TY - JOUR A1 - Deng, Chunchu A1 - Reinhard, Sebastian A1 - Hennlein, Luisa A1 - Eilts, Janna A1 - Sachs, Stefan A1 - Doose, Sören A1 - Jablonka, Sibylle A1 - Sauer, Markus A1 - Moradi, Mehri A1 - Sendtner, Michael T1 - Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus-response in spinal muscular atrophy JF - Translational Neurodegeneration N2 - Background: Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA. Methods: Using super-resolution microscopy, proximity ligation assay (PLA) and live imaging of cultured motoneurons from a mouse model of SMA, we investigated the dynamics of the axonal ER and ribosome distribution and activation. Results: We observed that the dynamic remodeling of ER was impaired in axon terminals of Smn-deficient motoneurons. In addition, in axon terminals of Smn-deficient motoneurons, ribosomes failed to respond to the brain-derived neurotrophic factor stimulation, and did not undergo rapid association with the axonal ER in response to extracellular stimuli. Conclusions: These findings implicate impaired dynamic interplay between the ribosomes and ER in axon terminals of motoneurons as a contributor to the pathophysiology of SMA and possibly also other motoneuron diseases. KW - spinal muscular atrophy KW - BDNF stimulation KW - dynamics of ribosomal assembly KW - presynaptic ER dynamics Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300649 SN - 2047-9158 VL - 11 IS - 1 ER - TY - JOUR A1 - Schlegel, Jan A1 - Sauer, Markus T1 - Hochaufgelöste Visualisierung einzelner Moleküle auf ganzen Zellen JF - BIOspektrum N2 - Biological systems are dynamic and three-dimensional but many techniques allow only static and two-dimensional observation of cells. We used three-dimensional (3D) lattice light-sheet single-molecule localization microscopy (dSTORM) to investigate the complex interactions and distribution of single molecules in the plasma membrane of whole cells. Different receptor densities of the adhesion receptor CD56 at different parts of the cell highlight the importance and need of three-dimensional observation and analysis techniques. KW - Visualisierung KW - Moleküle KW - Zellen Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232365 SN - 0947-0867 VL - 7 ER - TY - JOUR A1 - Reinhard, Sebastian A1 - Helmerich, Dominic A. A1 - Boras, Dominik A1 - Sauer, Markus A1 - Kollmannsberger, Philip T1 - ReCSAI: recursive compressed sensing artificial intelligence for confocal lifetime localization microscopy JF - BMC Bioinformatics N2 - 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. KW - compressed sensing KW - AI KW - SMLM KW - FLIMbee KW - dSTORM Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299768 VL - 23 IS - 1 ER - TY - JOUR A1 - Wäldchen, Sina A1 - Lehmann, Julian A1 - Klein, Teresa A1 - van de Linde, Sebastian A1 - Sauer, Markus T1 - Light-induced cell damage in live-cell super-resolution microscopy JF - Scientific Reports N2 - Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. KW - optical reconstruction microscopy KW - tag fusion proteins KW - localization microscopy KW - photodynamic therapy KW - diffraction limit KW - illumination microscopy KW - structured illumination KW - fluorescent probes KW - in vitro KW - dynamics Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145207 VL - 5 IS - 15348 ER - TY - JOUR A1 - Dannhäuser, Sven A1 - Mrestani, Achmed A1 - Gundelach, Florian A1 - Pauli, Martin A1 - Komma, Fabian A1 - Kollmannsberger, Philip A1 - Sauer, Markus A1 - Heckmann, Manfred A1 - Paul, Mila M. T1 - Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation JF - Frontiers in Cellular Neuroscience N2 - Introduction Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. Methods We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Results and discussion Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13\(^{GFSTF}\) 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13\(^{GFSTF}\), Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13\(^{GFSTF}\) subclusters that move toward the AZ center during PHP with unaltered Unc-13\(^{GFSTF}\) protein levels. KW - active zone KW - Unc-13 KW - MiMIC KW - presynaptic homeostasis KW - nanoarchitecture KW - localization microscopy KW - STORM KW - HDBSCAN Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299440 SN - 1662-5102 VL - 16 ER - TY - JOUR A1 - Ehmann, Nadine A1 - Sauer, Markus A1 - Kittel, Robert J. T1 - Super-resolution microscopy of the synaptic active zone JF - Frontiers in Cellular Neuroscience N2 - 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 KW - excitation-secretion coupling KW - Ca\(^{2+}\) channels KW - structure-function relationships KW - super-resolution microscopy KW - active zone KW - presynaptic calcium KW - neurotransmitter release Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148997 VL - 9 IS - 7 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - 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. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 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 - Berberich, Andreas A1 - Kurz, Andreas A1 - Reinhard, Sebastian A1 - Paul, Torsten Johann A1 - Burd, Paul Ray A1 - Sauer, Markus A1 - Kollmannsberger, Philip T1 - Fourier Ring Correlation and anisotropic kernel density estimation improve deep learning based SMLM reconstruction of microtubules JF - Frontiers in Bioinformatics N2 - Single-molecule super-resolution microscopy (SMLM) techniques like dSTORM can reveal biological structures down to the nanometer scale. The achievable resolution is not only defined by the localization precision of individual fluorescent molecules, but also by their density, which becomes a limiting factor e.g., in expansion microscopy. Artificial deep neural networks can learn to reconstruct dense super-resolved structures such as microtubules from a sparse, noisy set of data points. This approach requires a robust method to assess the quality of a predicted density image and to quantitatively compare it to a ground truth image. Such a quality measure needs to be differentiable to be applied as loss function in deep learning. We developed a new trainable quality measure based on Fourier Ring Correlation (FRC) and used it to train deep neural networks to map a small number of sampling points to an underlying density. Smooth ground truth images of microtubules were generated from localization coordinates using an anisotropic Gaussian kernel density estimator. We show that the FRC criterion ideally complements the existing state-of-the-art multiscale structural similarity index, since both are interpretable and there is no trade-off between them during optimization. The TensorFlow implementation of our FRC metric can easily be integrated into existing deep learning workflows. KW - dSTORM KW - deep learning–artificial neural network (DL-ANN) KW - single molecule localization microscopy KW - microtubule cytoskeleton KW - super-resolution Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261686 VL - 1 ER - TY - JOUR A1 - Dugar, Gaurav A1 - Svensson, Sarah L. A1 - Bischler, Thorsten A1 - Waldchen, Sina A1 - Reinhardt, Richard A1 - Sauer, Markus A1 - Sharma, Cynthia M. T1 - The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni JF - Nature Communications N2 - 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. KW - bacterial genetics KW - cell signalling KW - translation KW - Campylobacter jejuni Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173201 VL - 7 ER -