Single molecule studies of SNARE-induced vesicle fusion

描述（由申请人提供）：神经递质释放是最受调节的膜融合事件之一。与构成囊泡运输不同，突触囊泡被募集到突触前膜，但不容易融合。相反，平均有10个囊泡稳定地停靠在被称为活动区的突触区域，等待动作电位的产生。突触囊泡融合与动作电位到达后的Ca2+内流密切相关。胞吐作用在Ca2+到达的大约0.2毫秒内被触发。尽管已经发现许多关键因子对Ca2+依赖性神经递质释放至关重要，如SNAREs、synaptotagmin、complexin、Munc18和Munc13，但突触囊泡膜融合和Ca2+触发的分子机制仍不清楚。在这个更新的应用中，我们希望继续我们的努力，通过单分子和单粒子荧光成像显微镜实验的组合来破译分子机制。根据我们的假设，SNAREs和辅助蛋白不能完全组装，因此，在Ca2+信号到达之前不会触发融合，我们设计了标记策略来监测参与该过程的蛋白质和蛋白质复合物的不同构象状态。我们还开发了荧光报告器，提供了系统从囊泡对接到膜接触，最后到融合过程中含量和脂质交换的信息。这些标记策略以及单分子和单囊泡实验装置是本研究的基础。我们的实验是一个突破，因为我们第一次观察到Ca2+触发后的快速融合动力学。我们的新分析现在使我们能够确定所涉及的因素的作用，它们的化学计量（例如，快速Ca2+触发融合需要多少SNARE复合物和synaptotagmin分子？），提出并测试这些影响其功能的因素的突变体，然后将这些突变体用作神经元培养实验的工具，以解剖它们在体内的作用。此外，我们最近证明了使用标记抗体对突触蛋白进行超分辨率成像的能力，达到了前所未有的定位精度。这种体内成像方法将使我们能够成像突触蛋白沿轴突的分布和相关性，因此它将补充我们的重建和单分子生物物理研究。

DESCRIPTION (provided by applicant): Neurotransmitter release is one of the most regulated membrane fusion events. Unlike constitutive vesicle trafficking, synaptic vesicles are recruited to the presynaptic membrane, but do not readily fuse. Instead, an average of ten vesicles are stably docked at a region of the synapse termed the active zone awaiting an action potential. Synaptic vesicle fusion is closely associated with the Ca2+ influx that follows arrival of an action potential. Exocytosis is triggered within approximately 0.2 msec of the Ca2+ arrival. Although many key factors have been found to be essential for Ca2+-dependent neurotransmitter release, such as SNAREs, synaptotagmin, complexin, Munc18, and Munc13, the molecular mechanism of synaptic vesicle membrane fusion and Ca2+- triggering remains unclear. In this renewal application we wish to continue our efforts to decipher the molecular mechanism by using a combination of single molecule and single particle fluorescence imaging microscopy experiments. Driven by our hypothesis that SNAREs and auxiliary proteins do not fully assemble and, thus, do not trigger fusion until a Ca2+ signal arrives, we have designed labeling strategies to monitor distinct conformational states of the protein and protein-complexes involved in the process. We have also developed fluorescent reporters that provide information about content and lipid exchange as the system proceeds from vesicle docking, to membrane contact, and, finally, to fusion. These labeling strategies and the single molecule and single vesicle experimental setups are the cornerstones for the proposed research. Our assays are a breakthrough, since for the first time, we observe fast fusion kinetics upon Ca2+ triggering. Our new assay now enables us to determine the role of the factors involved, their stoichiometry (e.g., how many SNARE complexes and synaptotagmin molecules are required for fast Ca2+ triggered fusion?), to propose and test mutants of these factors that affect their function in the assay, and then to use these mutants as tools for experiments with neuronal cultures to dissect their role in vivo. Furthermore, we have recently demonstrated the ability of super-resolution imaging of synaptic proteins by using labeled antibodies to an unprecedented degree of localization accuracy. This in vivo imaging approach will enable us to image the distribution and correlation of synaptic proteins along axons, and so it will complement our reconstitution and single molecule biophysical studies.