journal}, journal = {The FASEB Journal}, keywords = {vladimir}, month = {jun}, 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.
• 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 (2018). 9 815-.
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  By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.

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

  %0 Journal Article %1 bortlein2018neutral %A Börtlein, Charlene %A Draeger, Annette %A Schoenauer, Roman %A Kuhlemann, Alexander %A Sauer, Markus %A Schneider-Schaulies, Sibylle %A Avota, Elita %D 2018 %J Frontiers in Immunology %P 815-- %T The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling %U https://www.frontiersin.org/article/10.3389/fimmu.2018.00815 %V 9 %X By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca2+ mobilization and T cell proliferation. NSM deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70 and PKCζ, and functional immune synapses (IS) are organized and stabilized via MTOC polarization.
• 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 (2018). 7(1) 99-.
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  Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.

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

  %0 Journal Article %1 heil2018sharpening %A Heil, Hannah S. %A Schreiber, Benjamin %A Götz, Ralph %A Emmerling, Monika %A Dabauvalle, Marie-Christine %A Krohne, Georg %A Höfling, Sven %A Kamp, Martin %A Sauer, Markus %A Heinze, Katrin G. %D 2018 %J Light: Science & Applications %N 1 %P 99-- %R 10.1038/s41377-018-0104-z %T Sharpening emitter localization in front of a tuned mirror %U https://doi.org/10.1038/s41377-018-0104-z %V 7 %X Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.

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

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

  %0 Journal Article %1 sauer2017singlemolecule %A Sauer, Markus %A Heilemann, Mike %B Chemical Reviews %D 2017 %I American Chemical Society %J Chem. Rev. %R 10.1021/acs.chemrev.6b00667 %T Single-Molecule Localization Microscopy in Eukaryotes %U http://dx.doi.org/10.1021/acs.chemrev.6b00667 %X Super-resolution fluorescence imaging by photoactivation or photoswitching of single fluorophores and position determination (single-molecule localization microscopy, SMLM) provides microscopic images with subdiffraction spatial resolution. This technology has enabled new insights into how proteins are organized in a cellular context, with a spatial resolution approaching virtually the molecular level. A unique strength of SMLM is that it delivers molecule-resolved information, along with super-resolved images of cellular structures. This allows quantitative access to cellular structures, for example, how proteins are distributed and organized and how they interact with other biomolecules. Ultimately, it is even possible to determine protein numbers in cells and the number of subunits in a protein complex. SMLM thus has the potential to pave the way toward a better understanding of how cells function at the molecular level. In this review, we describe how SMLM has contributed new knowledge in eukaryotic biology, and we specifically focus on quantitative biological data extracted from SMLM images.
• Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain Forero, Andrea; Rivero, Olga; Wäldchen, Sina; Ku, Hsing-Ping; Kiser, Dominik P.; Gärtner, Yvonne; Pennington, Laura S.; Waider, Jonas; Gaspar, Patricia; Jansch, Charline; Edenhofer, Frank; Resink, Thérèse J.; Blum, Robert; Sauer, Markus; Lesch, Klaus-Peter in Frontiers in Cellular Neuroscience (2017). 11 307-.
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  Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.

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

  %0 Journal Article %1 forero2017cadherin13 %A Forero, Andrea %A Rivero, Olga %A Wäldchen, Sina %A Ku, Hsing-Ping %A Kiser, Dominik P. %A Gärtner, Yvonne %A Pennington, Laura S. %A Waider, Jonas %A Gaspar, Patricia %A Jansch, Charline %A Edenhofer, Frank %A Resink, Thérèse J. %A Blum, Robert %A Sauer, Markus %A Lesch, Klaus-Peter %D 2017 %J Frontiers in Cellular Neuroscience %P 307-- %T Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain %U https://www.frontiersin.org/article/10.3389/fncel.2017.00307 %V 11 %X Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system. Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency. Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mouse embryos display increased cell densities in the DR at E13.5, E17.5 and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5. Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that CDH13 deficiency affects the cell density of the developing DR and the posterior innervation of the PFC, and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
• 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, (H. J. Bellen, Hrsg.) (2017). 6 e28360-.
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  Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

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

  %0 Journal Article %1 scholz2017mechanodependent %A Scholz, Nicole %A Guan, Chonglin %A Nieberler, Matthias %A Grotemeyer, Alexander %A Maiellaro, Isabella %A Gao, Shiqiang %A Beck, Sebastian %A Pawlak, Matthias %A Sauer, Markus %A Asan, Esther %A Rothemund, Sven %A Winkler, Jana %A Prömel, Simone %A Nagel, Georg %A Langenhan, Tobias %A Kittel, Robert J %D 2017 %E Bellen, Hugo J %I eLife Sciences Publications, Ltd %J eLife %P e28360-- %R 10.7554/eLife.28360 %T Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons %U https://doi.org/10.7554/eLife.28360 %V 6 %X Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
• Cyanine Conformational Restraint in the Far-Red Range Michie, Megan S.; Götz, Ralph; Franke, Christian; Bowler, Matthew; Kumari, Nikita; Magidson, Valentin; Levitus, Marcia; Loncarek, Jadranka; Sauer, Markus; Schnermann, Martin J. in J. Am. Chem. Soc. (2017). 139(36) 12406–12409.
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  Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

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

  %0 Journal Article %1 michie2017cyanine %A Michie, Megan S. %A Götz, Ralph %A Franke, Christian %A Bowler, Matthew %A Kumari, Nikita %A Magidson, Valentin %A Levitus, Marcia %A Loncarek, Jadranka %A Sauer, Markus %A Schnermann, Martin J. %B Journal of the American Chemical Society %D 2017 %I American Chemical Society %J J. Am. Chem. Soc. %N 36 %P 12406--12409 %R 10.1021/jacs.7b07272 %T Cyanine Conformational Restraint in the Far-Red Range %U http://dx.doi.org/10.1021/jacs.7b07272 %V 139 %X Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.
• 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 (2017). 114(7) 1448–1458.
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  Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.

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

  %0 Journal Article %1 bruhlmann2017parallel %A Brühlmann, David %A Sokolov, Michael %A Butté, Alessandro %A Sauer, Markus %A Hemberger, Jürgen %A Souquet, Jonathan %A Broly, Hervé %A Jordan, Martin %D 2017 %J Biotechnology and Bioengineering %N 7 %P 1448--1458 %R 10.1002/bit.26269 %T Parallel experimental design and multivariate analysis provides efficient screening of cell culture media supplements to improve biosimilar product quality %U http://dx.doi.org/10.1002/bit.26269 %V 114 %X Rational and high-throughput optimization of mammalian cell culture media has a great potential to modulate recombinant protein product quality. We present a process design method based on parallel design-of-experiment (DoE) of CHO fed-batch cultures in 96-deepwell plates to modulate monoclonal antibody (mAb) glycosylation using medium supplements. To reduce the risk of losing valuable information in an intricate joint screening, 17 compounds were separated into five different groups, considering their mode of biological action. The concentration ranges of the medium supplements were defined according to information encountered in the literature and in-house experience. The screening experiments produced wide glycosylation pattern ranges. Multivariate analysis including principal component analysis and decision trees was used to select the best performing glycosylation modulators. Subsequent D-optimal quadratic design with four factors (three promising compounds and temperature shift) in shake tubes confirmed the outcome of the selection process and provided a solid basis for sequential process development at a larger scale. The glycosylation profile with respect to the specifications for biosimilarity was greatly improved in shake tube experiments: 75% of the conditions were equally close or closer to the specifications for biosimilarity than the best 25% in 96-deepwell plates. Biotechnol. Bioeng. 2017;114: 1448–1458. © 2017 Wiley Periodicals, Inc.
• 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 (2017). 252(Supplement C) 32–42.
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  Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.

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

  %0 Journal Article %1 bruhlmann2017culture %A Brühlmann, David %A Muhr, Anais %A Parker, Rebecca %A Vuillemin, Thomas %A Bucsella, Blanka %A Kalman, Franka %A Torre, Serena %A La Neve, Fabio %A Lembo, Antonio %A Haas, Tobias %A Sauer, Markus %A Souquet, Jonathan %A Broly, Hervé %A Hemberger, Jürgen %A Jordan, Martin %D 2017 %J Journal of Biotechnology %N Supplement C %P 32--42 %R https://doi.org/10.1016/j.jbiotec.2017.04.026 %T Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation %U http://www.sciencedirect.com/science/article/pii/S016816561730189X %V 252 %X Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also. Up to six-fold increases of high mannose species were observed with increasing raffinose concentrations in the medium of shaken 96-deepwell plates and shake tubes when culturing two different CHO cell lines in two different media. The findings were confirmed in a pH-, oxygen- and CO2-controlled environment in lab-scale 3.5-L bioreactors. To circumvent detrimental effects on cell growth and productivity at high raffinose concentrations, the media osmolality was adjusted to reach the same value independently of the supplement concentration. Interestingly, raffinose predominantly enhanced mannose 5 glycans, and to a considerably smaller degree, mannose 6. While the underlying mechanism is still not fully understood, minor effects on the nucleotide sugar levels have been observed and transcriptomics analysis revealed that raffinose supplementation altered the expression levels of a number of glycosylation related genes. Among many genes, galactosyltransferase was downregulated and sialyltransferase upregulated. Our results highlight the potential of cell culture medium supplementation to modulate product quality.
• 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 (2017). 7(1) 6138-.
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  Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.

