) This proposal will resolve fundamental issues concerning the function of cysteine string proteins (csps). Csps are structurally unique components of synaptic vesicle membranes. Work in the previous funding period established that csps are essential for the normal evoked release of neurotransmitter at Drosophila nerve endings. The available evidence indicates that csps either constitute a novel regulatory link between synaptic vesicles and presynaptic Ca channels, or they participate in some later step in the exocytotic cascade (downstream of Ca entry). To clarify the role(s) of csps, the investigator plans to extend her studies of Drosophila csp mutants. In addition, she will use the Xenopus nerve-muscle co-culture system which give one extraordinary latitude to manipulate and study presynaptic events at a vertebrate synapse. The specific issues that she will address include: (i) Is Ca influx altered a nerve endings of Drosophila csp mutants? The PI recently succeeded in loading Ca-sensing dyes into Drosophila nerve endings, and this gives her the capability of determining whether the temperature-sensitive block of secretion transmitter release in csp mutants is due to impaired Ca entry; (ii) Are csps essential for evoked transmitter secretion at Xenopus synapses in culture? Preliminary data, obtained using antibodies to perturb csp function, answers this question affirmatively. This work will be advanced in several directions by use of other csp perturbants (antibodies and peptides), as well as recording of presynaptic Ca currents (which can be achieved in this preparation). These physiological investigations are complemented by rigorous biochemical studies of the action and specificity of the csp perturbants, give an unparalleled opportunity to resolve cellular and molecular details of csp function; (iii) Is there another csp isoform? Recent evidence was obtained for a second csp gene in Drosophila. They will clone the cDNA for this csp isoform and test the hypothesis that this csp normally is involved in constitutive secretion. Taken together, these studies will refine appreciably our knowledge of the function of these important proteins. These results will be important for efforts to understand the secretory cascade and membrane trafficking at the nerve ending. And, at a practical level, by analogy with the devastating impact of csp mutations on survival, behavior and synaptic function in Drosophila, it is likely that this work may help us eventually to identify and correct csp disorders in man.
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