Single-molecule manipulation of SNAREs

 描述(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Munc18-1、复合蛋白、突触素、神经营养因子和α-SNAP如何调控突触陷阱的组装和拆解。SNARE蛋白通过多个中间体组装，推动含有神经递质的囊泡与突触前质膜的融合。关键调控蛋白针对这些中间体激活和/或抑制SNARE组装，使突触胞吞作用由动作电位诱导的钙内流触发。融合后，神经营养因子和α-SNAP分解完全组装的SNARE复合体，以回收SNARE蛋白，这是频繁的神经元通讯所必需的。精确、快速的陷阱组装和分解是钙触发的突触胞吐的核心，是我们思维、记忆、情感和行动的基础。这一过程中的故障会导致各种神经疾病和神经退行性疾病。然而，人们对受调控的陷阱组装和拆解的机制知之甚少。我们开发了一种基于高分辨率光钳的独特的单分子操纵方法来研究陷阱的动态组装和拆卸。我们首次鉴定了几种陷阱组装的中间体，并表征了与这些中间体相关的能量、动力学以及完全组装的陷阱复合体，为研究陷阱的调控铺平了一条新的道路。我们将扩展我们的单分子方法来解决几个关键问题：(1)Munc18-1在SNAR组装和拆解中起什么重要作用？(2)复合素和突触素是否以及如何以钙依赖的方式激活和/或钳制SNAR组装？(3)Nsf和α-SNAP如何识别和拆解完全组装的SNAR复合体，而不识别膜融合所必需的部分组装的SNAR中间体？我们的研究将为调控陷阱的组装和解体、钙触发的胞吐作用以及相关疾病的机制提供独特的见解。

 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.