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

  %0 Journal Article %1 ziegler2017pathogen %A Ziegler, Sabrina %A Weiss, Esther %A Schmitt, Anna-Lena %A Schlegel, Jan %A Burgert, Anne %A Terpitz, Ulrich %A Sauer, Markus %A Moretta, Lorenzo %A Sivori, Simona %A Leonhardt, Ines %A Kurzai, Oliver %A Einsele, Hermann %A Loeffler, Juergen %D 2017 %J Scientific Reports %N 1 %P 6138-- %R 10.1038/s41598-017-06238-4 %T CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells %U https://doi.org/10.1038/s41598-017-06238-4 %V 7 %X Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
• 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, T. Müller-Reichert, P. Verkade (Hrsg.) (2017). (Bd. 140) 21–47.
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  Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.

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

  %0 Book Section %1 markert2017chapter %A Markert, Sebastian M. %A Bauer, Vivien %A Muenz, Thomas S. %A Jones, Nicola G. %A Helmprobst, Frederik %A Britz, Sebastian %A Sauer, Markus %A Rössler, Wolfgang %A Engstler, Markus %A Stigloher, Christian %B Correlative Light and Electron Microscopy III %D 2017 %E Müller-Reichert, Thomas %E Verkade, Paul %I Academic Press %N Supplement C %P 21--47 %R https://doi.org/10.1016/bs.mcb.2017.03.004 %T Chapter 2 - 3D subcellular localization with superresolution array tomography on ultrathin sections of various species %U http://www.sciencedirect.com/science/article/pii/S0091679X17300481 %V 140 %X Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D. We established protocols for the application of superresolution AT on ultrathin plastic sections of Caenorhabditis elegans, Trypanosoma brucei, and brain tissue of Cataglyphis fortis and Apis mellifera. All steps are described in detail from sample preparation to 3D reconstruction, including species-specific modifications. We thus showcase the versatility of our protocol and give some examples for biological questions that can be answered with this technique. We offer a step-by-step recipe for superresolution AT that can be easily applied for C. elegans, T. brucei, C. fortis, and A. mellifera and adapted for other model systems.
• Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy Burgert, Anne; Schlegel, Jan; Bécam, Jérôme; Doose, Sören; Bieberich, Erhard; Schubert-Unkmeir, Alexandra; Sauer, Markus in Angewandte Chemie (2017).
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  The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

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

  %0 Journal Article %1 burgert2017characterization %A Burgert, Anne %A Schlegel, Jan %A Bécam, Jérôme %A Doose, Sören %A Bieberich, Erhard %A Schubert-Unkmeir, Alexandra %A Sauer, Markus %D 2017 %J Angewandte Chemie %R 10.1002/ange.201700570 %T Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201700570 %X The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50–60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.
• Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities Memmel, Simon; Sisario, Dmitri; Zöller, Caren; Fiedler, Vanessa; Katzer, Astrid; Heiden, Robin; Becker, Nicholas; Eing, Lorenz; Ferreira, Fábio L.R.; Zimmermann, Heiko; Sauer, Markus; Flentje, Michael; Sukhorukov, Vladimir L.; Djuzenova, Cholpon S. in Oncotarget (2017). 8 45298–45310.
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  High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

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

  %0 Journal Article %1 memmel2017migration %A Memmel, Simon %A Sisario, Dmitri %A Zöller, Caren %A Fiedler, Vanessa %A Katzer, Astrid %A Heiden, Robin %A Becker, Nicholas %A Eing, Lorenz %A Ferreira, Fábio L.R. %A Zimmermann, Heiko %A Sauer, Markus %A Flentje, Michael %A Sukhorukov, Vladimir L. %A Djuzenova, Cholpon S. %D 2017 %J Oncotarget %P 45298–45310 %T Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities %U https://doi.org/10.18632/oncotarget.16847 %V 8 %X High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell linespecific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.
• Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging Lukeš, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Levet, Florian; Benda, Aleš; Letschert, Sebastian; Sauer, Markus; Brdička, Tomáš; Lasser, Theo; Cebecauer, Marek in Nature Communications (2017). 8(1) 1731-.
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  Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.

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

  %0 Journal Article %1 lukes2017quantifying %A Lukeš, Tomáš %A Glatzová, Daniela %A Kvíčalová, Zuzana %A Levet, Florian %A Benda, Aleš %A Letschert, Sebastian %A Sauer, Markus %A Brdička, Tomáš %A Lasser, Theo %A Cebecauer, Marek %D 2017 %J Nature Communications %N 1 %P 1731-- %R 10.1038/s41467-017-01857-x %T Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging %U https://doi.org/10.1038/s41467-017-01857-x %V 8 %X Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
• Incorporation studies of clickable ceramides in Jurkat cell plasma membranes Walter, T.; Schlegel, J.; Burgert, A.; Kurz, A.; Seibel, J.; Sauer, M. in Chem. Commun. (2017). 53(51) 6836–6839.
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  The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.

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

  %0 Journal Article %1 walter2017incorporation %A Walter, T. %A Schlegel, J. %A Burgert, A. %A Kurz, A. %A Seibel, J. %A Sauer, M. %D 2017 %I The Royal Society of Chemistry %J Chem. Commun. %N 51 %P 6836--6839 %R 10.1039/C7CC01220A %T Incorporation studies of clickable ceramides in Jurkat cell plasma membranes %U http://dx.doi.org/10.1039/C7CC01220A %V 53 %X The incorporation properties of ceramide analogues for click chemistry in Jurkat T cells were investigated. The analogues varied in the acyl chain length and the position of the functional group for click chemistry. Fluorescence microscopy studies including anisotropy and quenching experiments showed significant differences in the accessibility of the functional group indicating different incorporation properties into the plasma membrane.
• Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska; Dalby, Nils Ole; Doose, Sören; Sauer, Markus; Kneussel, Matthias; Strømgaard, Kristian in Nature Chemical Biology (2017). 13 153-.
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  γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

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

  %0 Journal Article %1 maric2016gephyrinbinding %A Maric, Hans Michael %A Hausrat, Torben Johann %A Neubert, Franziska %A Dalby, Nils Ole %A Doose, Sören %A Sauer, Markus %A Kneussel, Matthias %A Strømgaard, Kristian %D 2017 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nature Chemical Biology %P 153-- %T Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission %U https://doi.org/10.1038/nchembio.2246 %V 13 %X γ-Aminobutyric acid type A and glycine receptors are the major mediators of fast synaptic inhibition in the human central nervous system and are established drug targets. However, all drugs targeting these receptors bind to the extracellular ligand-binding domain of the receptors, which inherently is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor–gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate that the gephyrin super-binding peptides act as acute intracellular modulators of fast synaptic inhibition by modulating receptor clustering, thus being conceptually novel modulators of inhibitory neurotransmission.

• Photometry unlocks 3D information from 2D localization microscopy data Franke, Christian; Sauer, Markus; van de Linde, Sebastian in Nat Meth (2016). advance online publication
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  We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

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

  %0 Journal Article %1 franke2016photometry %A Franke, Christian %A Sauer, Markus %A van de Linde, Sebastian %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Photometry unlocks 3D information from 2D localization microscopy data %U http://dx.doi.org/10.1038/nmeth.4073 %V advance online publication %X We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.
• A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells Collenburg, Lena; Walter, Tim; Burgert, Anne; Müller, Nora; Seibel, Jürgen; Japtok, Lukasz; Kleuser, Burkhard; Sauer, Markus; Schneider-Schaulies, Sibylle in The Journal of Immunology (2016). 196(9) 3951–3962.
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  Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.

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

  %0 Journal Article %1 collenburg2016functionalized %A Collenburg, Lena %A Walter, Tim %A Burgert, Anne %A Müller, Nora %A Seibel, Jürgen %A Japtok, Lukasz %A Kleuser, Burkhard %A Sauer, Markus %A Schneider-Schaulies, Sibylle %D 2016 %J The Journal of Immunology %N 9 %P 3951--3962 %T A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells %U http://www.jimmunol.org/content/196/9/3951.abstract %V 196 %X Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
• Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian in Neurophotonics (2016). 3(4) 041802–041802.
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  Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.

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

  %0 Journal Article %1 markert2016filling %A Markert, Sebastian Matthias %A Britz, Sebastian %A Proppert, Sven %A Lang, Marietta %A Witvliet, Daniel %A Mulcahy, Ben %A Sauer, Markus %A Zhen, Mei %A Bessereau, Jean-Louis %A Stigloher, Christian %D 2016 %J Neurophotonics %N 4 %P 041802--041802 %T Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome %U http://dx.doi.org/10.1117/1.NPh.3.4.041802 %V 3 %X Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.
• The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni Dugar, Gaurav; Svensson, Sarah L.; Bischler, Thorsten; Wäldchen, Sina; Reinhardt, Richard; Sauer, Markus; Sharma, Cynthia M. in Nature Communications (2016). 7 11667-.
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  The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.

