Project 1- Reconstitution of SNAR-mediated Fusion with Native and Artificial Mem

突触囊泡的胞外膜融合是由SNARS突触趋化蛋白、SNAP-25和 Synaptobrevin/VAMP，它们以拉链的方式在膜之间反式组装，从而拉动 这些膜结合在一起，克服了聚变的能量障碍。此外，调节蛋白 包括突触素、Munc18和复合蛋白，它们与SNARs和参与的SNARs相互作用 膜，从而控制圈套的反应性和形成钙依赖的特殊特征 突触小泡的胞吐作用。虽然拉链模型在直觉上是令人满意的，并得到了 证据，蛋白质-蛋白质相互作用的精确序列和调控的作用部位 沿着融合途径的蛋白质仍然只有部分了解和有争议的讨论。 在这里，我们使用天然分泌囊泡和由纯化蛋白重组的脂质体的体外融合来 分离融合途径的部分反应，以了解其结构、动力学和化学计量学 参与的蛋白质的中间状态，并确定影响动力学的参数 每一种反应。在之前的资助期间，我们解决了神经元陷阱的细胞结构。 具有连接体和跨膜区的复合体，并表征突触素的相互作用 用含SNARE的脂质体。此外，我们还开发了测量核聚变的精细化分析方法。 中间体包括荧光互相关和低温电子显微镜，研究了 突触囊泡与含有SNARE的脂质体，并干扰在部分或 完全拉链状态，从而获得融合途径的中间状态。我们现在建议 利用这些成果来描述圈套介导的融合的中间步骤 并确定调节蛋白synaptoagmin 1、Complin 1和 Munc18-1作用于神经元陷阱催化的膜融合。在第一个具体目标中，我们计划 阐明陷阱组装和拉链如何与膜对接、半融合和融合相关联 使用天然和人造的囊泡。在第二个具体目标中，我们将确定陷阱中的步骤 核化和拉链由调节蛋白synaptopagmin、Muncls和 络合。该方案将与项目2和项目3密切合作执行，在这些项目中 采取了互补的办法。我们希望能获得有关 神经元胞吐的分子机制，这是其他方法所不能获得的。

Exocytotic membrane fusion of synaptic vesicles is driven by the SNAREs syntaxin, SNAP-25, and synaptobrevin/VAMP, which assemble in trans between the membranes in a zipper-like fashion, thus pulling the membranes together and overcoming the energy barrier for fusion. In addition, regulatory proteins including synaptotagmin, Munc18, and complexin interact both with the SNAREs and the participating membranes, thus controlling SNARE reactivity and shaping the specific features of cacium-dependent exocytosis of synaptic vesicles. While the zippering model is intuitively satisfying and confirmed by a large body of evidence, the precise sequence of protein-protein interactions and the site of action of the regulatory proteins along the fusion pathway is still only partially understood and controversially discussed. Here we use in-vitro fusion of native secretory vesicles and liposomes reconstituted with purified proteins to isolate partial reactions of the fusion pathway, to understand the structure, dynamics and stoichiometries of the intermediate states of the participating proteins, and to determine the parameters affecting kinetics of each reaction. In the previous funding period, we solved the ci7stal structure of the neuronal SNARE complex with its linkers and transmembrane domains and characterized the interaction of synaptotagmin with SNARE-containing liposomes. Moreover, we developed refined assays for measuring fusion intermediates including fluorescence cross-correlation and cryo electron microscopy, studied the fusion of synaptic vesicles with SNARE-containing liposomes, and interfered with SNARE assembly at the partially or fully zippered state, thus gaining access to intermediate states of the fusion pathway. We now propose to take advantage of these achievements to characterize the intermediate steps in SNARE-mediated fusion and to determine the precise step at which the regulatory proteins synaptotagmin 1, complexin 1, and Munc18-1 operate on membrane fusion catalyzed by neuronal SNAREs. In the first specific aim, we plan to elucidate how SNARE assembly and zippering is connected to membrane docking, hemifusion, and fusion using native and artificial vesicles. In the second specific aim, we will determine the steps in SNARE nucleation and zippering that are acted upon by the regulatory proteins synaptotagmin, MunclS, and complexin. The program will be carried out in close collaboration with Projects 2 and 3 in which complementary approaches are pursued. We expect to obtain essential mechanistic information about the molecular mechanism of neuronal exocytosis that is not obtainable by other approaches..