Single-molecule manipulation of SNAREs

 描述（由申请人提供）：我的建议的目的是了解钙触发突触胞吐的分子机制。我将使用单分子方法来研究Munc 18 -1，复合蛋白，突触结合蛋白，NSF和α-SNAP如何调节突触SNARE组装和拆卸。SNARE蛋白通过多种中间体组装，以驱动含有神经递质的囊泡与突触前质膜的融合。关键调节蛋白靶向这些中间体以激活和/或抑制SNARE组装，从而使突触胞吐能够由动作电位诱导的钙内流触发。融合后，NSF和α-SNAP分解完全组装的SNARE复合物以回收SNARE蛋白，这是频繁的神经元通信所必需的。精确、快速的SNARE组装和拆卸是钙触发的突触胞吐的核心，也是我们思考、记忆、情感和行动的基础。这一过程中的功能障碍会导致各种神经系统疾病和神经退行性疾病。然而，调节SNARE组装和拆卸的机制知之甚少。我们已经开发了一种独特的单分子操作方法的基础上，高分辨率的光镊研究动态SNARE组装和拆卸。我们已经确定了SNARE组装的几个中间体，并首次表征了与这些中间体以及完全组装的SNARE复合物相关的能量和动力学，为研究SNARE调控铺平了新的道路。我们将扩展我们的单分子方法来解决几个关键问题：（1）Munc 18 -1在SNARE组装和拆卸中的重要作用是什么？(2)复合蛋白和突触结合蛋白是否以及如何以钙依赖的方式激活和/或钳制SNARE组装？(3)NSF和α-SNAP如何识别和分解完全组装的SNARE复合物，而不是膜融合所必需的部分组装的SNARE中间体？我们的研究将提供独特的见解调节SNARE组装和拆卸，钙触发的胞吐作用和相关疾病的机制。

 DESCRIPTION (provided by applicant): The objective of my proposal is to understand the molecular mechanism of calcium-triggered synaptic exocytosis. I will use single-molecule approaches to investigate how Munc18-1, complexin, synaptotagmin, NSF, and α-SNAP regulate synaptic SNARE assembly and disassembly. SNARE proteins assemble via multiple intermediates to drive fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane. Key regulatory proteins target these intermediates to activate and/or inhibit SNARE assembly, enabling synaptic exocytosis to be triggered by the calcium influx induced by an action potential. After fusion, NSF and α-SNAP disassemble the fully assembled SNARE complexes to recycle SNARE proteins, which is required for frequent neuron communication. The precise, rapid SNARE assembly and disassembly is central to calcium-triggered synaptic exocytosis and underlies our thinking, memory, emotions, and actions. Malfunctions in this process cause various neurological disorders and neurodegenerative diseases. Yet, the mechanism underlying the regulated SNARE assembly and disassembly is poorly understood. We have developed a unique single-molecule manipulation approach based on high-resolution optical tweezers to investigate dynamic SNARE assembly and disassembly. We have identified several intermediates of SNARE assembly and characterized the energies, and kinetics associated with these intermediates as well as the fully assembled SNARE complex for the first time, paving a new road to study SNARE regulation. We will expand our single-molecule approach to address several key questions: (1) What is the essential role of Munc18-1 in SNARE assembly and disassembly? (2) Whether and how complexin and synaptotagmin activate and/or clamp SNARE assembly in a calcium-dependent manner? (3) How NSF and α-SNAP recognize and disassemble only the fully assembled SNARE complexes, but not the partially assembled SNARE intermediates essential for membrane fusion? Our research will provide unique insights into the mechanisms of regulated SNARE assembly and disassembly, calcium-triggered exocytosis, and associated diseases.