Single Molecule Studies of SNARE-Induced Vesicle Fusion

描述(申请人提供)：在真核细胞胞质中维持不同细胞器是生存所必需的。这些细胞器之间的物质交换需要两个磷脂膜的合并。所有已知的细胞膜融合形式，包括突触囊泡融合，都涉及一个高度保守的蛋白质家族，称为SNARES(可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体)。已知辅助SNARE结合蛋白可调节膜融合事件，如钙离子结合蛋白synaptopagmin。参与这一过程的一些关键分子的晶体结构已经被解决，例如突触陷阱复合体和突触素的细胞质结构域。体外大量脂质体-脂质体融合实验已经证实，SNARES和synaptopagmin构成了一种最小的、尽管效率低下的融合机制，但它们几乎没有揭示融合过程中蛋白质和脂类之间的顺序和空间相互作用的潜在分子机制。我们建议用单分子荧光方法研究钙离子触发突触囊泡融合的分子机制。我们以前的工作为拟议的研究提供了框架。具体地说，我们建议研究蛋白质-蛋白质、蛋白质-脂质相互作用和融合之间的关系，研究SNARE蛋白翻译后修饰和脂质/胆固醇组成对融合的影响，研究SNARE和突触素在对接的膜之间的相互作用，并研究ATPase N-乙基马来酸乙酯敏感因子(NSF)分解SNARE复合体的分子机制。这些体外研究将得到使用PC12细胞的体内研究的补充。我们预计，我们的体外系统可以扩展到包括其他因素，以获得一个重组系统，最终可能接近神经元中突触小泡融合机制的特性。这样的系统可以作为新药发现的一个有效的模型系统。

DESCRIPTION (provided by applicant): The maintenance of distinct organelles within the eukaryotic cytosol is essential for survival. The exchange of material between these organelles requires the merger of two phospholipids membranes. All known forms of intracellular membrane fusion, including synaptic vesicle fusion, involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive factor Attachment Protein Receptors). Auxiliary SNARE binding proteins are known to regulate membrane fusion events, such as the Ca2+ binding protein synaptotagmin. Crystal structures of some of the key players involved in this process have been solved, such as the synaptic SNARE complex and the cytoplasmic domain of synaptotagmin. In vitro bulk liposome-liposome fusion experiments have established that SNARES and synaptotagmin constitute a minimal, albeit inefficient, fusion machinery, but they have done little to reveal the underlying molecular mechanism, both in terms of sequential and spatial interactions between proteins and lipids during fusion. We propose to study the molecular mechanism of Ca2+-triggered synaptic vesicle fusion by single molecule fluorescence methods. Our previous work has provided the framework for the proposed studies. Specifically, we propose to study correlations between protein-protein, protein-lipid interactions and fusion, to study the effect of post-translational modifications of SNARE proteins and of the lipid/cholesterol composition on fusion, to study the interactions of SNAREs and synaptotagmin at the interface between docked membranes, and to study the molecular mechanism of SNARE complex disassembly by the ATPase N- ethylmalemeide-sensitive factor (NSF). These in vitro studies will be complemented by in vivo studies using PC12 cells. We anticipate that our in vitro system can be extended to include other factors in order to obtain a reconstituted system that may eventually approach the properties of the synaptic vesicle fusion machinery in the neuron. Such a system could serve as an efficient model system for novel drug discovery.