@article{ZhanStanciauskasStigloheretal.2014, author = {Zhan, Hong and Stanciauskas, Ramunas and Stigloher, Christian and Dizon, Kevin K. and Jospin, Maelle and Bessereau, Jean-Luis and Pinaud, Fabien}, title = {In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans}, series = {Nature Communications}, volume = {5}, journal = {Nature Communications}, number = {4974}, doi = {10.1038/ncomms5974}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121125}, year = {2014}, abstract = {Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca(2+) channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.}, language = {en} } @article{RozyckaWojtasJakobetal.2014, author = {Rozycka, Miroslawa and Wojtas, Magdalena and Jakob, Michal and Stigloher, Christian and Grzeszkowiak, Mikolaj and Mazur, Maciej and Ozyhar, Andrzej}, title = {Intrinsically Disordered and Pliable Starmaker-Like Protein from Medaka (Oryzias latipes) Controls the Formation of Calcium Carbonate Crystals}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {12}, issn = {1932-6203}, doi = {10.1371/journal.pone.0114308}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114251}, year = {2014}, abstract = {Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.}, language = {en} } @article{FrankMarcudeOliveiraAlmeidaPetersenetal.2015, author = {Frank, Benjamin and Marcu, Ana and de Oliveira Almeida Petersen, Antonio Luis and Weber, Heike and Stigloher, Christian and Mottram, Jeremy C. and Scholz, Claus J{\"u}rgen and Schurigt, Uta}, title = {Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210}, series = {Parasites \& Vectors}, volume = {8}, journal = {Parasites \& Vectors}, number = {404}, doi = {10.1186/s13071-015-0974-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124997}, year = {2015}, abstract = {Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.}, language = {en} } @article{MarkertBritzProppertetal.2016, 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}, title = {Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome}, series = {Neurophotonics}, volume = {3}, journal = {Neurophotonics}, number = {4}, doi = {10.1117/1.NPh.3.4.041802}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187292}, pages = {041802}, year = {2016}, 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.}, language = {en} } @article{KupperStigloherFeldhaaretal.2016, author = {Kupper, Maria and Stigloher, Christian and Feldhaar, Heike and Gross, Roy}, title = {Distribution of the obligate endosymbiont Blochmannia floridanus and expression analysis of putative immune genes in ovaries of the carpenter ant Camponotus floridanus}, series = {Arthropod Structure \& Development}, volume = {45}, journal = {Arthropod Structure \& Development}, number = {5}, doi = {10.1016/j.asd.2016.09.004}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187482}, pages = {475-487}, year = {2016}, abstract = {The bacterial endosymbiont Blochmannia floridanus of the carpenter ant Camponotus floridanus contributes to its hosts' ontogeny via nutritional upgrading during metamorphosis. This primary endosymbiosis is essential for both partners and vertical transmission of the endosymbionts is guaranteed by bacterial infestation of oocytes. Here we present a detailed analysis of the presence and localisation of B. floridanus in the ants' ovaries obtained by FISH and TEM analyses. The most apical part of the germarium harbouring germ-line stem cells (GSCs) is not infected by the bacteria. The bacteria are detectable for the first time in lower parts of the germarium when cystocytes undergo the 4th and 5th division and B. floridanus infects somatic cells lying under the basal lamina surrounding the ovarioles. With the beginning of cystocyte differentiation, the endosymbionts are exclusively transported from follicle cells into the growing oocytes. This infestation of the oocytes by bacteria very likely involves exocytosis endocytosis processes between follicle cells and the oocytes. Nurse cells were never found to harbour the endosymbionts. Furthermore we present first gene expression data in C floridanus ovaries. These data indicate a modulation of immune gene expression which may facilitate tolerance towards the endosymbionts and thus may contribute to their transovarial transmission.