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

  %0 Journal Article %1 dugar2016csrafliw %A Dugar, Gaurav %A Svensson, Sarah L. %A Bischler, Thorsten %A Wäldchen, Sina %A Reinhardt, Richard %A Sauer, Markus %A Sharma, Cynthia M. %D 2016 %I The Author(s) %J Nature Communications %P 11667-- %T The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni %U http://dx.doi.org/10.1038/ncomms11667 %V 7 %X The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.
• Super-resolution imaging of plasma membrane proteins with click chemistry Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Soeren; Sauer, Markus in Frontiers in Cell and Developmental Biology (2016). 4 98-.
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  Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.

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

  %0 Journal Article %1 mateosgil2016superresolution %A Mateos-Gil, Pablo %A Letschert, Sebastian %A Doose, Soeren %A Sauer, Markus %D 2016 %J Frontiers in Cell and Developmental Biology %P 98-- %T Super-resolution imaging of plasma membrane proteins with click chemistry %U http://journal.frontiersin.org/article/10.3389/fcell.2016.00098 %V 4 %X Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.
• Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling Yadav, Preeti; Selvaraj, Bhuvaneish T.; Bender, Florian L. P.; Behringer, Marcus; Moradi, Mehri; Sivadasan, Rajeeve; Dombert, Benjamin; Blum, Robert; Asan, Esther; Sauer, Markus; Julien, Jean-Pierre; Sendtner, Michael in Acta Neuropathologica (2016). 132(1) 93–110.
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  In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

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

  %0 Journal Article %1 yadav2016neurofilament %A Yadav, Preeti %A Selvaraj, Bhuvaneish T. %A Bender, Florian L. P. %A Behringer, Marcus %A Moradi, Mehri %A Sivadasan, Rajeeve %A Dombert, Benjamin %A Blum, Robert %A Asan, Esther %A Sauer, Markus %A Julien, Jean-Pierre %A Sendtner, Michael %D 2016 %J Acta Neuropathologica %N 1 %P 93--110 %R 10.1007/s00401-016-1564-y %T Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling %U http://dx.doi.org/10.1007/s00401-016-1564-y %V 132 %X In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3–stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
• Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates Alexander, Mertsch; Sebastian, Letschert; Elisabeth, Memmel; Markus, Sauer; Jürgen, Seibel (2016). 347-.
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  The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.

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

  %0 Generic %1 alexander2016synthesis %A Alexander, Mertsch %A Sebastian, Letschert %A Elisabeth, Memmel %A Markus, Sauer %A Jürgen, Seibel %B Zeitschrift für Naturforschung C %D 2016 %P 347-- %R 10.1515/znc-2016-0123 %T Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates %U https://www.degruyter.com/view/j/znc.2016.71.issue-9-10/znc-2016-0123/znc-2016-0123.xml %V 71 %X The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.
• OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors Jung, L A; Gebhardt, A; Koelmel, W; Ade, C P; Walz, S; Kuper, J; von Eyss, B; Letschert, S; Redel, C; d’Artista, L; Biankin, A; Zender, L; Sauer, M; Wolf, E; Evan, G; Kisker, C; Eilers, M in Oncogene (2016). 36 1911-.
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  MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

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

  %0 Journal Article %1 jung2016omomyc %A Jung, L A %A Gebhardt, A %A Koelmel, W %A Ade, C P %A Walz, S %A Kuper, J %A von Eyss, B %A Letschert, S %A Redel, C %A d'Artista, L %A Biankin, A %A Zender, L %A Sauer, M %A Wolf, E %A Evan, G %A Kisker, C %A Eilers, M %D 2016 %I Macmillan Publishers Limited, part of Springer Nature. %J Oncogene %P 1911-- %T OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors %U http://dx.doi.org/10.1038/onc.2016.354 %V 36 %X MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.
• Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition Werner, Christian; Pauli, Martin; Doose, Sören; Weishaupt, Andreas; Haselmann, Holger; Grünewald, Benedikt; Sauer, Markus; Heckmann, Manfred; Toyka, Klaus V.; Asan, Esther; Sommer, Claudia; Geis, Christian in Brain (2016). 139(2) 365–379.
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  See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.

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

  %0 Journal Article %1 werner2016human %A Werner, Christian %A Pauli, Martin %A Doose, Sören %A Weishaupt, Andreas %A Haselmann, Holger %A Grünewald, Benedikt %A Sauer, Markus %A Heckmann, Manfred %A Toyka, Klaus V. %A Asan, Esther %A Sommer, Claudia %A Geis, Christian %D 2016 %J Brain %N 2 %P 365--379 %T Human autoantibodies to amphiphysin induce defective presynaptic vesicle dynamics and composition %U http://dx.doi.org/10.1093/brain/awv324 %V 139 %X See Irani (doi:10.1093/awv364) for a scientific commentary on this article. Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.
• Multi-target spectrally resolved fluorescence lifetime imaging microscopy Niehorster, Thomas; Loschberger, Anna; Gregor, Ingo; Kramer, Benedikt; Rahn, Hans-Jurgen; Patting, Matthias; Koberling, Felix; Enderlein, Jorg; Sauer, Markus in Nat Meth (2016). advance online publication
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  We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

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

  %0 Journal Article %1 niehorster2016multitarget %A Niehorster, Thomas %A Loschberger, Anna %A Gregor, Ingo %A Kramer, Benedikt %A Rahn, Hans-Jurgen %A Patting, Matthias %A Koberling, Felix %A Enderlein, Jorg %A Sauer, Markus %D 2016 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %T Multi-target spectrally resolved fluorescence lifetime imaging microscopy %U http://dx.doi.org/10.1038/nmeth.3740 %V advance online publication %X We introduce a pattern-matching technique for efficient identification of fluorophore ratios in complex multidimensional fluorescence signals using reference fluorescence decay and spectral signature patterns of individual fluorescent probes. Alternating pulsed laser excitation at three different wavelengths and time-resolved detection on 32 spectrally separated detection channels ensures efficient excitation of fluorophores and a maximum gain of fluorescence information. Using spectrally resolved fluorescence lifetime imaging microscopy (sFLIM), we were able to visualize up to nine different target molecules simultaneously in mouse C2C12 cells. By exploiting the sensitivity of fluorescence emission spectra and the lifetime of organic fluorophores on environmental factors, we carried out fluorescence imaging of three different target molecules in human U2OS cells with the same fluorophore. Our results demonstrate that sFLIM can be used for super-resolution multi-target imaging by stimulated emission depletion (STED).

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

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

  %0 Journal Article %1 Schücker17022015 %A Schücker, Katharina %A Holm, Thorge %A Franke, Christian %A Sauer, Markus %A Benavente, Ricardo %D 2015 %J Proceedings of the National Academy of Sciences %N 7 %P 2029-2033 %R 10.1073/pnas.1414814112 %T Elucidation of synaptonemal complex organization by super-resolution imaging with isotropic resolution %U http://www.pnas.org/content/112/7/2029.abstract %V 112 %X Synaptonemal complexes (SCs) are meiosis-specific multiprotein complexes that are essential for synapsis, recombination, and segregation of homologous chromosomes, but the molecular organization of SCs remains unclear. We used immunofluorescence labeling in combination with super-resolution imaging and average position determination to investigate the molecular architecture of SCs. Combination of 2D super-resolution images recorded from different areas of the helical ladder-like structure allowed us to reconstruct the 3D molecular organization of the mammalian SC with isotropic resolution. The central element is composed of two parallel cables at a distance of ∼100 nm, which are oriented perpendicular to two parallel cables of the lateral element arranged at a distance of ∼220 nm. The two parallel cable elements form twisted helical structures that are connected by transversal filaments by their N and C termini. A single-cell preparation generates sufficient localizations to compile a 3D model of the SC with nanometer precision.
• Super-resolution microscopy of the synaptic active zone Ehmann, Nadine; Sauer, Markus; Kittel, Robert J in Frontiers in Cellular Neuroscience (2015). 9(7)
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  Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.

