@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{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{GrobTritscherGruebeletal.2021, author = {Grob, Robin and Tritscher, Clara and Gr{\"u}bel, Kornelia and Stigloher, Christian and Groh, Claudia and Fleischmann, Pauline N. and R{\"o}ssler, Wolfgang}, title = {Johnston's organ and its central projections in Cataglyphis desert ants}, series = {Journal of Comparative Neurology}, volume = {529}, journal = {Journal of Comparative Neurology}, number = {8}, doi = {10.1002/cne.25077}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-225679}, pages = {2138 -- 2155}, year = {2021}, abstract = {The Johnston's organ (JO) in the insect antenna is a multisensory organ involved in several navigational tasks including wind-compass orientation, flight control, graviception, and, possibly, magnetoreception. Here we investigate the three dimensional anatomy of the JO and its neuronal projections into the brain of the desert ant Cataglyphis, a marvelous long-distance navigator. The JO of C. nodus workers consists of 40 scolopidia comprising three sensory neurons each. The numbers of scolopidia slightly vary between different sexes (female/male) and castes (worker/queen). Individual scolopidia attach to the intersegmental membrane between pedicel and flagellum of the antenna and line up in a ring-like organization. Three JO nerves project along the two antennal nerve branches into the brain. Anterograde double staining of the antennal afferents revealed that JO receptor neurons project to several distinct neuropils in the central brain. The T5 tract projects into the antennal mechanosensory and motor center (AMMC), while the T6 tract bypasses the AMMC via the saddle and forms collaterals terminating in the posterior slope (PS) (T6I), the ventral complex (T6II), and the ventrolateral protocerebrum (T6III). Double labeling of JO and ocellar afferents revealed that input from the JO and visual information from the ocelli converge in tight apposition in the PS. The general JO anatomy and its central projection patterns resemble situations in honeybees and Drosophila. The multisensory nature of the JO together with its projections to multisensory neuropils in the ant brain likely serves synchronization and calibration of different sensory modalities during the ontogeny of navigation in Cataglyphis.}, language = {en} } @article{MarkertSkoruppaYuetal.2020, author = {Markert, Sebastian M. and Skoruppa, Michael and Yu, Bin and Mulcahy, Ben and Zhen, Mai and Gao, Shangbang and Sendtner, Michael and Stigloher, Christian}, title = {Overexpression of an ALS-associated FUS mutation in C. elegans disrupts NMJ morphology and leads to defective neuromuscular transmission}, series = {Biology Open}, volume = {9}, journal = {Biology Open}, doi = {10.1242/bio.055129}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230662}, year = {2020}, abstract = {The amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.}, language = {en} } @article{KramerMeyerNatusStigloheretal.2021, author = {Kramer, Susanne and Meyer-Natus, Elisabeth and Stigloher, Christian and Thoma, Hanna and Schnaufer, Achim and Engstler, Markus}, title = {Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {3}, doi = {10.1093/nar/gkaa1142}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230647}, year = {2021}, abstract = {Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.}, 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{BazihizinaBoehmMessereretal.2022, author = {Bazihizina, Nadia and B{\"o}hm, Jennifer and Messerer, Maxim and Stigloher, Christian and M{\"u}ller, Heike M. and Cuin, Tracey Ann and Maierhofer, Tobias and Cabot, Joan and Mayer, Klaus F. X. and Fella, Christian and Huang, Shouguang and Al-Rasheid, Khaled A. S. and Alquraishi, Saleh and Breadmore, Michael and Mancuso, Stefano and Shabala, Sergey and Ache, Peter and Zhang, Heng and Zhu, Jian-Kang and Hedrich, Rainer and Scherzer, S{\"o}nke}, title = {Stalk cell polar ion transport provide for bladder-based salinity tolerance in Chenopodium quinoa}, series = {New Phytologist}, volume = {235}, journal = {New Phytologist}, number = {5}, doi = {10.1111/nph.18205}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-287222}, pages = {1822 -- 1835}, year = {2022}, abstract = {Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder. Under salt stress, sodium (Na\(^{+}\)), chloride (Cl\(^{-}\)), potassium (K\(^{+}\)) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller. In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder-directed solute flow. RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.}, language = {en} } @article{BritzMarkertWitvlietetal.2021, author = {Britz, Sebastian and Markert, Sebastian Matthias and Witvliet, Daniel and Steyer, Anna Maria and Tr{\"o}ger, Sarah and Mulcahy, Ben and Kollmannsberger, Philip and Schwab, Yannick and Zhen, Mei and Stigloher, Christian}, title = {Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy}, series = {Frontiers in Neuroanatomy}, volume = {15}, journal = {Frontiers in Neuroanatomy}, issn = {1662-5129}, doi = {10.3389/fnana.2021.732520}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249622}, year = {2021}, abstract = {At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.}, language = {en} } @article{SputhPanzerStigloheretal.2021, author = {Sputh, Sebastian and Panzer, Sabine and Stigloher, Christian and Terpitz, Ulrich}, title = {Superaufgel{\"o}ste Mikroskopie: Pilze unter Beobachtung}, series = {BIOspektrum}, volume = {27}, journal = {BIOspektrum}, number = {4}, issn = {1868-6249}, doi = {10.1007/s12268-021-1592-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270014}, pages = {380-382}, year = {2021}, abstract = {The diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).}, language = {de} }