9604889 Zinsmaier The signaling pathway of depolarization-dependent neurotransmitter release is a product of trafficking synaptic vesicles through repeated cycles of secretion, membrane recycling, and vesicle genesis. Each stage of this cycle requires the cooperative action of proteins. This proposal studies the cysteine-string protein (csp) intrinsic to synaptic vesicles. The loss of csp protein in Drosophila mutants progressively reduces evoked transmitter release at higher temperatures, but not spontaneous release. Such a failure of neurotransmitter release in csp mutants could be due to a defect at any stage of the vesicle cycle. To gain a better understanding of csp function this proposal will determine which stage of the vesicle cycle requires csp function by imaging the dynamics of the synaptic vesicle cycle in the csp mutant flies. From earlier studies it has been proposed, that csp might serve as a cofactor to catalytically recruit an unknown protein to cooperate protein interactions critical for evoked neurotransmitter release. To explore this working hypothesis the proposal plans to identify protein domains of csp which are critical for its function by genetic engineering. In addition, a strategy has been designed to determine the molecular components that interact with csp combining the purification of interacting proteins and the isolation of mutations interacting with the csp mutation. The identification of critical parts of the csp protein and its interacting partners will provide the basis to develop more specific ideas to directly elucidate csp function. Given the numerous signaling pathways conserved from flies to mammals, Drosophila will serve as a model system to illuminate basic mechanisms of normal and abnormal synaptic transmission.