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

  %0 Journal Article %1 10.3389/fncel.2015.00007 %A Ehmann, Nadine %A Sauer, Markus %A Kittel, Robert J %D 2015 %J Frontiers in Cellular Neuroscience %N 7 %R 10.3389/fncel.2015.00007 %T Super-resolution microscopy of the synaptic active zone %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00007/abstract %V 9 %X Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins.
• Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium Schneider, Johannes; Klein, Teresa; Mielich-Süss, Benjamin; Koch, Gudrun; Franke, Christian; Kuipers, Oscar P.; Kovács, Ákos T.; Sauer, Markus; Lopez, Daniel in PLoS Genet (2015). 11(4) e1005140-.
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  <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>

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

  %0 Journal Article %1 schneider2015spatiotemporal %A Schneider, Johannes %A Klein, Teresa %A Mielich-Süss, Benjamin %A Koch, Gudrun %A Franke, Christian %A Kuipers, Oscar P. %A Kovács, Ákos T. %A Sauer, Markus %A Lopez, Daniel %D 2015 %I Public Library of Science %J PLoS Genet %N 4 %P e1005140-- %T Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium %U http://dx.doi.org/10.1371%2Fjournal.pgen.1005140 %V 11 %X <title>Author Summary</title> <p>Cellular membranes organize proteins related to signal transduction, protein sorting and membrane trafficking into the so-called lipid rafts. It has been proposed that the functional diversity of lipid rafts would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known due in part to the complexity that entails the manipulation of eukaryotic cells. The recent discovery that bacteria organize many cellular processes in membrane microdomains (FMMs), functionally similar to the eukaryotic lipid rafts, prompted us to explore FMMs diversity in the bacterial model <italic>Bacillus subtilis</italic>. We show that diversification of FMMs occurs in cells and gives rise to functionally distinct microdomains, which compartmentalize distinct signal transduction pathways and regulate the expression of different genetic programs. We discovered that FMMs diversification does not occur randomly. Cells sequentially regulate the specialization of the FMMs during cell growth to ensure an effective and diverse activation of signaling processes.</p>
• Artifacts in single-molecule localization microscopy Burgert, Anne; Letschert, Sebastian; Doose, Sören; Sauer, Markus in Histochemistry and Cell Biology (2015). 144 123–131.
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  Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

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

  %0 Journal Article %1 burgert2015artifacts %A Burgert, Anne %A Letschert, Sebastian %A Doose, Sören %A Sauer, Markus %B 2 %D 2015 %J Histochemistry and Cell Biology %P 123-131 %T Artifacts in single-molecule localization microscopy %U http://dx.doi.org/10.1007/s00418-015-1340-4 %V 144 %X Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.
• Quantitative Super-Resolution Microscopy of Nanopipette-Deposited Fluorescent Patterns Hennig, Simon; van de Linde, Sebastian; Bergmann, Stephan; Huser, Thomas; Sauer, Markus in ACS Nano (2015). 9(8) 8122–8130.
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  We describe a method for the deposition of minute amounts of fluorophore-labeled oligonucleotides with high local precision in conductive and transparent solid layers of poly(vinyl alcohol) (PVA) doped with glycerin and cysteamine (PVA-G-C layers). Deposition of negatively charged fluorescent molecules was accomplished with a setup based on a scanning ion conductance microscope (SICM) using nanopipettes with tip diameters of ?100 nm by using the ion flux flowing between two electrodes through the nanopipette. To investigate the precision of the local deposition process, we performed in situ super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM). Exploiting the single-molecule sensitivity and reliability of dSTORM, we determine the number of fluorescent molecules deposited in single spots. The correlation of applied charge and number of deposited molecules enables the quantification of delivered molecules by measuring the charge during the delivery process. We demonstrate the reproducible deposition of 3?168 fluorescent molecules in single spots and the creation of fluorescent structures. The fluorescent structures are highly stable and can be reused several times.

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

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

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

  %0 Journal Article %1 waldchen2015lightinduced %A Wäldchen, Sina %A Lehmann, Julian %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2015 %I Nature Publishing Group %J Scientific Reports %P 15348-- %R 10.1038/srep15348 %T Light-induced cell damage in live-cell super-resolution microscopy %U http://dx.doi.org/10.1038/srep15348 %V 5 %X Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(−2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(−2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
• Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy Grimm, Jonathan B.; Klein, Teresa; Kopek, Benjamin G.; Shtengel, Gleb; Hess, Harald F.; Sauer, Markus; Lavis, Luke D. in Angewandte Chemie (2015). n/a-n/a.
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  The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.

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

  %0 Journal Article %1 grimm2015synthesis %A Grimm, Jonathan B. %A Klein, Teresa %A Kopek, Benjamin G. %A Shtengel, Gleb %A Hess, Harald F. %A Sauer, Markus %A Lavis, Luke D. %D 2015 %I WILEY-VCH Verlag %J Angewandte Chemie %P n/a--n/a %R 10.1002/ange.201509649 %T Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy %U http://dx.doi.org/10.1002/ange.201509649 %X The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a “caged” Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules.
• A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer von Eyss, Björn; Jaenicke, Laura A; Kortlever, Roderik M; Royla, Nadine; Wiese, Katrin E; Letschert, Sebastian; McDuffus, Leigh-Anne; Sauer, Markus; Rosenwald, Andreas; Evan, Gerard I; Kempa, Stefan; Eilers, Martin in Cancer Cell (2015). 28(6) 743–757.
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  Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.

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

  %0 Journal Article %1 voneyss2015mycdriven %A von Eyss, Björn %A Jaenicke, Laura A %A Kortlever, Roderik M %A Royla, Nadine %A Wiese, Katrin E %A Letschert, Sebastian %A McDuffus, Leigh-Anne %A Sauer, Markus %A Rosenwald, Andreas %A Evan, Gerard I %A Kempa, Stefan %A Eilers, Martin %D 2015 %J Cancer Cell %N 6 %P 743--757 %R http://dx.doi.org/10.1016/j.ccell.2015.10.013 %T A MYC-Driven Change in Mitochondrial Dynamics Limits YAP/TAZ Function in Mammary Epithelial Cells and Breast Cancer %U http://www.sciencedirect.com/science/article/pii/S1535610815003906 %V 28 %X Summary In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.
• Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy Andronic, Joseph; Shirakashi, Ryo; Pickel, Simone U.; Westerling, Katherine M.; Klein, Teresa; Holm, Thorge; Sauer, Markus; Sukhorukov, Vladimir L. in PLoS ONE (2015). 10(3) e0119990.
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  Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

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

  %0 Journal Article %1 10.1371/journal.pone.0119990 %A Andronic, Joseph %A Shirakashi, Ryo %A Pickel, Simone U. %A Westerling, Katherine M. %A Klein, Teresa %A Holm, Thorge %A Sauer, Markus %A Sukhorukov, Vladimir L. %D 2015 %I Public Library of Science %J PLoS ONE %N 3 %P e0119990 %R 10.1371/journal.pone.0119990 %T Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy %U http://dx.doi.org/10.1371%2Fjournal.pone.0119990 %V 10 %X Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ~3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
• Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation Paul, Mila M; Pauli, Martin; Ehmann, Nadine; Hallermann, Stefan; Sauer, Markus; Kittel, Robert J; Heckmann, Manfred in Frontiers in Cellular Neuroscience (2015). 9(29)
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  The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.

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

  %0 Journal Article %1 10.3389/fncel.2015.00029 %A Paul, Mila M %A Pauli, Martin %A Ehmann, Nadine %A Hallermann, Stefan %A Sauer, Markus %A Kittel, Robert J %A Heckmann, Manfred %D 2015 %J Frontiers in Cellular Neuroscience %N 29 %R 10.3389/fncel.2015.00029 %T Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2015.00029/abstract %V 9 %X The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
• Tailoring recombinant protein quality by rational media design Brühlmann, David; Jordan, Martin; Hemberger, Jürgen; Sauer, Markus; Stettler, Matthieu; Broly, Hervé in Biotechnology Progress (2015). 31(3) 615–629.
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  Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015

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

  %0 Journal Article %1 bruhlmann2015tailoring %A Brühlmann, David %A Jordan, Martin %A Hemberger, Jürgen %A Sauer, Markus %A Stettler, Matthieu %A Broly, Hervé %D 2015 %J Biotechnology Progress %N 3 %P 615--629 %R 10.1002/btpr.2089 %T Tailoring recombinant protein quality by rational media design %U http://dx.doi.org/10.1002/btpr.2089 %V 31 %X Clinical efficacy and safety of recombinant proteins are closely associated with their structural characteristics. The major quality attributes comprise glycosylation, charge variants (oxidation, deamidation, and C- & N-terminal modifications), aggregates, low-molecular-weight species (LMW), and misincorporation of amino acids in the protein backbone. Cell culture media design has a great potential to modulate these quality attributes due to the vital role of medium in mammalian cell culture. The purpose of this review is to provide an overview of the way both classical cell culture medium components and novel supplements affect the quality attributes of recombinant therapeutic proteins expressed in mammalian hosts, allowing rational and high-throughput optimization of mammalian cell culture media. A selection of specific and/or potent inhibitors and activators of oligosaccharide processing as well as components affecting multiple quality attributes are presented. Extensive research efforts in this field show the feasibility of quality engineering through media design, allowing to significantly modulate the protein function. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:615–629, 2015
• Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus in Nano Lett. (2015). 15(2) 1374–1381.
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  Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

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

  %0 Journal Article %1 hennig2015instant %A Hennig, Simon %A van de Linde, Sebastian %A Lummer, Martina %A Simonis, Matthias %A Huser, Thomas %A Sauer, Markus %B Nano Letters %D 2015 %I American Chemical Society %J Nano Lett. %N 2 %P 1374--1381 %R 10.1021/nl504660t %T Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes %U http://dx.doi.org/10.1021/nl504660t %V 15 %X Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.
• Quantifying molecular colocalization in live cell fluorescence microscopy Humpert, Fabian; Yahiatène, Idir; Lummer, Martina; Sauer, Markus; Huser, Thomas in Journal of Biophotonics (2015). 8(1-2) 124–132.
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  One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

