TY - JOUR A1 - Ziegler, Sabrina A1 - Weiss, Esther A1 - Schmitt, Anna-Lena A1 - Schlegel, Jan A1 - Burgert, Anne A1 - Terpitz, Ulrich A1 - Sauer, Markus A1 - Moretta, Lorenzo A1 - Sivori, Simona A1 - Leonhardt, Ines A1 - Kurzai, Oliver A1 - Einsele, Hermann A1 - Loeffler, Juergen T1 - CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells JF - Scientific Reports N2 - 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. KW - pattern recognition receptors KW - fungal infection KW - Aspergillus fumigatus KW - natural killer cells Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170637 VL - 7 IS - 6138 ER - TY - JOUR A1 - Memmel, Simon A1 - Sisario, Dmitri A1 - Zöller, Caren A1 - Fiedler, Vanessa A1 - Katzer, Astrid A1 - Heiden, Robin A1 - Becker, Nicholas A1 - Eing, Lorenz A1 - Ferreira, Fábio L.R. A1 - Zimmermann, Heiko A1 - Sauer, Markus A1 - Flentje, Michael A1 - Sukhorukov, Vladimir L. A1 - Djuzenova, Cholpon S. T1 - Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities JF - Oncotarget N2 - 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/mTOR-inhibitor 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 line-specific 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. KW - chemotherapy KW - glioblastoma multiforme KW - migration KW - treatment Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170719 VL - 8 IS - 28 ER - TY - JOUR A1 - Lukeš, Tomáš A1 - Glatzová, Daniela A1 - Kvíčalová, Zuzana A1 - Levet, Florian A1 - Benda, Aleš A1 - Letschert, Sebastian A1 - Sauer, Markus A1 - Brdička, Tomáš A1 - Lasser, Theo A1 - Cebecauer, Marek T1 - Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging JF - Nature Communications N2 - 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. KW - biology KW - fluorescence imaging KW - imaging the immune system KW - super-resolution microscopy Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172993 VL - 8 ER - TY - JOUR A1 - Ferero, Andrea A1 - Rivero, Olga A1 - Wäldchen, Sina A1 - Ku, Hsing-Ping A1 - Kiser, Dominik P. A1 - Gärtner, Yvonne A1 - Pennington, Laura S. A1 - Waider, Jonas A1 - Gaspar, Patricia A1 - Jansch, Charline A1 - Edenhofer, Frank A1 - Resink, Thérèse J. A1 - Blum, Robert A1 - Sauer, Markus A1 - Lesch, Klaus-Peter T1 - Cadherin-13 Deficiency Increases Dorsal Raphe 5-HT Neuron Density and Prefrontal Cortex Innervation in the Mouse Brain JF - Frontiers in Cellular Neuroscience N2 - 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 mice display increased cell densities in the DR at embryonic stages 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 prefrontal cortex (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. KW - serotonin KW - cadherin-13 (CDH13) KW - T-cadherin KW - neurodevelopment KW - psychiatric disorders KW - radial glia KW - dorsal raphe KW - prefrontal cortex Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170313 VL - 11 IS - 307 ER - TY - JOUR A1 - Becam, Jérôme A1 - Walter, Tim A1 - Burgert, Anne A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Seibel, Jürgen A1 - Schubert-Unkmeir, Alexandra T1 - Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria JF - Scientific Reports N2 - Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae. KW - ceramide analogs KW - Neisseria KW - ceramide Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159367 VL - 7 ER - TY - JOUR A1 - Scholz, Nicole A1 - Guan, Chonglin A1 - Nieberler, Matthias A1 - Grotmeyer, Alexander A1 - Maiellaro, Isabella A1 - Gao, Shiqiang A1 - Beck, Sebastian A1 - Pawlak, Matthias A1 - Sauer, Markus A1 - Asan, Esther A1 - Rothemund, Sven A1 - Winkler, Jana A1 - Prömel, Simone A1 - Nagel, Georg A1 - Langenhan, Tobias A1 - Kittel, Robert J T1 - Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons JF - eLife N2 - 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. KW - Latrophilin KW - adhesion GPCR KW - dCIRL KW - sensory physiology KW - metabotropic signalling KW - mechanotransduction Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170520 VL - 6 IS - e28360 ER -