TY - THES A1 - Sauer, Markus T1 - DNA-Bindungseigenschaften von Mitgliedern der p53 Familie T1 - DNA binding properties of members of the p53 family N2 - Ein sehr wichtiger Tumorsuppressor ist der Transkriptionsfaktor p53, der Zellschicksals-Entscheidungen wie Zellzyklus-Arrest und programmierten Zelltod (Apoptose) kontrolliert. Die Wirkung von p53 und von seinen Familienmitgliedern p63 und p73 beruht überwiegend auf der Fähigkeit, als Transkriptionsfaktoren die Genexpression zu regulieren. Die DNA-Bindung an Promotoren von Zielgenen ist dabei von grundlegender Bedeutung und wird durch die hoch konservierte zentrale DNA-Bindungs-Domäne und den Carboxy-Terminus bestimmt. In dieser Arbeit wurden die DNA-Bindungseigenschaften von p53 und verschiedener Carboxy-terminalen p73 Isoformen untersucht. In „electrophoretic mobility shift assay” (EMSA) Experimenten bildeten p53 und p73gamma nur schwache Sequenz-spezifische DNA-Komplexe, wohingegen p73alpha, beta und delta die DNA deutlich stärker banden. Die schwache DNA-Bindung von p53 und p73gamma kann durch mehrfach positiv geladene Carboxy-Termini erklärt werden, die über eine Sequenz-unabhängige DNA-Bindung ein Gleiten entlang der DNA ermöglichen. Die Deletion der Carboxy-terminalen Domäne (CTD) von p53 („p53delta30“) verstärkte dementsprechend die Sequenz-spezifische DNA-Bindung in vitro und seine Übertragung auf p73alpha („p73alpha+30“) schwächte sie ab. Mittels „fluorescence recovery after photobleaching“ (FRAP) Experimenten konnte in lebenden Zellen eine Verminderung der intra-nukleären Mobilität von p53 und p73alpha+30 durch die CTD gezeigt werden, die aus der Sequenz-unabhängigen DNA-Bindung resultiert. Zusätzlich reduzierte die CTD die Sequenz-spezifische DNA-Bindung von p53 an den p21 (CDKN1A) Promotor. Das Spektrum der regulierten Zielgene wurde in einer Genom-weiten Genexpressions-Analyse nicht durch die CTD verändert, sondern maßgeblich durch das Protein-Rückgrat von p53 beziehungsweise p73 bestimmt. Allerdings verminderte die CTD das Ausmaß der Transkriptions-Regulation und hemmte die Induktion von Zellzyklus-Arrest und Apoptose. Die mehrfach positiv geladene CTD in p53 besitzt demzufolge eine negativ regulatorische Wirkung, die in den wichtigsten p73 Isoformen alpha, beta und delta fehlt. Die zentrale DNA-Bindungs-Domäne trägt durch elektrostatische Wechselwirkungen zwischen H1-Helices (Aminosäurereste 177 bis 182) unterschiedlicher p53 Monomere zu kooperativer DNA-Bindung und zu Zellschicksals-Entscheidungen bei. Anhand von Mutanten, die unterschiedlich starke H1-Helix-Interaktionen ermöglichen, konnte gezeigt werden, dass starke Interaktionen die Bindung an Promotoren von pro-apoptotischen Genen verstärkte, wohingegen die Bindung an anti-apoptotische und Zellzyklus-blockierende Gene unabhängig von der Interaktions-Stärke war. Diese Unterschiede in der Promotor-Bindung ließen sich nicht auf eine veränderte zelluläre Lokalisation der Mutanten zurückführen, da alle Mutanten überwiegend nukleär lokalisiert waren. Eine an Serin 183 Phosphorylierungs-defekte Mutante von p53 bildete stabile DNA-Komplexe, entsprechend einer Mutante mit starker H1-Helix-Interaktion, und trans-aktivierte pro-apoptotische Promotoren stärker als Mutanten, die Phosphorylierung von p53 an Serin 183 simulieren. Da zusätzlich bekannt ist, dass Serin 183 mit der H1-Helix wechselwirkt, könnte diese Phosphorylierung einen physiologischen Mechanismus zur Regulation der H1-Helix-Interaktion und damit des Zellschicksals darstellen. Zusammenfassend ließ sich zeigen, dass sowohl die Interaktions-Stärke zweier DNA-Bindungs-Domänen als auch die elektrische Ladung des Carboxy-Terminus die DNA-Bindungseigenschaften von p53 Familienmitgliedern bestimmen und so Zellschicksals-Entscheidungen der p53 Familie beeinflussen. N2 - A very important tumour suppressor is the transcription factor p53 that controls cell fate decisions like cell cycle arrest and programmed cell death (apoptosis). The effects of p53 and its family members p63 and p73 are mainly based on their transcription factor activities to regulate gene expression. The DNA binding to promoters of target genes is of fundamental importance for their functionality and is determined by the highly conserved core DNA binding domain and the carboxy-terminus. In this thesis the DNA binding properties of p53 and different carboxy-terminal p73 isoforms were examined. In electrophoretic mobility shift assays (EMSA) p53 and p73gamma formed only weak sequence-specific protein-DNA-complexes while p73alpha, beta and delta bound