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

9604889 Zinsmaier去极化依赖性神经递质释放的信号通路是突触小泡通过反复的分泌、膜循环和小泡发生的循环运输的产物。这个循环的每个阶段都需要蛋白质的协同作用。该方案研究突触小泡固有的半胱氨酸串蛋白(CSP)。果蝇突变体中CSP蛋白的丢失逐渐减少了在较高温度下诱发的递质释放，但不是自发释放。CSP突变体中神经递质释放的失败可能是由于囊泡周期的任何阶段的缺陷造成的。为了更好地了解CSP的功能，这一建议将通过成像CSP突变果蝇的突触小泡周期的动力学来确定囊泡周期的哪个阶段需要CSP功能。根据早期的研究，CSP可能作为辅因子催化招募未知蛋白质来协调对诱发的神经递质释放至关重要的蛋白质相互作用。为了探索这一工作假说，该提案计划通过基因工程确定CSP的蛋白质结构域，这些结构域对CSP的功能至关重要。此外，还设计了一种策略来确定与CSP相互作用的分子成分，结合相互作用蛋白的纯化和与CSP突变相互作用的突变的分离。鉴定CSP蛋白的关键部分及其相互作用伙伴将为发展更具体的思路来直接阐明CSP的功能提供基础。考虑到从苍蝇到哺乳动物的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.