Dissecting the endocytic functions of Synaptotagmin

描述（由申请人提供）：Synaptotagmin I （Syt I）是一种钙结合突触囊泡蛋白，作为神经递质释放的钙传感器。几项研究也表明Syt I参与突触囊泡循环，但其在神经递质释放中的关键作用限制了对Syt I在内吞作用中的功能的研究。Davis实验室最近开发了一种在体内突触囊泡释放后急性灭活Syt I的方法；该技术与内吞作用的实时成像相结合，为内吞作用过程中Syt I功能的研究提供了一种强有力的新方法。本文的实验将这些技术与超微结构分析相结合，以确定Syt I在胞吞作用中的作用。首先，突触前末端的电子显微镜将用于评估内吞过程中Syt I急性失活的影响。这些研究将有助于确定Syt I在内吞循环中的作用。其次，Syt I突变和缺失结构将在果蝇运动神经元中表达，以确定其内吞活性所需的Syt I蛋白部分。最后，这些分析工具将应用于stoned，一个具有syl样表型的基因，以确定它们的蛋白质产物在内吞作用中是否以及如何合作。

DESCRIPTION (provided by applicant): Synaptotagmin I (Syt I) is a calcium-binding synaptic vesicle protein that functions as the calcium sensor for neurotransmitter release. Several studies have also implicated Syt I in synaptic vesicle recycling, but its critical role in neurotransmitter release has limited study of Syt I function during endocytosis. The Davis laboratory has recently developed a method to acutely inactivate Syt I following synaptic vesicle release in vivo; this technique, when combined with live imaging of endocytosis, provides a powerful new approach for the study of Syt I function during endocytosis. The experiments presented here will combine these techniques with ultrastructural analyses to define the role of Syt I in endocytosis. First, electron microscopy of the presynaptic terminal will be used to assess the effects of acute inactivation of Syt I during endocytosis. These studies will help determine where in the endocytic cycle Syt I functions. Second, Syt I mutation and deletion constructs will be expressed in fly motor neurons, to identify the portions of the Syt I protein that are required for its endocytic activity. Finally, these tools of analysis will be applied to stoned, a gene with a syt l-like phenotype, to determine if and how their protein products cooperate during endocytosis.