Defining the 'late' role for UNC-18 in synaptic vesicle exocytosis

描述（由申请人提供）：SM蛋白是细胞中所有膜融合步骤所必需的，但其作用机制尚不清楚。例如，对突触处的SM蛋白（Unc 18蛋白）的研究表明，这些蛋白可以促进或抑制突触囊泡融合。这些相互矛盾的结果可能是由于Unc 18的多种结合模式。Unc 18可以以闭合形式结合突触融合蛋白，或者它可以结合突触融合蛋白的开放形式的N-末端。我们的初步数据表明，C。与闭合形式的突触融合蛋白Unc 18的结合对于其融合功能不是必需的。目前的工作模型是Unc 18与syntaxin的N-末端肽结合，通过SNARE复合物相互作用介导Unc 18在融合中的作用。该模型预测，首先，在C.因此，线虫中的N-肽和SNARE的相互作用将是保守的，并且在N-18和突触融合蛋白的结合区域中的突变将破坏N-肽和SNARE的相互作用。我将使用体内下拉实验来测定这些突变的特异性。其次，我将确定这些突变是否会破坏体内融合。具体来说，我将使用电子显微镜来表征囊泡对接在突触前末梢;我将测量囊泡启动通过量化的大小容易释放池;并且，我将使用在体内电生理学来测量单囊泡融合事件（微型EPSC）和钙诱发事件的动力学。该分析将确定Unc 18与组装的核心复合物相互作用的功能意义，并可能阐明Unc 18在突触囊泡胞吐中的融合促进作用。

DESCRIPTION (provided by applicant): SM proteins are required for all membrane fusion steps in a cell, yet the mechanism of their action is not known. For example, studies of the SM protein at the synapse - the Unc18 proteins - suggest that these proteins can either promote or inhibit synaptic vesicle fusion. These conflicting results may be due to the multiple binding modes of Unc18. Unc18 can bind syntaxin in a closed form or it can bind to the N-terminus of the open form of syntaxin. Our preliminary data indicate that binding of the C. elegans Unc18 to the closed form of syntaxin is not essential for its function in fusion. The current working model is that Unc18 binding to the N-terminal peptide of syntaxin mediating the role of Unc18 in fusion via SNARE complex interaction. This model predicts, first, that the binding of UNC-18 to syntaxin in C. elegans will be conserved, and that mutations in the binding regions of UNC-18 and syntaxin will disrupt the N-peptide and SNARE interactions. I will assay the specificity of these mutations by using in vivo pull down experiments. Second, I will determine if these mutations disrupt fusion in vivo. Specifically, I will use electron microscopy to characterize vesicle docking at presynaptic terminals; I will measure vesicle priming by quantifying the size of the readily releasable pool; And, I will use in vivo electrophysiology to measure the kinetics of single vesicle fusion events (miniature EPSCs) and calcium evoked events. This analysis will determine the functional significance of Unc18's interaction with the assembled core complex and may elucidate the fusion-promoting role of Unc18 in synaptic vesicle exocytosis.