considerably stronger to DNA. A highly positively charged carboxy-terminus can explain the weak DNA binding of p53 and p73gamma by enabling sequence-nonspecific DNA binding leading to sliding on DNA. According to this the deletion of the carboxy-terminal domain (CTD) of p53 („p53delta30“) reinforced DNA binding in vitro, and its fusion to p73alpha („p73alpha+30“) attenuated it. In living cells the CTD reduced intranuclear mobility of p53 and p73alpha+30 in fluorescence recovery after photobleaching (FRAP) experiments by mediating sequence-nonspecific binding to DNA. In addition, the CTD reduced sequence-specific occupancy of the p21 (CDKN1A) promoter by p53 in vivo. In an unbiased genome-wide gene expression analysis the spectrum of target genes was not changed by the presence of the CTD, but mainly determined by the p53 and p73 protein backbone, respectively. However, the CTD diminished the level of target gene activation and inhibited the induction of cell cycle arrest and apoptosis. As a result, the highly positively charged carboxy-terminus of p53 exhibits a negative regulatory effect that is missing in the most important p73 isoforms alpha, beta and delta. The core DNA binding domain adds to cooperative DNA binding and cell fate decisions by electrostatic interactions between H1 helices (residues 177 to 182) of different p53 monomers. Strong H1 helix interactions increased binding to promoters of pro-apoptotic genes, whereas binding to anti-apoptotic and proliferation inhibiting genes was independent of the interaction strength as shown by mutants with different strengths of the H1 helix interactions. These differences in promoter binding were not caused by different cellular localizations of the mutants as they were all predominantly localized to the nucleus. A serine 183 phosphorylation-defective mutant of p53 formed stable protein-DNA-complexes, comparable to a mutant with strong H1 helix interactions, and trans-activated pro-apoptotic promoters stronger than mutants that mimicked p53 phosphorylated on serine 183. Due to the fact that serine 183 interacts with the H1 helix, these data suggest that phosphorylation of serine 183 is a physiological mechanism to regulate H1 helix interactions and thereby cell fate decisions. In summary, it was shown that both the interaction strength of two DNA binding domains and the electrostatic charge of the CTD define the DNA binding properties of p53 family members and thereby influence cell fate decisions of the p53 family. KW - Protein p53 KW - DNS-Bindung KW - Protein p73 KW - Transkriptionsfaktor KW - Apoptosis KW - FRAP KW - Gleiten KW - Carboxy-Terminus KW - H1-Helix KW - FRAP KW - sliding KW - carboxy terminus KW - H1 helix Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-35083 ER - TY - THES A1 - Sauer, Markus T1 - Mixed-Reality for Enhanced Robot Teleoperation T1 - Mixed-Reality zur verbesserten Fernbedienung von Robotern N2 - In den letzten Jahren ist die Forschung in der Robotik soweit fortgeschritten, dass die Mensch-Maschine Schnittstelle zunehmend die kritischste Komponente für eine hohe Gesamtperformanz von Systemen zur Navigation und Koordination von Robotern wird. In dieser Dissertation wird untersucht wie Mixed-Reality Technologien für Nutzerschnittstellen genutzt werden können, um diese Gesamtperformanz zu erhöhen. Hierzu werden Konzepte und Technologien entwickelt, die durch Evaluierung mit Nutzertest ein optimiertes und anwenderbezogenes Design von Mixed-Reality Nutzerschnittstellen ermöglichen. Er werden somit sowohl die technische Anforderungen als auch die menschlichen Faktoren für ein konsistentes Systemdesign berücksichtigt. Nach einer detaillierten Problemanalyse und der Erstellung eines Systemmodels, das den Menschen als Schlüsselkomponente mit einbezieht, wird zunächst die Anwendung der neuartigen 3D-Time-of-Flight Kamera zur Navigation von Robotern, aber auch für den Einsatz in Mixed-Reality Schnittstellen analysiert und optimiert. Weiterhin wird gezeigt, wie sich der Netzwerkverkehr des Videostroms als wichtigstes Informationselement der meisten Nutzerschnittstellen für die Navigationsaufgabe auf der Netzwerk Applikationsebene in typischen Multi-Roboter Netzwerken mit dynamischen Topologien und Lastsituation optimieren lässt. Hierdurch ist es möglich in sonst in sonst typischen Ausfallszenarien den Videostrom zu erhalten und die Bildrate zu stabilisieren. Diese fortgeschrittenen Technologien werden dann auch dem entwickelten Konzept der generischen 3D Mixed Reality Schnittselle eingesetzt. Dieses Konzept