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

  %0 Journal Article %1 humpert2015quantifying %A Humpert, Fabian %A Yahiatène, Idir %A Lummer, Martina %A Sauer, Markus %A Huser, Thomas %D 2015 %I WILEY-VCH Verlag %J Journal of Biophotonics %N 1-2 %P 124--132 %R 10.1002/jbio.201300146 %T Quantifying molecular colocalization in live cell fluorescence microscopy %U http://dx.doi.org/10.1002/jbio.201300146 %V 8 %X One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

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

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

  %0 Journal Article %1 ANIE:ANIE201406045 %A Letschert, Sebastian %A Göhler, Antonia %A Franke, Christian %A Bertleff-Zieschang, Nadja %A Memmel, Elisabeth %A Doose, Sören %A Seibel, Jürgen %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Angewandte Chemie International Edition %P n/a--n/a %R 10.1002/anie.201406045 %T Super-Resolution Imaging of Plasma Membrane Glycans %U http://dx.doi.org/10.1002/anie.201406045 %X Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane-associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super-resolution fluorescence imaging was used to visualize plasma membrane glycans with single-molecule sensitivity. Our results demonstrate a homogeneous distribution of N-acetylmannosamine (ManNAc)-, N-acetylgalactosamine (GalNAc)-, and O-linked N-acetylglucosamine (O-GlcNAc)-modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.
• How to switch a fluorophore: from undesired blinking to controlled photoswitching van de Linde, Sebastian; Sauer, Markus in Chem. Soc. Rev. 2014 (2014). 43(4) 1076–1087.
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  Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.

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

  %0 Journal Article %1 C3CS60195A %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I The Royal Society of Chemistry %J Chem. Soc. Rev. 2014 %P 1076-1087 %R 10.1039/C3CS60195A %T How to switch a fluorophore: from undesired blinking to controlled photoswitching %U http://dx.doi.org/10.1039/C3CS60195A %V 43(4) %X Molecular optical photoswitches based on fluorescent proteins and organic dyes are fundamental for super-resolution fluorescence imaging and tracking methods. Precise control of switching{,} bio-labeling compatibility{,} and high brightness make photoswitches broadly applicable. This review emphasizes the design and development of photoswitches and the requirements they need to fulfill for their successful application in single-molecule localization microscopy. Furthermore{,} we discuss recent developments in improving the photoswitching performance with a special focus on organic dyes.
• A Blueprint for Cost-Efficient Localization Microscopy Holm, Thorge; Klein, Teresa; Löschberger, Anna; Klamp, Tobias; Wiebusch, Gerd; van de Linde, Sebastian; Sauer, Markus in ChemPhysChem (2014). 15(4) 651–654.
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  Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.

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

  %0 Journal Article %1 CPHC:CPHC201300739 %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Klamp, Tobias %A Wiebusch, Gerd %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J ChemPhysChem %N 4 %P 651--654 %R 10.1002/cphc.201300739 %T A Blueprint for Cost-Efficient Localization Microscopy %U http://dx.doi.org/10.1002/cphc.201300739 %V 15 %X Crystal clear: We introduce a miniaturized localization microscopy setup based on cost-effective components. We demonstrate its feasibility for subdiffraction resolution fluorescence imaging in resolving different cellular nanostructures. The setup can be used advantageously in practical courses for training students in super-resolution fluorescence microscopy.
• Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM) Whelan, Donna R.; Holm, Thorge; Sauer, Markus; Bell, Toby D. M. in Australian Journal of Chemistry (2014). 67 179–183.
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  The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

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

  %0 Journal Article %1 whelan2014focus %A Whelan, Donna R. %A Holm, Thorge %A Sauer, Markus %A Bell, Toby D. M. %B 2 %D 2014 %J Australian Journal of Chemistry %P 179-183 %T Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM) %U http://www.publish.csiro.au/paper/CH13499 %V 67 %X The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.
• Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx Löschberger, Anna; Niehörster, Thomas; Sauer, Markus in Biotechnology Journal (2014). n/a-n/a.
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  Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.

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

  %0 Journal Article %1 BIOT:BIOT201400026 %A Löschberger, Anna %A Niehörster, Thomas %A Sauer, Markus %D 2014 %I WILEY-VCH Verlag %J Biotechnology Journal %P n/a--n/a %R 10.1002/biot.201400026 %T Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx %U http://dx.doi.org/10.1002/biot.201400026 %X Reactive oxygen species (ROS), including hydrogen peroxide, are known to cause structural damage not only in living, but also in fixed, cells. Copper-catalyzed azide–alkyne cycloaddition (click chemistry) is known to produce ROS. Therefore, fluorescence imaging of cellular structures, such as the actin cytoskeleton, remains challenging when combined with click chemistry protocols. In addition, the production of ROS substantially weakens the fluorescence signal of fluorescent proteins. This led us to develop ClickOx, which is a new click chemistry protocol for improved conservation of the actin structure and better conservation of the fluorescence signal of green fluorescent protein (GFP)-fusion proteins. Herein we demonstrate that efficient oxygen removal by addition of an enzymatic oxygen scavenger system (ClickOx) considerably reduces ROS-associated damage during labeling of nascent DNA with ATTO 488 azide by Cu(I)-catalyzed click chemistry. Standard confocal and super-resolution fluorescence images of phalloidin-labeled actin filaments and GFP/yellow fluorescent protein-labeled cells verify the conservation of the cytoskeleton microstructure and fluorescence intensity, respectively. Thus, ClickOx can be used advantageously for structure preservation in conventional and most notably in super-resolution microscopy methods.
• High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons Andreska, Thomas; Aufmkolk, Sarah; Sauer, Markus; Blum, Robert in Frontiers in Cellular Neuroscience (2014). 8(107)
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  In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.

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

  %0 Journal Article %1 10.3389/fncel.2014.00107 %A Andreska, Thomas %A Aufmkolk, Sarah %A Sauer, Markus %A Blum, Robert %D 2014 %J Frontiers in Cellular Neuroscience %N 107 %R 10.3389/fncel.2014.00107 %T High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons %U http://www.frontiersin.org/cellular_neuroscience/10.3389/fncel.2014.00107/abstract %V 8 %X In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.
• The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress Mehlitz, Adrian; Karunakaran, Karthika; Herweg, Jo-Ana; Krohne, Georg; van de Linde, Sebastian; Rieck, Elke; Sauer, Markus; Rudel, Thomas in Cellular Microbiology (2014). n/a-n/a.
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  Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.

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

  %0 Journal Article %1 CMI:CMI12278 %A Mehlitz, Adrian %A Karunakaran, Karthika %A Herweg, Jo-Ana %A Krohne, Georg %A van de Linde, Sebastian %A Rieck, Elke %A Sauer, Markus %A Rudel, Thomas %D 2014 %J Cellular Microbiology %P n/a--n/a %R 10.1111/cmi.12278 %T The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress %U http://dx.doi.org/10.1111/cmi.12278 %X Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER–vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER–vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER–SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER–SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.
• Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging Proppert, Sven; Wolter, Steve; Holm, Thorge; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus in Opt. Express (2014). 22(9) 10304–10316.
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  In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentallysimple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches andcompare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.

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

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

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

  %0 Journal Article %1 ehmann2014quantitative %A Ehmann, Nadine %A van de Linde, Sebastian %A Alon, Amit %A Ljaschenko, Dmitrij %A Keung, Xi Zhen %A Holm, Thorge %A Rings, Annika %A DiAntonio, Aaron %A Hallermann, Stefan %A Ashery, Uri %A Heckmann, Manfred %A Sauer, Markus %A Kittel, Robert J. %D 2014 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Commun %T Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states %U http://dx.doi.org/10.1038/ncomms5650 %V 5 %X The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.
• PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching Sauer, Markus; Neuweiler, Hannes in Methods Mol Biol - Fluorescence Spectroscopy and Microscopy, Y. Engelborghs, A. J. Visser (Hrsg.) (2014). (Bd. 1076) 597–615.
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  Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples.

