Studie des Vesikelzyklus während der Exo-und Endozytose mit optischen Methoden

Im zentralen Nervensystem tritt die Kommunikation zwischen Nervenzellen mittels chemischer Botenstoffe (Neurotransmitter) hauptsächlich an den Synapsen auf. Bei Stimulierung werden die Botenstoffe durch Ca2+-abhängige Exozytose präsynaptischer Vesikel in den synaptischen Spalt freigesetzt, wo sie postsynaptische Rezeptoren aktivieren. Um den Pool an Vesikeln nach der Freisetzung von Botenstoffen wieder aufzufüllen, werden Vesikelkomponenten nach der Exozytose wieder sortiert und durch kompensatorische Endozytose in die Zelle aufgenommen. In dieser Studie werden sowohl exogene cypHer-basierte und genetisch kodierte GFP-basierte pH-Reporter verwendet, um den Vesikelzyklus in kultivierten hippocampalen Neuronen der Ratte zu untersuchen. Durch die Verwendung cyHher-konjugierter Antikörper gegen die luminalen Domänen von Vesikelproteinen (z.B. Syt1) wird der Zyklus endogener Vesikelproteine visualisiert. Zusätzlich ermöglicht die Verwendung von CypHer-Antikörpern die Überwachung von in der Zellmembran gestrandeten Vesikelproteinen, welche den sogenanntenIn the central nervous system, communication between nerve cells mainly occurs at synapses via chemical neurotransmitters. Upon stimulation the transmitters are released by Ca2+-triggered exocytosis of synaptic vesicles from presynaptic terminal to the synaptic cleft, where they activate postsynaptic receptors. In order to repopulate the vesicle pool for further rounds of release, after exocytosis vesicle components need to be resorted and retrieved from the surface by compensatory endocytosis. In this study, both exogenous cypHer-based and genetically encoded GFP-based pH-switchable reporters are used to investigate the vesicle cycling in cultured rat hippocampal neurons. Using cypHer-conjugated antibodies against luminal domains of vesicle proteins (e.g. Syt1), visualizing cycling of endogenous vesicle proteins has been achieved. In addition, by monitoring surface-stranded vesicle proteins, constituting the so-called readily retrievable pool, using cypHer-antibody, it was clearly demonstrated that upon stimulation stained Syt1 residing on the presynaptic membrane rather than freshly exocytosed unlabeled ones are preferentially retrieved to form fresh vesicles, indicating an active participation of the surface pool in the vesicle cycling. Furthermore, the existence of this surface pool is also corroborated by the observation of surface-stranded Syt1 at the synaptic periphery, which is generally identified as the site of endocytosis. These evidences indicate that exocytosis and subsequent compensatory endocytosis of vesicles are driven in parallel from the vesicle pool and the surface pool, respectively. Since the surface pool is repopulated by newly exocytosed vesicle components, fast translocation of vesicle proteins from presynaptic center towards the periphery is expected. To explore potential roles of this translocation in synaptic transmission, dynamin-inhibitor Dynasore was applied to induce protein crowding by blocking endocytosis. Under influence of this drug a clear release depression was observed during high frequency stimulation, which was, however, absent under control conditions. Based on this observation, it is concluded that functional block of previously used release sites rather than insufficient vesicle supply accounts for the fast release depression after blocking endocytosis. This finding implies an important role of dynamin for sustained synaptic transmission at high rates beyond its well-known role in mediating fission, a late step of endocytosis