Investigating the regulation of SNARE mediated membrane fusion by complexin and synaptotagmin on a single vesicle level using TIRF microscopy

神经元内的钙依赖性突触囊泡融合是通过形成可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)复合体来实现的。在对接和随后的融合过程中，通过将囊泡相关的v型圈套与靶膜上的同源t型圈套配对，使囊泡和靶膜紧密相连。在这个严格调控的过程中，络合素被报道既是SNAREpin组装的促进者，也是SNAREpin组装的抑制者，它促进了组装过程，但随后夹住了一半的融合。在刺激下，复杂蛋白钳被认为是通过钙感受器突触素释放的。然而，络合素和突触素与SNARE复合体的确切功能和相互作用仍存在争议。在这项提议中，我的目标是使用TIRF(全内反射荧光)显微镜和微流控流动系统相结合的高时间/空间分辨率分析来研究复杂蛋白和突触素在SNARE介导的融合中的作用。在这项试验中，将监测单个囊泡与平面支撑双层的对接和融合，以提供关于对接速率、融合速率以及对接和融合之间的延迟的信息。为了研究它们的结构-功能关系，我将确定不同的自然存在的络合蛋白和突触素的异构体和突变的影响。此外，还将分析膜脂组成对融合能力的影响。这些结果将得到动态光散射和表面等离子体共振的支持。阐明突触囊泡融合的详细机制最终可能有助于改善神经退行性疾病和其他神经元疾病的治疗和治疗选择。

Calcium dependent synaptic vesicle fusion in neurons is mediated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. During the docking and subsequent fusion process, vesicle and target membrane are brought into close proximity by pairing of vesicle-associated v-SNAREs with cognate t-SNAREs on target membranes. In this tightly regulated process, complexin has been reported to act as both a promotor and inhibitor of SNAREpin assembly by facilitating the assembly process, but then clamping fusion halfway. Upon stimulation, the complexin clamp is believed to be released through the calcium sensor synaptotagmin. However, the exact function and interaction of complexin and synaptotagmin with the SNARE complex remain controversial. In this proposal, I aim to investigate the role of complexin and synaptotagmin during SNARE-mediated fusion using a high temporal/spatial resolution assay that combines TIRF (total internal reflection fluorescence) microscopy with a microfluidic flow system. In this assay docking and fusion of single vesicles with a planar supported bilayer will be monitored to provide information about the docking rate, fusion rate, and delay between docking and fusion. To study their structure-function relationships, I will determine the effect of different naturally occurring isoforms and mutations of complexin and synaptotagmin. In addition, I will analyze the influence of the membrane lipid composition towards fusion capability. These results will be supported by dynamic light scattering and surface plasmon resonance. Elucidating the detailed mechanism of synaptic vesicle fusion may ultimately contribute to improved treatment and therapy options for neurodegenerative diseases and other neuronal disorders.