TY - JOUR A1 - Paul, Mila M. A1 - Pauli, Martin A1 - Ehmann, Nadine A1 - Hallermann, Stefan A1 - Sauer, Markus A1 - Kittel, Robert J. A1 - Heckmann, Manfred T1 - Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation JF - Frontiers in Cellular Neuroscience N2 - The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp\(^{69}\)). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp\(^{69}\) AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (syt\(^{KD}\)) in wildtype (wt), brp\(^{69}\) and rab3 null mutants (rab3\(^{rup}\)), where Brp is concentrated at a small number of AZs. At wt and rab3\(^{rup}\) synapses, syt\(^{KD}\) lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, syt\(^{KD}\) did not alter EPSC amplitude at brp\(^{69}\) synapses, but shortened delay and rise time. In fact, following syt\(^{KD}\), these kinetic properties were strikingly similar in wt and brp\(^{69}\), which supports the notion that Syt protracts release at brp\(^{69}\) synapses. To gain insight into this surprising role of Syt at brp\(^{69}\) AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At tonic type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after syt\(^{KD}\). Notably, syt\(^{KD}\) boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that syt\(^{KD}\) increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt. KW - neuromuscular junction KW - Bruchpilot KW - synaptic delay KW - dSTORM KW - synaptotagmin KW - presynaptic differentiation KW - neurotransmitter release KW - active zone KW - synaptic transmission KW - fluorescent probes Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148988 VL - 9 IS - 29 ER - TY - THES A1 - Ehmann, Nadine T1 - Linking the active zone ultrastructure to function in Drosophila T1 - Struktur-Funktions-Beziehungen an der aktiven Zone in Drosophila N2 - Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication. Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains. This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron. N2 - Kommunikation zwischen Nervenzellen ist von grundlegender Bedeutung für die Hirnfunktion. An chemischen Synapsen findet diese an hoch spezialisierten interzellulären Kontaktstellen statt, den aktiven Zonen, welche die Voraussetzung für präzise Neurotransmission schaffen und somit die synaptische Kommunikation gewährleisten. In aktiven Zonen befindet sich eine Vielzahl von Proteinen dicht gepackt, die Geschwindigkeit, Genauigkeit und Plastizität der Signaltransduktion vermitteln. Bisher ist es jedoch unklar, in welcher Weise die molekularen Organisationsprinzipien dieser Proteine die Funktion der aktiven Zone beeinflussen. Teilweise ist dies dem Auflösungsvermögen konventioneller Lichtmikroskopie geschuldet, das nicht ausreicht um die Architektur der aktiven Zone im Nanometer Bereich aufzuklären. Unlängst jedoch haben neue Methoden der hochaufgelösten Fluoreszenzmikroskopie ihren Weg in die Neurowissenschaften gefunden. Diese sind in der Lage die Lücke zwischen optischer Lichtmikroskopie und Elektronenmikroskopie zu schließen, ohne die Identität der Proteinspezies aus den Augen zu verlieren. Besonderes Interesse kommt hierbei sogenannten Lokalisationsmikroskopie Techniken zu. Diese können neben der Darstellung molekularer Organisationen im Idealfall sogar quantitative Informationen über die absolute Anzahl bestimmter Moleküle in subzellulären Bereichen liefern. In der vorliegenden Arbeit wurde eine Methode entwickelt, die auf klassischer Immunohistochemie beruht und dSTORM (direct stochastic optical reconstruction microscopy) nutzt, um die endogene Proteinorganisation in situ zu quantifizieren. Fokussierend auf Brp (Bruchpilot), einem Protein an der aktiven Zone von Drosophila melanogaster, zeigen die Ergebnisse, dass die Zytomatrix an der aktiven Zone modular aufgebaut ist, wobei jedes Modul ~137 Brp Moleküle umfasst. Diese sind zum Großteil in etwa 15 Gruppen mit je 7 Untereinheiten angeordnet. Um auf einen quantitativen Zusammenhang zwischen der Ultrastruktur der aktiven Zone und ihrer Funktion zu schließen, wurden Drosophila Mutanten eingesetzt und mittels Elektrophysiologie funktionell untersucht. Die Ergebnisse veranschaulichen, dass sich spezifische Eigenschaften von Kurzzeitplastizität in der präzisen Anordnung von Brp widerspiegeln, was Rückschlüsse auf verschiedene Ursprünge synaptischer Depression zulässt. Darüber hinaus beschrieben dSTORM Experimente erstmals, dass ein funktioneller Gradient entlang des Motoneurons mit der graduellen Veränderung der Anzahl von Bruchpilotmolekülen pro aktive Zone korreliert. KW - Taufliege KW - Elektrophysiologie KW - Fluoreszenzmikroskopie KW - Synapse KW - Drosophila KW - active zone KW - structure-function relationships KW - super-resolution microscopy KW - electrophysiology KW - Synapses KW - Microscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118186 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 -