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

  %0 Book Section %1 year={2014} %A Sauer, Markus %A Neuweiler, Hannes %B Methods Mol Biol - Fluorescence Spectroscopy and Microscopy %D 2014 %E Engelborghs, Yves %E Visser, Antonie J.W.G. %I Humana Press %P 597-615 %R 10.1007/978-1-62703-649-8_27 %T PET-FCS: Probing Rapid Structural Fluctuations of Proteins and Nucleic Acids by Single-Molecule Fluorescence Quenching %U http://dx.doi.org/10.1007/978-1-62703-649-8_27 %V 1076 %X Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van der Waals contact and can be used as reporter for structural fluctuations at the 1-nm scale in proteins, peptides, and nucleic acids. The combination of PET with fluorescence correlation spectroscopy (FCS) establishes a powerful method (PET-FCS) to study equilibrium dynamics at the single-molecule level on time scales from nano- to milliseconds. We delineate the fundamentals of PET-based fluorescence quenching, reporter engineering, instrumental and experimental design, and provide examples. %@ 978-1-62703-648-1
• Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus in Journal of Cell Science (2014). 127(20) 4351–4355.
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  Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

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

  %0 Journal Article %1 Löschberger15102014 %A Löschberger, Anna %A Franke, Christian %A Krohne, Georg %A van de Linde, Sebastian %A Sauer, Markus %D 2014 %J Journal of Cell Science %N 20 %P 4351-4355 %R 10.1242/jcs.156620 %T Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution %U http://jcs.biologists.org/content/127/20/4351.abstract %V 127 %X Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.
• Timing Protein Assembly in Neurons Sauer, Markus in Chemistry & Biology (2014). 21(6) 703–704.
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  Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.

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

  %0 Journal Article %1 sauer2014timing %A Sauer, Markus %D 2014 %J Chemistry & Biology %N 6 %P 703--704 %R https://doi.org/10.1016/j.chembiol.2014.06.001 %T Timing Protein Assembly in Neurons %U http://www.sciencedirect.com/science/article/pii/S1074552114001847 %V 21 %X Integration of two fluorescence imaging methods enables tracking of the formation of fibrillar Aβ peptide amyloid aggregates in neurons, as discussed by Esbjörner and colleagues in this issue of Chemistry & Biology. This approach has the potential to fundamentally improve our understanding of the onset and therapeutic intervention of neurodegenerative diseases.
• Eight years of single-molecule localization microscopy Klein, Teresa; Proppert, Sven; Sauer, Markus in Histochemistry and Cell Biology (2014). 141(6) 561–575.
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  Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.

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

  %0 Journal Article %1 klein2014eight %A Klein, Teresa %A Proppert, Sven %A Sauer, Markus %D 2014 %J Histochemistry and Cell Biology %N 6 %P 561--575 %R 10.1007/s00418-014-1184-3 %T Eight years of single-molecule localization microscopy %U https://doi.org/10.1007/s00418-014-1184-3 %V 141 %X Super-resolution imaging by single-molecule localization (localization microscopy) provides the ability to unravel the structural organization of cells and the composition of biomolecular assemblies at a spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. Constant improvements in fluorescent probes, efficient and specific labeling techniques as well as refined data analysis and interpretation strategies further improved localization microscopy. Today, it allows us to interrogate how the distribution and stoichiometry of interacting proteins in subcellular compartments and molecular machines accomplishes complex interconnected cellular processes. Thus, it exhibits potential to address fundamental questions of cell and developmental biology. Here, we briefly introduce the history, basic principles, and different localization microscopy methods with special focus on direct stochastic optical reconstruction microscopy (dSTORM) and summarize key developments and examples of two- and three-dimensional localization microscopy of the last 8 years.
• Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity Bielopolski, Noa; Lam, Alice D.; Bar-On, Dana; Sauer, Markus; Stuenkel, Edward L.; Ashery, Uri in Journal of Biological Chemistry (2014). 289 17087–17099.
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  Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

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

  %0 Journal Article %1 bielopolski2014differential %A Bielopolski, Noa %A Lam, Alice D. %A Bar-On, Dana %A Sauer, Markus %A Stuenkel, Edward L. %A Ashery, Uri %B 24 %D 2014 %J Journal of Biological Chemistry %P 17087-17099 %T Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity %U http://www.jbc.org/content/289/24/17087.abstract %V 289 %X Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537–578 or 897–917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

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

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

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

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

  %0 Journal Article %1 van de Linde20138 %A van de Linde, Sebastian %A Aufmkolk, Sarah %A Franke, Christian %A Holm, Thorge %A Klein, Teresa %A Löschberger, Anna %A Proppert, Sven %A Wolter, Steve %A Sauer, Markus %D 2013 %J Chemistry & Biology %N 1 %P 8 - 18 %R 10.1016/j.chembiol.2012.11.004 %T Investigating Cellular Structures at the Nanoscale with Organic Fluorophores %U http://www.sciencedirect.com/science/article/pii/S1074552112004280 %V 20 %X Super-resolution fluorescence imaging can provide insights into cellular structure and organization with a spatial resolution approaching virtually electron microscopy. Among all the different super-resolution methods single-molecule-based localization microscopy could play an exceptional role in the future because it can provide quantitative information, for example, the absolute number of biomolecules interacting in space and time. Here, small organic fluorophores are a decisive factor because they exhibit high fluorescence quantum yields and photostabilities, thus enabling their localization with nanometer precision. Besides past progress, problems with high-density and specific labeling, especially in living cells, and the lack of suited standards and long-term continuous imaging methods with minimal photodamage render the exploitation of the full potential of the method currently challenging.
• Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics Seibel, Jürgen; König, Simone; Göhler, Antonia; Doose, Sören; Memmel, Elisabeth; Bertleff, Nadja; Sauer, Markus in Expert Rev Proteomics (2013). 10(1) 25–31.
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  Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.

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

  %0 Journal Article %1 seibel2013investigating %A Seibel, Jürgen %A König, Simone %A Göhler, Antonia %A Doose, Sören %A Memmel, Elisabeth %A Bertleff, Nadja %A Sauer, Markus %B Expert Review of Proteomics %D 2013 %I Expert Reviews %J Expert Rev Proteomics %N 1 %P 25--31 %R 10.1586/epr.12.72 %T Investigating infection processes with a workflow from organic chemistry to biophysics: the combination of metabolic glycoengineering, super-resolution fluorescence imaging and proteomics %U http://dx.doi.org/10.1586/epr.12.72 %V 10 %X Infectious diseases continue to be one of the major threats to public health. In the initial events of infection, glycoproteins of human cells interact with surface proteins of bacteria or viruses, the so-called environmental adhesins. In order to pinpoint the driving forces during infection, it is necessary to study the adhesive properties of human cell surface glycoproteins with regard to their primary amino acid sequence and post-translational modifications. The authors discuss how recent developments in seemingly independent fields of the natural sciences, bio-organic synthesis, biophysical visualization and bioanalysis, open the door for a promising interdisciplinary approach to study human infection processes. The use of special synthesized carbohydrate labels, in combination with new super-resolution imaging approaches, allows access to both mapping and identification of cell surface glycoproteins well below the diffraction limit. The methodology will clarify which surface molecules are involved in bacterial adherence with potential implications for bacterial and viral infection prevention.
• Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes Andronic, Joseph; Bobak, Nicole; Bittner, Stefan; Ehling, Petra; Kleinschnitz, Christoph; Herrmann, Alexander M.; Zimmermann, Heiko; Sauer, Markus; Wiendl, Heinz; Budde, Thomas; Meuth, Sven G.; Sukhorukov, Vladimir L. in Biochimica et Biophysica Acta (BBA) - Biomembranes (2013). 1828(2) 699–707.
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  Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.

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

  %0 Journal Article %1 Andronic2013699 %A Andronic, Joseph %A Bobak, Nicole %A Bittner, Stefan %A Ehling, Petra %A Kleinschnitz, Christoph %A Herrmann, Alexander M. %A Zimmermann, Heiko %A Sauer, Markus %A Wiendl, Heinz %A Budde, Thomas %A Meuth, Sven G. %A Sukhorukov, Vladimir L. %D 2013 %J Biochimica et Biophysica Acta (BBA) - Biomembranes %N 2 %P 699 - 707 %R 10.1016/j.bbamem.2012.09.028 %T Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes %U http://www.sciencedirect.com/science/article/pii/S0005273612003495 %V 1828 %X Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated KV1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K2P) channels in the RVD of human CD4+ T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K2P5.1 and K2P18.1 as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K2P5.1 and K2P18.1 on the RVD was comparable to that of KV1.3. K2P9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K2P channels.
• Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus in Sci. Rep. (2013). 3
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  There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.

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

  %0 Journal Article %1 klamp2013highly %A Klamp, Tobias %A Camps, Marta %A Nieto, Benjamin %A Guasch, Francesc %A Ranasinghe, Rohan T. %A Wiedemann, Jens %A Petrášek, Zdeněk %A Schwille, Petra %A Klenerman, David %A Sauer, Markus %D 2013 %I Macmillan Publishers Limited. All rights reserved %J Sci. Rep. %T Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay %U http://dx.doi.org/10.1038/srep01852 %V 3 %X There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude.
• Localization microscopy coming of age: from concepts to biological impact Sauer, Markus in Journal of Cell Science (2013). 126(16) 3505–3513.
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  Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.

