EuroSYNAPSE - Spatio-temporal organization of the synaptic membrane for synaptic vesicle protein recycling

• Antragsteller: Professor Volker Haucke, Ph.D.; Professor Dr. Jürgen Klingauf
• Fachliche Zuordnung: Biochemie
• Förderung: Förderung von 2009 bis 2012
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 128368325

The presynaptic terminal contains clusters of synaptic vesicles (SVs), key organelles of chemical neurotransmission (1). Proteomics data indicate that SVs comprise distinct sets of proteins and lipids present in defined stoichiometries (2) which must be maintained during repetitive rounds of exo- and endocytosis. How this precise sorting of SV membrane proteins is accomplished molecularly is not well understood. SV components could remain clustered during their exo-endocytic itinerary as suggested by high-resolution stimulated emission-depletion (STED) microscopy (3), or be reclustered at the cell surface (4) by individual sorting via recognition of sorting determinants by cargo-specific adaptor proteins such as AP-2, stonin2/stoned B (5, 6), AP180 and perhaps others. SV exo- and endocytosis appear to be temporally coupled but spatially segregated between the active zone (sites of fusion) and the surrounding endocytic or periactive zone. The tight temporal coupling between SV exo- and endocytosis (7) suggests that SV cargo protein sorting and recycling likely involves precise control of the localization and dynamics of endocytic proteins or protein complexes as they partition between sites of fusion (active zone) and endocytosis (periactive zone) (8). How the spatio-temporal dynamics of endocytic proteins at the nerve terminal are controlled is largely unknown but likely involves membrane-associated multidomain scaffolding proteins organizing the periactive zone (7, 9). While recent years have witnessed enormous progress in the identification and characterization of the cytomatrix assembled at the active zone (CAZ, also termed presynaptic grid) comparably little is known about the structural components and the functional organization of the periactive zone that surrounds it (7). From a simplified perspective the periactive zone might be viewed as a three-layered structure comprised of a pool of surface stranded SV cargo, dynamic assemblies of endocytic proteins, and largely immobile scaffolds such as the giant protein piccolo. However, the precise underlying mechanisms for this organization have not been unravelled. Movement of endocytic proteins to and from the periactive zone during SV cycling is accompanied by the concomitant assembly of clathrin-coated structures. This process involves the coordinated formation of a regulated network of protein-protein and protein-lipid interactions. The endocytic network appears to be organized around central hubs, i.e. factors that at a given time or within a defined space display a disproportionately high number of interactions (10). As maturation of the endocytic vesicle progresses, the network proceeds from a SV cargo and phosphatidylinositol (4,5)-bisphosphate (PIP2)- to an AP-2-, and finally to a clathrin-centered state (6, 10). A number of studies indicate that this progression involves the regulated relocalization of endocytic proteins or complexes thereof to sites of SV endocytosis (1, 8). Ultrastructural and light microscopy data in combination with dominant-negative approaches have revealed that molecularly similar components such as the SH3 domain containing proteins amphiphysin, syndapin, endophilin, and intersectin are recruited to the periactive zone and play non-overlapping functional roles in SV recycling. Based on combined genetic, biochemical, and functional studies endocytic proteins might be grouped into functional protein modules, i.e. macromolecular complexes defined by their physical interactions within a characteristic time domain and by their spatial sequestration (11). For example, the scaffolding protein intersectin physically and functionally interacts with eps15, dynamin, and synaptojanin. Mutants of eps15 and intersectin in D. melanogaster phenotypically resemble each other (12) and both proteins co-migrate from the centre of synaptic boutons to the periactive zone in response to activity, suggesting that intersectin together with eps15 is part of a functional module that regulates localization and activity of dynamin and synaptojanin at late stages of clathrin-mediated SV endocytosis (Figure 1B). At present we do not understand how such movement is accomplished at the molecular level but interactions with the actin cytoskeleton are likely to be involved. As endocytic proteins can be associated with several modules intersectin and eps15 via their EH domains are also part of a functional module containing the synaptotagmin-specific sorting adaptor stonedB/ stonin2, its interactor GIT1 (our own unpublished data), AP180, and the AP-2 complex implicated in retrieval of surface-stranded pools of SV cargo from the presynaptic plasmalemma (Figure 1A). Thus, functional endocytic protein modules may be viewed as dynamic components of the periactive zone that are under tight spatio-temporal control. How such regulatory control is accomplished and how it is synchronized with the migration of the vesicle membrane and associated components after fusion remains unknown (9), largely because current methodology allows either subcellular imaging without access to the time domain (i.e. by conventional electron microscopy) or fast live cell imaging based on light microscopic techniques that do not provide sufficient spatial resolution. The work described in this proposal aims to tackle these problems and will thus enable us to gain unprecedented insights into the spatio-temporal organization of the synaptic membrane for clathrin-mediated SV protein recycling.

• DFG-Verfahren: Sachbeihilfen