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

使用 TIRF 显微镜研究复合蛋白和突触结合蛋白在单个囊泡水平上对 SNARE 介导的膜融合的调节

• 批准号: 217141997
• 负责人: Dr. Andrea Gohlke
• 金额: --
• 依托单位: Department of Cell Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2011-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/217141997?language=en
• 关键词: Investigating; regulation; SNARE; mediated; membrane

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.

神经元中的钙依赖性突触囊泡融合是由可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的形成介导的。在对接和随后的融合过程中，囊泡和靶膜通过囊泡相关的v-SNARE与靶膜上的同源t-SNARE配对而紧密接近。据报道，在这个严格调控的过程中，复合蛋白既可以促进SNAREpin组装，也可以抑制SNAREpin组装，促进组装过程，但在中途抑制融合。在刺激时，复合蛋白钳被认为通过钙传感器突触结合蛋白释放。然而，复杂蛋白和突触结合蛋白与SNARE复合物的确切功能和相互作用仍然存在争议。在这个建议中，我的目标是调查复杂和synaptotagmin在SNARE介导的融合过程中的作用，使用高时间/空间分辨率的分析，结合TIRF（全内反射荧光）显微镜与微流体流动系统。在该测定中，将监测单个囊泡与平面支持的双层的对接和融合，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，我将确定不同的天然存在的异构体和复合蛋白和突触结合蛋白的突变的影响。此外，我将分析膜脂组成对融合能力的影响。这些结果将支持动态光散射和表面等离子体共振。阐明突触囊泡融合的详细机制可能最终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。