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

  %0 Journal Article %1 Sauer15082013 %A Sauer, Markus %D 2013 %J Journal of Cell Science %N 16 %P 3505-3513 %R 10.1242/jcs.123612 %T Localization microscopy coming of age: from concepts to biological impact %U http://jcs.biologists.org/content/126/16/3505.abstract %V 126 %X Super-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.
• Methylene blue- and thiol-based oxygen depletion for super-resolution imaging Schäfer, Philip; van de Linde, Sebastian; Lehmann, Julian; Sauer, Markus; Doose, Sören in Analytical Chemistry (2013). 85(6) 3393–3400.
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  Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

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

  %0 Journal Article %1 doi:10.1021/ac400035k %A Schäfer, Philip %A van de Linde, Sebastian %A Lehmann, Julian %A Sauer, Markus %A Doose, Sören %D 2013 %J Analytical Chemistry %N 6 %P 3393 - 3400 %R 10.1021/ac400035k %T Methylene blue- and thiol-based oxygen depletion for super-resolution imaging %U http://pubs.acs.org/doi/abs/10.1021/ac400035k %V 85 %X Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions aiming at designing an oxygen scavenger system as alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as β-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere β-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.
• Elements of Transcriptional Machinery Are Compatible among Plants and Mammals Wolf, Annette; Akrap, Nina; Marg, Berenice; Galliardt, Helena; Heiligentag, Martyna; Humpert, Fabian; Sauer, Markus; Kaltschmidt, Barbara; Kaltschmidt, Christian; Seidel, Thorsten in PLOS ONE (2013). 8(1) e53737-.
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  In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.

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

  %0 Journal Article %1 wolf2013elements %A Wolf, Annette %A Akrap, Nina %A Marg, Berenice %A Galliardt, Helena %A Heiligentag, Martyna %A Humpert, Fabian %A Sauer, Markus %A Kaltschmidt, Barbara %A Kaltschmidt, Christian %A Seidel, Thorsten %D 2013 %I Public Library of Science %J PLOS ONE %N 1 %P e53737-- %T Elements of Transcriptional Machinery Are Compatible among Plants and Mammals %U https://doi.org/10.1371/journal.pone.0053737 %V 8 %X In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-κB in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-κB such as its inhibitor IκB isoform α that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-κB in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-κB signaling pathways. The system successfully enabled the controlled manipulation of NF-κB activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e.g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
• A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants Lummer, Martina; Humpert, Fabian; Wiedenlübbert, Matthias; Sauer, Markus; Schüttpelz, Mark; Staiger, Dorothee in Molecular Plant (2013). 6(5) 1518–1530.
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  Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.

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

  %0 Journal Article %1 lummer2013reversibly %A Lummer, Martina %A Humpert, Fabian %A Wiedenlübbert, Matthias %A Sauer, Markus %A Schüttpelz, Mark %A Staiger, Dorothee %D 2013 %J Molecular Plant %N 5 %P 1518--1530 %R https://doi.org/10.1093/mp/sst040 %T A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants %U http://www.sciencedirect.com/science/article/pii/S1674205214602240 %V 6 %X Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolution fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluorescent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ‘positively switchable’ PADRON C-s with the ‘negatively switchable’ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localization analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each photoactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7:rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants. Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells. Synthetic, codon-optimized reversibly photoswitchable fluorescent proteins rsFastLIME-s, bsDRONPA-s, and PADRONC-s show high-level expression in both plants and mammalian cells. Repeated on-switching allows collecting extended series of images, thus enabling live cell imaging with high temporal and spatial resolution.

• Superresolution Optical Fluctuation Imaging (SOFI) Dertinger, Thomas; Colyer, Ryan; Vogel, Robert; Heilemann, Mike; Sauer, Markus; Enderlein, Jörg; Weiss, Shimon in Advances in Experimental Medicine and Biology, (E. Zahavy; A. Ordentlich; S. Yitzhaki; A. Shafferman, Hrsg.) (2012). 733 17–21.
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  Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.

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

  %0 Journal Article %1 springerlink:10.1007/978-94-007-2555-3_2 %A Dertinger, Thomas %A Colyer, Ryan %A Vogel, Robert %A Heilemann, Mike %A Sauer, Markus %A Enderlein, Jörg %A Weiss, Shimon %B Nano-Biotechnology for Biomedical and Diagnostic Research %D 2012 %E Zahavy, Eran %E Ordentlich, Arie %E Yitzhaki, Shmuel %E Shafferman, Avigdor %I Springer Netherlands %J Advances in Experimental Medicine and Biology %P 17-21 %T Superresolution Optical Fluctuation Imaging (SOFI) %U http://dx.doi.org/10.1007/978-94-007-2555-3_2 %V 733 %X Superresolution microscopy has shifted the limits for fluorescence microscopy in cell ­biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed “superresolution optical fluctuation ­imaging” (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances. %@ 978-94-007-2555-3
• Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution Löschberger, Anna; van de Linde, Sebastian; Dabauvalle, Marie-Christine; Rieger, Bernd; Heilemann, Mike; Krohne, Georg; Sauer, Markus in Journal of Cell Science (2012). 125(3) 570–575.
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  One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

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

  %0 Journal Article %1 Löschberger01022012 %A Löschberger, Anna %A van de Linde, Sebastian %A Dabauvalle, Marie-Christine %A Rieger, Bernd %A Heilemann, Mike %A Krohne, Georg %A Sauer, Markus %D 2012 %J Journal of Cell Science %N 3 %P 570-575 %R 10.1242/jcs.098822 %T Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution %U http://jcs.biologists.org/content/125/3/570.abstract %V 125 %X One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.
• Live-Cell Super-Resolution Imaging with Synthetic Fluorophores van de Linde, Sebastian; Heilemann, Mike; Sauer, Markus in Annual Review of Physical Chemistry (2012). 63(1) null.
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  Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

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

  %0 Journal Article %1 doi:10.1146/annurev-physchem-032811-112012 %A van de Linde, Sebastian %A Heilemann, Mike %A Sauer, Markus %D 2012 %J Annual Review of Physical Chemistry %N 1 %P null %R 10.1146/annurev-physchem-032811-112012 %T Live-Cell Super-Resolution Imaging with Synthetic Fluorophores %U http://www.annualreviews.org/doi/abs/10.1146/annurev-physchem-032811-112012 %V 63 %X Super-resolution imaging methods now can provide spatial resolution that is well below the diffraction limit approaching virtually molecular resolution. They can be applied to biological samples and provide new and exciting views on the structural organization of cells and the dynamics of biomolecular assemblies on wide timescales. These revolutionary developments come with novel requirements for fluorescent probes, labeling techniques, and data interpretation strategies. Synthetic fluorophores have a small size, are available in many colors spanning the whole spectrum, and can easily be chemically modified and used for stoichiometric labeling of proteins in live cells. Because of their brightness, their photostability, and their ability to be operated as photoswitchable fluorophores even in living cells under physiological conditions, synthetic fluorophores have the potential to substantially accelerate the broad application of live-cell super-resolution imaging methods. Expected final online publication date for the Annual Review of Physical Chemistry Volume 63 is March 31, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
• Fluorescence localization microscopy: The transition from concept to biological research tool Owen, Dylan M.; Sauer, Markus; Gaus, Katharina in cib (2012). 5(1942-0889) 0–1.
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  Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.

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

  %0 Journal Article %1 owen2012fluorescence %A Owen, Dylan M. %A Sauer, Markus %A Gaus, Katharina %D 2012 %I Landes Bioscience Inc. %J cib %N 1942-0889 %P 0---1 %T Fluorescence localization microscopy: The transition from concept to biological research tool %U http://www.landesbioscience.com/journals/cib/article/20348/ %V 5 %X Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.
• Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters Bar-On, Dana; Wolter, Steve; van de Linde, Sebastian; Heilemann, Mike; Nudelman, German; Nachliel, Esther; Gutman, Menachem; Sauer, Markus; Ashery, Uri in Journal of Biological Chemistry (2012).
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  Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

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

  %0 Journal Article %1 Bar-On14062012 %A Bar-On, Dana %A Wolter, Steve %A van de Linde, Sebastian %A Heilemann, Mike %A Nudelman, German %A Nachliel, Esther %A Gutman, Menachem %A Sauer, Markus %A Ashery, Uri %D 2012 %J Journal of Biological Chemistry %R 10.1074/jbc.M112.353250 %T Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters %U http://www.jbc.org/content/early/2012/06/14/jbc.M112.353250.abstract %X Key synaptic proteins from the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy (dSTORM) together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and non-clustered molecules at the membrane of PC12 cells with single-molecule precision. dSTORM images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The non-clustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Though, syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.
• Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images WOLTER, S.; SAUER, M. in Journal of Microscopy (2012). 245(1) 109–109.
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  @article{JMI:JMI3562, author = {WOLTER, S. and SAUER, M.}, journal = {Journal of Microscopy}, keywords = {sauer}, number = 1, pages = {109–109}, publisher = {Blackwell Publishing Ltd}, title = {Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images}, volume = 245, year = 2012 }

  %0 Journal Article %1 JMI:JMI3562 %A WOLTER, S. %A SAUER, M. %D 2012 %I Blackwell Publishing Ltd %J Journal of Microscopy %N 1 %P 109--109 %R 10.1111/j.1365-2818.2011.03562.x %T Follow-up to paper by S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, entitled Real-Time Computation of Subdiffraction-Resolution Fluorescence Images %U http://dx.doi.org/10.1111/j.1365-2818.2011.03562.x %V 245
• Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies Terpitz, Ulrich; Letschert, Sebastian; Bonda, Ulrich; Spahn, Christoph; Guan, Chonglin; Sauer, Markus; Zimmermann, Ulrich; Bamberg, Ernst; Zimmermann, Dirk; Sukhorukov, Vladimir in Journal of Membrane Biology (2012). 1–12.
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  The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.

