@article{KouhestaniGeisAlsourietal.2021, author = {Kouhestani, Dina and Geis, Maria and Alsouri, Saed and Bumm, Thomas G. P. and Einsele, Hermann and Sauer, Markus and Stuhler, Gernot}, title = {Variant signaling topology at the cancer cell-T-cell interface induced by a two-component T-cell engager}, series = {Cellular \& Molecular Immunology}, volume = {18}, journal = {Cellular \& Molecular Immunology}, doi = {10.1038/s41423-020-0507-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-241189}, pages = {1568-1570}, year = {2021}, abstract = {No abstract available.}, language = {en} } @article{KunzGoetzGaoetal.2020, author = {Kunz, Tobias C. and G{\"o}tz, Ralph and Gao, Shiqiang and Sauer, Markus and Kozjak-Pavlovic, Vera}, title = {Using Expansion Microscopy to Visualize and Characterize the Morphology of Mitochondrial Cristae}, series = {Frontiers in Cell and Developmental Biology}, volume = {8}, journal = {Frontiers in Cell and Developmental Biology}, issn = {2296-634X}, doi = {10.3389/fcell.2020.00617}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208296}, year = {2020}, 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.}, language = {en} } @article{BalakrishnanHemmenChoudhuryetal.2022, author = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.}, title = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence}, series = {Communications Biology}, volume = {5}, journal = {Communications Biology}, number = {1}, doi = {10.1038/s42003-022-03106-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301140}, year = {2022}, abstract = {G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbr{\"u}ck model.}, language = {en} } @article{HaakeHaackSchaeferetal.2023, author = {Haake, Markus and Haack, Beatrice and Sch{\"a}fer, Tina and Harter, Patrick N. and Mattavelli, Greta and Eiring, Patrick and Vashist, Neha and Wedekink, Florian and Genssler, Sabrina and Fischer, Birgitt and Dahlhoff, Julia and Mokhtari, Fatemeh and Kuzkina, Anastasia and Welters, Marij J. P. and Benz, Tamara M. and Sorger, Lena and Thiemann, Vincent and Almanzar, Giovanni and Selle, Martina and Thein, Klara and Sp{\"a}th, Jacob and Gonzalez, Maria Cecilia and Reitinger, Carmen and Ipsen-Escobedo, Andrea and Wistuba-Hamprecht, Kilian and Eichler, Kristin and Filipski, Katharina and Zeiner, Pia S. and Beschorner, Rudi and Goedemans, Renske and Gogolla, Falk Hagen and Hackl, Hubert and Rooswinkel, Rogier W. and Thiem, Alexander and Romer Roche, Paula and Joshi, Hemant and P{\"u}hringer, Dirk and W{\"o}ckel, Achim and Diessner, Joachim E. and R{\"u}diger, Manfred and Leo, Eugen and Cheng, Phil F. and Levesque, Mitchell P. and Goebeler, Matthias and Sauer, Markus and Nimmerjahn, Falk and Schuberth-Wagner, Christine and Felten, Stefanie von and Mittelbronn, Michel and Mehling, Matthias and Beilhack, Andreas and van der Burg, Sjoerd H. and Riedel, Angela and Weide, Benjamin and Dummer, Reinhard and Wischhusen, J{\"o}rg}, title = {Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-39817-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357333}, year = {2023}, abstract = {Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.}, language = {en} } @article{BoertleinDraegerSchoenaueretal.2018, author = {B{\"o}rtlein, Charlene and Draeger, Annette and Schoenauer, Roman and Kuhlemann, Alexander and Sauer, Markus and Schneider-Schaulies, Sybille and Avota, Elita}, title = {The neutral sphingomyelinase 2 is required to polarize and sustain T Cell receptor signaling}, series = {Frontiers in Immunology}, volume = {9}, journal = {Frontiers in Immunology}, number = {815}, doi = {10.3389/fimmu.2018.00815}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176572}, year = {2018}, 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 Ca\(^{2+}\) 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 are organized and stabilized via MTOC polarization.}, language = {en} } @article{DugarSvenssonBischleretal.2016, author = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Waldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.}, title = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni}, series = {Nature Communications}, volume = {7}, journal = {Nature Communications}, doi = {10.1038/ncomms11667}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173201}, year = {2016}, 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.}, language = {en} } @article{GeigerKerstingSchlegeletal.2022, author = {Geiger, Nina and Kersting, Louise and Schlegel, Jan and Stelz, Linda and F{\"a}hr, Sofie and Diesendorf, Viktoria and Roll, Valeria and Sostmann, Marie and K{\"o}nig, Eva-Maria and Reinhard, Sebastian and Brenner, Daniela and Schneider-Schaulies, Sibylle and Sauer, Markus and Seibel, J{\"u}rgen and Bodem, Jochen}, title = {The acid ceramidase is a SARS-CoV-2 host factor}, series = {Cells}, volume = {11}, journal = {Cells}, number = {16}, issn = {2073-4409}, doi = {10.3390/cells11162532}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286105}, year = {2022}, abstract = {SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.