Analysis of the Drosophila Cysteine-String Proteins and Their Role for Neurotransmission

小行星9604889 去极化依赖性神经递质释放的信号通路是通过分泌、膜再循环和囊泡发生的重复循环运输突触囊泡的产物。这个循环的每个阶段都需要蛋白质的协同作用。这项提议研究突触囊泡内的半胱氨酸串蛋白。 果蝇突变体中csp蛋白的缺失在较高温度下逐渐减少诱发的递质释放，但不是自发释放。 csp突变体中神经递质释放的这种失败可能是由于囊泡周期任何阶段的缺陷。 为了更好地理解csp的功能，本研究将通过对csp突变果蝇突触囊泡周期的动态成像来确定囊泡周期的哪个阶段需要csp的功能。 从早期的研究已经提出，csp可能作为一个辅因子，催化招募一个未知的蛋白质，以配合蛋白质相互作用的关键诱发神经递质释放。为了探索这一工作假设，该提案计划通过基因工程来鉴定csp的蛋白质结构域，这些结构域对其功能至关重要。此外，还设计了一种策略，结合相互作用蛋白质的纯化和与csp突变相互作用的突变的分离来确定与csp相互作用的分子组分。 csp蛋白及其相互作用伙伴的关键部分的鉴定将为开发更具体的想法以直接阐明csp功能提供基础。 鉴于从果蝇到哺乳动物都有许多保守的信号通路，果蝇将作为一个模型系统来阐明正常和异常突触传递的基本机制。

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.