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一个常见模型是，SNARE核心复合体的组装驱动要融合的膜的接近和合并。核心复合体的组装主要是在溶液中进行的，从与病毒融合蛋白的类似物中可以推断出细胞膜融合的许多特征。因此，为了了解突触前膜融合机如何在分子结构水平上工作，必须解决几个问题。膜表面上的陷阱组装反应与溶液中的一样吗？SNARE核心复合体的组装如何以及在多大程度上减少了要融合的两层膜之间的距离？T-SNARE受体的化学计量比如何 膜表面受调控，活性物种是什么？力是如何从复合体传递到两个融合膜的，以及需要在膜表面发生什么蛋白质和脂肪的结构转换才能使两个脂双层融合？辅助蛋白和特化膜脂在这一过程中起什么作用？本项目的目标是通过确定在膜表面和膜之间形成的圈套复合体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点 目的验证突触前膜融合的一般陷阱假说的几个子假说。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组膜制剂中评估SNARE复合体组装的各种中间体的动态结构；在特定目标3中将研究胆固醇和磷脂酰肌醇-4，5-二磷酸在SNARE复合体组装中的作用；在特定目标4中将在不同的脂质背景下确定在力量转导中潜在重要的SNARE结构域的结构。

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.