}, language = {en} } @article{PauliPaulProppertetal.2021, author = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and K{\"u}rzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sir{\´e}n, Anna-Leena}, title = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections}, series = {Communications Biology}, volume = {4}, journal = {Communications Biology}, doi = {10.1038/s42003-021-01939-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259830}, pages = {407}, year = {2021}, 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 mu m, 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 mu m 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. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.}, language = {en} } @article{EiringMcLaughlinMatikondaetal.2021, author = {Eiring, Patrick and McLaughlin, Ryan and Matikonda, Siddharth S. and Han, Zhongying and Grabenhorst, Lennart and Helmerich, Dominic A. and Meub, Mara and Beliu, Gerti and Luciano, Michael and Bandi, Venu and Zijlstra, Niels and Shi, Zhen-Dan and Tarasov, Sergey G. and Swenson, Rolf and Tinnefeld, Philip and Glembockyte, Viktorija and Cordes, Thorben and Sauer, Markus and Schnermann, Martin J.}, title = {Targetable conformationally restricted cyanines enable photon-count-limited applications}, series = {Angewandte Chemie Internationale Edition}, volume = {60}, journal = {Angewandte Chemie Internationale Edition}, number = {51}, doi = {10.1002/anie.202109749}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-256559}, pages = {26685-26693}, year = {2021}, abstract = {Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule F{\"o}rster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.}, language = {en} } @article{GrimmKleinKopeketal.2016, 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.}, title = {Synthesis of a far-red photoactivatable silicon-containing rhodamine for super-resolution microscopy}, series = {Angewandte Chemie International Edition}, volume = {55}, journal = {Angewandte Chemie International Edition}, number = {5}, doi = {10.1002/anie.201509649}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191069}, pages = {1723-1727}, year = {2016}, 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.}, language = {en} } @article{HaackBaikerSchlegeletal.2021, author = {Haack, Stephanie and Baiker, Sarah and Schlegel, Jan and Sauer, Markus and Sparwasser, Tim and Langenhorst, Daniela and Beyersdorf, Niklas}, title = {Superagonistic CD28 stimulation induces IFN-γ release from mouse T helper 1 cells in vitro and in vivo}, series = {European Journal of Immunology}, volume = {51}, journal = {European Journal of Immunology}, number = {3}, doi = {10.1002/eji.202048803}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-239028}, pages = {738 -- 741}, year = {2021}, abstract = {Like human Th1 cells, mouse Th1 cells also secrete IFN-γ 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-γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans.}, language = {en} } @article{SchlegelPetersDooseetal.2019, author = {Schlegel, Jan and Peters, Simon and Doose, S{\"o}ren and Schubert-Unkmeir, Alexandra and Sauer, Markus}, title = {Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites}, series = {Frontiers in Cell and Developmental Biology}, volume = {7}, journal = {Frontiers in Cell and Developmental Biology}, number = {194}, doi = {10.3389/fcell.2019.00194}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201639}, year = {2019}, 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.}, language = {en} } @article{EhmannSauerKittel2015, author = {Ehmann, Nadine and Sauer, Markus and Kittel, Robert J.}, title = {Super-resolution microscopy of the synaptic active zone}, series = {Frontiers in Cellular Neuroscience}, volume = {9}, journal = {Frontiers in Cellular Neuroscience}, number = {7}, doi = {10.3389/fncel.2015.00007}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148997}, year = {2015}, 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}, language = {en} } @article{TrinksReinhardDrobnyetal.2021, author = {Trinks, Nora and Reinhard, Sebastian and Drobny, Matthias and Heilig, Linda and L{\"o}ffler, J{\"u}rgen and Sauer, Markus and Terpitz, Ulrich}, title = {Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy}, series = {Communications Biology}, volume = {4}, journal = {Communications Biology}, number = {1}, doi = {10.1038/s42003-021-02669-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-264996}, year = {2021}, 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.