ermöglicht eine integrierte 3D Darstellung der verfügbaren Information, so dass räumliche Beziehungen von Informationen aufrechterhalten werden und somit die Anzahl der mentalen Transformationen beim menschlichen Bediener reduziert wird. Gleichzeitig werden durch diesen Ansatz auch immersive Stereo Anzeigetechnologien unterstützt, welche zusätzlich das räumliche Verständnis der entfernten Situation fördern. Die in der Dissertation vorgestellten und evaluierten Ansätze nutzen auch die Tatsache, dass sich eine lokale Autonomie von Robotern heute sehr robust realisieren lässt. Dies wird zum Beispiel zur Realisierung eines Assistenzsystems mit variabler Autonomie eingesetzt. Hierbei erhält der Fernbediener über eine Kraftrückkopplung kombiniert mit einer integrierten Augmented Reality Schnittstelle, einen Eindruck über die Situation am entfernten Arbeitsbereich, aber auch über die aktuelle Navigationsintention des Roboters. Die durchgeführten Nutzertests belegen die signifikante Steigerung der Navigationsperformanz durch den entwickelten Ansatz. Die robuste lokale Autonomie ermöglicht auch den in der Dissertation eingeführten Ansatz der prädiktiven Mixed-Reality Schnittstelle. Die durch diesen Ansatz entkoppelte Regelschleife über den Menschen ermöglicht es die Sichtbarkeit von unvermeidbaren Systemverzögerungen signifikant zu reduzieren. Zusätzlich können durch diesen Ansatz beide für die Navigation hilfreichen Blickwinkel in einer 3D-Nutzerschnittstelle kombiniert werden – der exozentrische Blickwinkel und der egozentrische Blickwinkel als Augmented Reality Sicht. N2 - With the progress in robotics research the human machine interfaces reach more and more the status of being the major limiting factor for the overall system performance of a system for remote navigation and coordination of robots. In this monograph it is elaborated how mixed reality technologies can be applied for the user interfaces in order to increase the overall system performance. Concepts, technologies, and frameworks are developed and evaluated in user studies which enable for novel user-centered approaches to the design of mixed-reality user interfaces for remote robot operation. Both the technological requirements and the human factors are considered to achieve a consistent system design. Novel technologies like 3D time-of-flight cameras are investigated for the application in the navigation tasks and for the application in the developed concept of a generic mixed reality user interface. In addition it is shown how the network traffic of a video stream can be shaped on application layer in order to reach a stable frame rate in dynamic networks. The elaborated generic mixed reality framework enables an integrated 3D graphical user interface. The realized spatial integration and visualization of available information reduces the demand for mental transformations for the human operator and supports the use of immersive stereo devices. The developed concepts make also use of the fact that local robust autonomy components can be realized and thus can be incorporated as assistance systems for the human operators. A sliding autonomy concept is introduced combining force and visual augmented reality feedback. The force feedback component allows rendering the robot's current navigation intention to the human operator, such that a real sliding autonomy with seamless transitions is achieved. The user-studies prove the significant increase in navigation performance by application of this concept. The generic mixed reality user interface together with robust local autonomy enables a further extension of the teleoperation system to a short-term predictive mixed reality user interface. With the presented concept of operation, it is possible to significantly reduce the visibility of system delays for the human operator. In addition, both advantageous characteristics of a 3D graphical user interface for robot teleoperation- an exocentric view and an augmented reality view – can be combined. T3 - Forschungsberichte in der Robotik = Research Notes in Robotics - 5 KW - Mobiler Roboter KW - Autonomer Roboter KW - Mensch-Maschine-Schnittstelle KW - Mixed Reality KW - Mensch-Roboter-Interaktion KW - Teleoperation KW - Benutzerschnittstelle KW - Robotik KW - Mensch-Maschine-System KW - Human-Robot-Interaction KW - Teleoperation KW - User Interface Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-55083 SN - 978-3-923959-67-9 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 - TY - JOUR A1 - Wolter, Steve A1 - Endesfelder, Ulrike A1 - Linde, Sebastian van de A1 - Heilemann, Mike A1 - Sauer, Markus T1 - Measuring localization performance of super-resolution algorithms on very active samples JF - Optics Express N2 - Super-resolution fluorescence imaging based on inglemolecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as threedimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer. Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85936 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 - Lando, David A1 - Endesfelder, Ulrike A1 - Berger, Harald A1 - Subramanian, Lakxmi A1 - Dunne, Paul D. A1 - McColl, James A1 - Klenerman, David A1 - Carr, Antony M. A1 - Sauer, Markus A1 - Allshire, Robin C. A1 - Heilemann, Mike A1 - Laue, Ernest D. T1 - Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast JF - Open Biology N2 - 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. KW - nucleosome KW - fission yeast KW - identification KW - propagation KW - CSE4, CENP-A KW - CENP-A KW - schizosaccaromyces-pombe KW - fluorescent protein KW - centomeres KW - superresolution KW - chromatin KW - centromere KW - ingle-molecule microscopy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134682 VL - 2 IS - 120078 ER - TY - JOUR A1 - Wolf, Annette A1 - Akrap, Nina A1 - Marg, Berenice A1 - Galliardt, Helena A1 - Heiligentag, Martyna A1 - Humpert, Fabian A1 - Sauer, Markus A1 - Kaltschmidt, Barbara A1 - Kaltschmidt, Christian A1 - Seidel, Thorsten T1 - Elements of Transcriptional Machinery Are Compatible among Plants and Mammals JF - PLoS ONE N2 - In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-\(\kappa\) B in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-\(\kappa\) B such as its inhibitor I\(\kappa\)B isoform \(\alpha\) that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-\(\kappa\)B in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-\(\kappa\)B signaling pathways. The system successfully enabled the controlled manipulation of NF-\(\kappa\)B activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e. g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway. KW - complexes KW - in vivo KW - DNA-binding KW - nuclear proe KW - gene expression KW - NF-KAPPA-B KW - RNA-binding protein KW - alpha KW - inflammation KW - homodimers Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131203 VL - 8 IS - 1 ER - TY - JOUR A1 - Klamp, Tobias A1 - Camps, Marta A1 - Nieto, Benjamin A1 - Guasch, Francesc A1 - Ranasinghe, Rohan T. A1 - Wiedemann, Jens A1 - Petrášek, Zdeněk A1 - Schwille, Petra A1 - Klenerman, David A1 - Sauer, Markus T1 - Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay JF - Scientific Reports N2 - There is an urgent need for rapid and highly sensitive detection of pathogen-derivedDNAin a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp targetDNA fragments of Micrococcus luteus in small sample volumes of 20 mL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of,5 pMand a linear detection range spanning three orders of magnitude. KW - laboratory techniques and procedures KW - diseases KW - infectious diseases KW - assay systems Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130500 VL - 3 IS - 1852 ER - TY - BOOK A1 - Bock, Stefanie A1 - Gauch, Fabian A1 - Giernat, Yannik A1 - Hillebrand, Frank A1 - Kozlova, Darja A1 - Linck, Lisa A1 - Moschall, Rebecca A1 - Sauer, Markus A1 - Schenk, Christian A1 - Ulrich, Kristina A1 - Bodem, Jochen T1 - HIV-1 : Lehrbuch von Studenten für Studenten T1 - HIV-1 : a textbook for students written by students N2 - Dies ist ein Lehrbuch über die HIV-1 Replikation, Pathogenese und Therapie. Es richtet sich an Studenten der Biologie und der Medizin, die etwas mehr über HIV erfahren wollen und stellt neben virologischen Themen auch die zellulären Grundlagen dar. Es umfasst den Viruseintritt, die reverse Transkription, Genom-Integration, Transkriptionsregualtion, die Kotrolle des Spleißens, der Polyadenylierung und des RNA-Exportes. Die Darstellung wird abgerundet mit Kapiteln zum intrazellulärem Transport, zu Nef und zum Virusassembly. In zwei weiteren Kapitel wird die HIV-1 Pathogenese und die Therapie besprochen. Zur Lernkontrolle sind den Kapiteln Fragen und auch Klausurfragen angefügt. KW - HIV KW - Retroviren KW - Lehrbuch KW - Viren KW - Virologie KW - Transkription KW - RNS KW - Therapie KW - Pathogenese KW - Epidemiologie KW - RNA-Export KW - Polyadenylierung KW - Reverse Transkription KW - Transkription Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78980 SN - 978-3-923959-90-7 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 -