PRESYNAPTIC FUSION COMPLEX ASSEMBLY ON MEMBRANES PROBED BY MICRO-FLUORESCENCE AND

突触前和其他细胞内膜融合机制的常见模型是SNARE核心复合物的组装驱动待融合的膜的接近和合并。核心复合物的组装主要在溶液中进行了研究，并且从与病毒融合蛋白的类比中推断出细胞内膜融合的许多特征。因此，必须解决几个问题，以了解突触前膜融合机器如何在分子结构水平上工作。SNARE组装反应在膜表面上与在溶液中相同吗？SNARE核心复合物的组装如何以及在多大程度上减少了两个将要融合的膜之间的距离？t-SNARE受体的化学计量是怎样的 调节膜表面的活性物质是什么？力是如何从复合物传递到两个融合的膜上的？在膜表面需要发生什么样的蛋白质和脂质的结构转换才能使两个脂质双层融合？在这个过程中，辅助蛋白和特殊的膜脂起着什么样的作用？这个项目的目标是通过确定SNARE复合物在膜表面和膜之间形成时的一般动态结构来回答这些问题。将开发新的显微镜和光谱方法来实现这一目标 的目标，并测试几个子假设的一般圈套假说的突触前膜融合。将追求四个具体目标：在目标1和2中，将分别在质膜片和重构膜制剂中评估SNARE复合物组装的各种中间体的动态结构;在具体目标3中将检查胆固醇和磷脂酰肌醇-4，5-二磷酸对SNARE复合物组装的作用;并且，在力转导中潜在重要的SNARE结构域的结构将在具体目标4中的各种脂质背景中确定。

A common model of the mechanism of presynaptic and other intracellular membrane fusions is that the assembly of the SNARE core complex drives the approach and merger of the membranes that are to be fused. The assembly of the core complex has been studied primarily in solution and numerous features of intracellular membrane fusion have been inferred from analogies with viral fusion proteins. Therefore, several questions must be addressed in order to understand how presynaptic membrane fusion machines work at the molecular structural level. Are the SNARE assembly reactions the same on membrane surfaces as in solution? How and to what extent does the assembly of the SNARE core complex reduce the distance between the two membranes that are to be fused? How are the stoichiometries of t-SNARE receptors regulated on membrane surfaces and what are the reactive species? How is the force transduced from the complex into the two fusing membranes and what structural transformations of protein and lipid need to occur at the membrane surfaces to get two lipid bilayers to fuse? What are the roles of accessory proteins and specialized membrane lipids in this process? The goal of this project is to answer these questions by determining the general dynamic architecture of the SNARE complex as it forms on the surface of and in between membranes. Novel microscopic and spectroscopic approaches will be developed to achieve this goal and to test several sub-hypotheses of the general SNARE hypothesis of presynaptic membrane fusion. Four specific aims will be pursued: In aims 1 and 2, the dynamic architectures of various intermediates of SNARE complex assembly will be assessed in plasma membrane sheet and reconstituted membrane preparations, respectively; the role of cholesterol and phosphatidylinositol-4,5-biphosphate on SNARE complex assembly will be examined in specific aim 3; and, the structures of SNARE domains that are potentially important in force transduction will be determined in various lipid backgrounds in specific aim 4.