TY - JOUR A1 - Andronic, Joseph A1 - Shirakashi, Ryo A1 - Pickel, Simone U. A1 - Westerling, Katherine M. A1 - Klein, Teresa A1 - Holm, Thorge A1 - Sauer, Markus A1 - Sukhorukov, Vladimir L. T1 - Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy JF - PLoS One N2 - Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells. KW - electrolytes KW - isotonic KW - membrane proteins KW - cell membranes KW - hypotonic KW - hypotonic solutions KW - tonicity KW - permeability Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126408 VL - 10 IS - 3 ER - TY - JOUR A1 - Owen, Dylan M. A1 - Sauer, Markus A1 - Gaus, Katharina T1 - Fluorescence localization microscopy JF - Communicative & Integrative Biology N2 - Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy. Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124416 VL - 5 IS - 4 ER - TY - JOUR A1 - Andreska, Thomas A1 - Aufmkolk, Sarah A1 - Sauer, Markus A1 - Blum, Robert T1 - High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons JF - Frontiers in Cellular Neuroscience N2 - In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity. KW - hippocampal neurons KW - synapse structure KW - presynapse KW - synaptic localization KW - BDNF Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119793 SN - 1662-5102 VL - 8 IS - 107 ER - TY - JOUR A1 - Proppert, Sven A1 - Wolter, Steve A1 - Holm, Thorge A1 - Klein, Theresa A1 - van de Linde, Sebastian A1 - Sauer, Markus T1 - Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging JF - Optics Express N2 - In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity. KW - three-dimensional microscopy KW - fluorescence microscopy KW - medical and biological imaging KW - superresolution Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119730 SN - 1094-4087 VL - 22 IS - 9 ER - TY - JOUR A1 - Vogel, Benjamin A1 - Löschberger, Anna A1 - Sauer, Markus A1 - Hock, Robert T1 - Cross-linking of DNA through HMGA1 suggests a DNA scaffold N2 - Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins. KW - DNA Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68865 ER -