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

  %0 Journal Article %1 springerlink:10.1007/s00232-012-9484-9 %A Terpitz, Ulrich %A Letschert, Sebastian %A Bonda, Ulrich %A Spahn, Christoph %A Guan, Chonglin %A Sauer, Markus %A Zimmermann, Ulrich %A Bamberg, Ernst %A Zimmermann, Dirk %A Sukhorukov, Vladimir %D 2012 %I Springer New York %J Journal of Membrane Biology %P 1-12 %T Dielectric Analysis and Multi-cell Electrofusion of the Yeast Pichia pastoris for Electrophysiological Studies %U http://dx.doi.org/10.1007/s00232-012-9484-9 %X The yeast Pichia pastoris has become the most favored eukaryotic host for heterologous protein expression. P. pastoris strains capable of overexpressing various membrane proteins are now available. Due to their small size and the fungal cell wall, however, P. pastoris cells are hardly suitable for direct electrophysiological studies. To overcome these limitations, the present study aimed to produce giant protoplasts of P. pastoris by means of multi-cell electrofusion. Using a P. pastoris strain expressing channelrhodopsin-2 (ChR2), we first developed an improved enzymatic method for cell wall digestion and preparation of wall-less protoplasts. We thoroughly analyzed the dielectric properties of protoplasts by means of electrorotation and dielectrophoresis. Based on the dielectric data of tiny parental protoplasts (2–4 μm diameter), we elaborated efficient electrofusion conditions yielding consistently stable multinucleated protoplasts of P. pastoris with diameters of up to 35 μm. The giant protoplasts were suitable for electrophysiological measurements, as proved by whole-cell patch clamp recordings of light-induced, ChR2-mediated currents, which was impossible with parental protoplasts. The approach presented here offers a potentially valuable technique for the functional analysis of low-signal channels and transporters, expressed heterologously in P. pastoris and related host systems.
• Live-Cell Super-Resolution Imaging Goes Multicolor Klein, Teresa; van de Linde, Sebastian; Sauer, Markus in ChemBioChem (2012). 13(13) 1861–1863.
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  New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.

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

  %0 Journal Article %1 CBIC:CBIC201200347 %A Klein, Teresa %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I WILEY-VCH Verlag %J ChemBioChem %N 13 %P 1861--1863 %R 10.1002/cbic.201200347 %T Live-Cell Super-Resolution Imaging Goes Multicolor %U http://dx.doi.org/10.1002/cbic.201200347 %V 13 %X New resolutions: The combined use of photoactivatable fluorescent proteins and synthetic fluorophores considerably expands our options for multicolor super-resolution fluorescence imaging and enables for the first time the simultaneous imaging of more than two proteins with subdiffraction optical resolution in living cells.
• rapidSTORM: accurate, fast open-source software for localization microscopy Wolter, Steve; Loschberger, Anna; Holm, Thorge; Aufmkolk, Sarah; Dabauvalle, Marie-Christine; van de Linde, Sebastian; Sauer, Markus in Nat Meth (2012). 9(11) 1040–1041.
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  To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.

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

  %0 Journal Article %1 wolter2012rapidstorm %A Wolter, Steve %A Loschberger, Anna %A Holm, Thorge %A Aufmkolk, Sarah %A Dabauvalle, Marie-Christine %A van de Linde, Sebastian %A Sauer, Markus %D 2012 %I Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. %J Nat Meth %N 11 %P 1040--1041 %T rapidSTORM: accurate, fast open-source software for localization microscopy %U http://dx.doi.org/10.1038/nmeth.2224 %V 9 %X To the Editor In recent years, there has been a dramatic increase in the use of localization microscopy: that is, super-resolution fluorescence imaging by stochastic photoswitching, photoactivation or other fluorescence-generating processes. The evaluation of localization microscopy data with a Gaussian model of the point-spread function (PSF) is a common and cogent technique, and Poissonian maximum-likelihood estimator (MLE) fitting of the Gaussian model has been shown to yield optimal precision. However, current software support is based on less precise approximations of MLE fitting or is slow, hardware dependent, closed source or unmaintained. Many advances have been made with unpublished or prototypical software packages, leaving established labs with homemade software and new labs with immense technology entry costs. This state has weakened the core strengths of localization microscopy: cost-efficiency and experimental simplicity.
• Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast Lando, David; Endesfelder, Ulrike; Berger, Harald; Subramanian, Lakxmi; Dunne, Paul D.; McColl, James; Klenerman, David; Carr, Antony M.; Sauer, Markus; Allshire, Robin C.; Heilemann, Mike; Laue, Ernest D. in Open Biology (2012). 2(7)
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  The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

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

  %0 Journal Article %1 LandoJuly2012 %A Lando, David %A Endesfelder, Ulrike %A Berger, Harald %A Subramanian, Lakxmi %A Dunne, Paul D. %A McColl, James %A Klenerman, David %A Carr, Antony M. %A Sauer, Markus %A Allshire, Robin C. %A Heilemann, Mike %A Laue, Ernest D. %D 2012 %J Open Biology %N 7 %R 10.1098/rsob.120078 %T Quantitative single-molecule microscopy reveals that CENP-ACnp1 deposition occurs during G2 in fission yeast %U http://rsob.royalsocietypublishing.org/content/2/7/120078.abstract %V 2 %X The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.
• Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments Yahiatène, Idir; Doose, Sören; Huser, Thomas; Sauer, Markus in Analytical Chemistry (2012). 84(6) 2729–2736.
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  We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.

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

  %0 Journal Article %1 doi:10.1021/ac2030283 %A Yahiatène, Idir %A Doose, Sören %A Huser, Thomas %A Sauer, Markus %D 2012 %J Analytical Chemistry %N 6 %P 2729-2736 %R 10.1021/ac2030283 %T Correlation-Matrix Analysis of Two-Color Coincidence Events in Single-Molecule Fluorescence Experiments %U http://pubs.acs.org/doi/abs/10.1021/ac2030283 %V 84 %X We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.

• Live-cell dSTORM with SNAP-tag fusion proteins Klein, Teresa; Loeschberger, Anna; Proppert, Sven; Wolter, Steve; van de Linde, Sebastian; Sauer, Markus in NATURE METHODS (2011). 8(1) 7–9.
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  In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.

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

  %0 Journal Article %1 Klein2011 %A Klein, Teresa %A Loeschberger, Anna %A Proppert, Sven %A Wolter, Steve %A van de Linde, Sebastian %A Sauer, Markus %C MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND %D 2011 %I NATURE PUBLISHING GROUP %J NATURE METHODS %N 1 %P 7-9 %T Live-cell dSTORM with SNAP-tag fusion proteins %U http://dx.doi.org/10.1038/nmeth0111-7b %V 8 %X In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione.
• Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments Noe, Frank; Doose, Sören; Daidone, Isabella; Löllmann, Marc; Sauer, Markus; Chodera, John D.; Smith, Jeremy C. in Proceedings of the National Academy of Sciences (2011).
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  There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprints� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.

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

  %0 Journal Article %1 Noe %A Noe, Frank %A Doose, Sören %A Daidone, Isabella %A Löllmann, Marc %A Sauer, Markus %A Chodera, John D. %A Smith, Jeremy C. %D 2011 %J Proceedings of the National Academy of Sciences %T Dynamical fingerprints for probing individual relaxation processes in biomolecular dynamics with simulations and kinetic experiments %U http://dx.doi.org/10.1073/pnas.1004646108 %X There is a gap between kinetic experiment and simulation in their views of the dynamics of complex biomolecular systems. Whereas experiments typically reveal only a few readily discernible exponential relaxations, simulations often indicate complex multistate behavior. Here, a theoretical framework is presented that reconciles these two approaches. The central concept is “dynamical fingerprintsâ€� which contain peaks at the time scales of the dynamical processes involved with amplitudes determined by the experimental observable. Fingerprints can be generated from both experimental and simulation data, and their comparison by matching peaks permits assignment of structural changes present in the simulation to experimentally observed relaxation processes. The approach is applied here to a test case interpreting single molecule fluorescence correlation spectroscopy experiments on a set of fluorescent peptides with molecular dynamics simulations. The peptides exhibit complex kinetics shown to be consistent with the apparent simplicity of the experimental data. Moreover, the fingerprint approach can be used to design new experiments with site-specific labels that optimally probe specific dynamical processes in the molecule under investigation.
• Direct stochastic optical reconstruction microscopy with standard fluorescent probes van de Linde, Sebastian; Loschberger, Anna; Klein, Teresa; Heidbreder, Meike; Wolter, Steve; Heilemann, Mike; Sauer, Markus in Nat. Protocols (2011). 6(7) 991–1009.
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  Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ~20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.

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