}, language = {en} } @article{JahnMarkertRyuetal.2016, author = {Jahn, Martin T. and Markert, Sebastian M. and Ryu, Taewoo and Ravasi, Timothy and Stigloher, Christian and Hentschel, Ute and Moitinho-Silva, Lucas}, title = {Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling}, series = {Scientific Reports}, volume = {6}, journal = {Scientific Reports}, number = {35860}, doi = {10.1038/srep35860}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167513}, year = {2016}, abstract = {Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.}, language = {en} } @article{KaltdorfSchulzeHelmprobstetal.2017, author = {Kaltdorf, Kristin Verena and Schulze, Katja and Helmprobst, Frederik and Kollmannsberger, Philip and Dandekar, Thomas and Stigloher, Christian}, title = {Fiji macro 3D ART VeSElecT: 3D automated reconstruction tool for vesicle structures of electron tomograms}, series = {PLoS Computational Biology}, volume = {13}, journal = {PLoS Computational Biology}, number = {1}, doi = {10.1371/journal.pcbi.1005317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172112}, year = {2017}, abstract = {Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. Both automatic and semi-automatic modes are explained including a tutorial.}, language = {en} } @article{HelmprobstLillesaarStigloher2017, author = {Helmprobst, Frederik and Lillesaar, Christina and Stigloher, Christian}, title = {Expression of sept3, sept5a and sept5b in the Developing and Adult Nervous System of the Zebrafish (Danio rerio)}, series = {Frontiers in Neuroanatomy}, volume = {11}, journal = {Frontiers in Neuroanatomy}, number = {6}, doi = {10.3389/fnana.2017.00006}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157625}, year = {2017}, abstract = {Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins are expressed in largely overlapping regions of the developing brain. However, the expression of sept5a is much more confined compared to sept3 and sept5b. In contrast, the expression of all the three analyzed septins is largely similar in the adult brain.}, language = {en} } @article{KaltdorfTheissMarkertetal.2018, author = {Kaltdorf, Kristin Verena and Theiss, Maria and Markert, Sebastian Matthias and Zhen, Mei and Dandekar, Thomas and Stigloher, Christian and Kollmannsberger, Philipp}, title = {Automated classification of synaptic vesicles in electron tomograms of C. elegans using machine learning}, series = {PLoS ONE}, volume = {13}, journal = {PLoS ONE}, number = {10}, doi = {10.1371/journal.pone.0205348}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176831}, pages = {e0205348}, year = {2018}, abstract = {Synaptic vesicles (SVs) are a key component of neuronal signaling and fulfil different roles depending on their composition. In electron micrograms of neurites, two types of vesicles can be distinguished by morphological criteria, the classical "clear core" vesicles (CCV) and the typically larger "dense core" vesicles (DCV), with differences in electron density due to their diverse cargos. Compared to CCVs, the precise function of DCVs is less defined. DCVs are known to store neuropeptides, which function as neuronal messengers and modulators [1]. In C. elegans, they play a role in locomotion, dauer formation, egg-laying, and mechano- and chemosensation [2]. Another type of DCVs, also referred to as granulated vesicles, are known to transport Bassoon, Piccolo and further constituents of the presynaptic density in the center of the active zone (AZ), and therefore are important for synaptogenesis [3]. To better understand the role of different types of SVs, we present here a new automated approach to classify vesicles. We combine machine learning with an extension of our previously developed vesicle segmentation workflow, the ImageJ macro 3D ART VeSElecT. With that we reliably distinguish CCVs and DCVs in electron tomograms of C. elegans NMJs using image-based features. Analysis of the underlying ground truth data shows an increased fraction of DCVs as well as a higher mean distance between DCVs and AZs in dauer larvae compared to young adult hermaphrodites. Our machine learning based tools are adaptable and can be applied to study properties of different synaptic vesicle pools in electron tomograms of diverse model organisms.}, language = {en} } @article{JarickBertscheStahletal.2018, author = {Jarick, Marcel and Bertsche, Ute and Stahl, Mark and Schultz, Daniel and Methling, Karen and Lalk, Michael and Stigloher, Christian and Steger, Mirco and Schlosser, Andreas and Ohlsen, Knut}, title = {The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, number = {13693}, doi = {10.1038/s41598-018-32109-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177333}, year = {2018}, abstract = {The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.}, language = {en} }