}, language = {en} } @article{SchneiderKleinMielichSuessetal.2015, author = {Schneider, Johannes and Klein, Teresa and Mielich-S{\"u}ss, Benjamin and Koch, Gudrun and Franke, Christian and Kuipers, Oskar P. and Kov{\´a}cs, {\´A}kos T. and Sauer, Markus and Lopez, Daniel}, title = {Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium}, series = {PLoS Genetics}, volume = {11}, journal = {PLoS Genetics}, number = {4}, doi = {10.1371/journal.pgen.1005140}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125577}, pages = {e1005140}, year = {2015}, abstract = {Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium.}, language = {en} } @article{JanschZieglerForeroetal.2021, author = {Jansch, Charline and Ziegler, Georg C. and Forero, Andrea and Gredy, Sina and W{\"a}ldchen, Sina and Vitale, Maria Rosaria and Svirin, Evgeniy and Z{\"o}ller, Johanna E. M. and Waider, Jonas and G{\"u}nther, Katharina and Edenhofer, Frank and Sauer, Markus and Wischmeyer, Erhard and Lesch, Klaus-Peter}, title = {Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly}, series = {Journal of Neural Transmission}, volume = {128}, journal = {Journal of Neural Transmission}, number = {2}, issn = {1435-1463}, doi = {10.1007/s00702-021-02303-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268519}, pages = {225-241}, year = {2021}, 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.}, language = {en} } @article{ReinhardHelmerichBorasetal.2022, author = {Reinhard, Sebastian and Helmerich, Dominic A. and Boras, Dominik and Sauer, Markus and Kollmannsberger, Philip}, title = {ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy}, series = {BMC Bioinformatics}, volume = {23}, journal = {BMC Bioinformatics}, number = {1}, doi = {10.1186/s12859-022-05071-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-299768}, year = {2022}, abstract = {Background Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method. Results Here we introduce ReCSAI, a compressed sensing neural network to reconstruct localizations for confocal dSTORM, together with a simulation tool to generate training data. We implemented and compared different artificial network architectures, aiming to combine the advantages of compressed sensing and deep learning. We found that a U-Net with a recursive structure inspired by iterative compressed sensing showed the best results on realistic simulated datasets with noise, as well as on real experimentally measured confocal lifetime scanning data. Adding a trainable wavelet denoising layer as prior step further improved the reconstruction quality. Conclusions Our deep learning approach can reach a similar reconstruction accuracy for confocal dSTORM as frame binning with traditional fitting without requiring the acquisition of multiple frames. In addition, our work offers generic insights on the reconstruction of sparse measurements from noisy experimental data by combining compressed sensing and deep learning. We provide the trained networks, the code for network training and inference as well as the simulation tool as python code and Jupyter notebooks for easy reproducibility.}, language = {en} } @article{WeissSchlegelTerpitzetal.2020, author = {Weiss, Esther and Schlegel, Jan and Terpitz, Ulrich and Weber, Michael and Linde, J{\"o}rg and Schmitt, Anna-Lena and H{\"u}nniger, Kerstin and Marischen, Lothar and Gamon, Florian and Bauer, Joachim and L{\"o}ffler, Claudia and Kurzai, Oliver and Morton, Charles Oliver and Sauer, Markus and Einsele, Hermann and Loeffler, Juergen}, title = {Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus}, series = {Frontiers in Immunology}, volume = {11}, journal = {Frontiers in Immunology}, issn = {1664-3224}, doi = {10.3389/fimmu.2020.02117}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212581}, year = {2020}, 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\(^{bright}\)CD16\(^{dim}\) 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.}, language = {en} } @article{LandoEndesfelderBergeretal.2012, 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.}, title = {Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast}, series = {Open Biology}, volume = {2}, journal = {Open Biology}, number = {120078}, doi = {10.1098/rsob.120078}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134682}, year = {2012}, 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-A\(^{Cnp1}\) 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-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.}, language = {en} } @article{LukešGlatzovaKvičalovaetal.2017, author = {Lukeš, Tom{\´a}š and Glatzov{\´a}, Daniela and Kv{\´i}čalov{\´a}, Zuzana and Levet, Florian and Benda, Aleš and Letschert, Sebastian and Sauer, Markus and Brdička, Tom{\´a}š and Lasser, Theo and Cebecauer, Marek}, title = {Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, doi = {10.1038/s41467-017-01857-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172993}, year = {2017}, 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.}, language = {en} }