Dissecting the endocytic functions of Synaptotagmin

描述(申请人提供)：突触素I(SYT I)是一种钙结合的突触囊泡蛋白，功能是神经递质释放的钙传感器。一些研究也发现SYT I参与了突触小泡的再循环，但其在神经递质释放中的关键作用限制了对SYT I在内吞作用中作用的研究。Davis实验室最近开发了一种方法，可以在体内突触小泡释放后迅速灭活SYT I；这项技术与内吞作用的实时成像相结合，为研究SYT I在内吞作用中的功能提供了一种强有力的新方法。本文介绍的实验将结合这些技术和超微结构分析来确定SYT I在内吞作用中的作用。首先，突触前末端的电子显微镜将被用来评估在内吞作用中SYT I的急性失活的影响。这些研究将有助于确定SYT I在内吞周期中的作用。其次，将在飞行运动神经元中表达SYT I突变和缺失结构，以确定其内吞活动所需的SYT I蛋白部分。最后，这些分析工具将被应用于具有类似L系统表型的stoned基因，以确定它们的蛋白质产物在内吞过程中是否以及如何协同作用。

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.