Direct single-molecule observation ofregulated SNARE assembly

项目摘要--Aleksander A.Rebane 钙离子引发的激素和神经递质胞吐的失衡会导致严重的疾病，包括 2型糖尿病和各种神经疾病，如帕金森氏症、精神分裂症和癫痫。 然而，人们对钙离子如何触发胞吐作用仍知之甚少。特别有趣的是突触的胞吐作用。 神经递质，它是对局部钙离子内流的反应，由动作的到来触发 轴突末端的电位。调节性胞吐中膜融合的核心机制 由可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体(SNARs)、络合蛋白和 突触集合素。圈套形成了一大类分子融合机器，它们通过强制关联 拉链形成盘绕的四螺旋结构，从而将相对的膜拉近 核聚变。打开和关闭融合需要额外的组件来调节突触传递。这 这项任务是通过络合素和突触素来完成的，它们抑制了不需要的自发突触胞吐 通过将陷阱拉链悬吊在半空中作为夹子。当钙从体内排出时，就会发生控制释放 夹住以恢复诱捕拉链并诱导神经递质释放。然而，确切的成分是 对于夹具以及夹紧和松开的机制，人们知之甚少。这很难做到 用传统的系综方法观察受调控的圈套组装途径上的分子事件 方法：研究方法。这些事件本质上是瞬时的，并且仅在膜的排斥力存在的情况下发生 武力。我们通过使用光学镊子将精确已知的拉力施加到单根上来解决这些困难 诱捕复杂分子以模拟膜斥力并稳定部分组装的 中间体，同时使用分子延伸测量来确定毫秒级的结构 时间尺度和纳米分辨率。我们用最先进的方法分析这些测量结果 推导出SNARE络合物组装中间体的构象、能量和动力学。在目标1中，我们将 分析SNAP-25 I67T和I67N两个SNARE复合体突变对严重急性呼吸综合征的影响 神经病，包括共济失调和智力残疾。我们将使用光学镊子来测量这些 突变改变了诱捕拉链的能量、动力学和途径。然后，我们将雇用 重组膜融合试验比较观察到的诱捕拉链的变化如何影响 圈套介导的膜融合。在目标2中，我们将直接观察络合蛋白、突触素、 诱捕装置上的钙。我们将调查陷阱组件是通过什么机构夹住的 络合素，以及突触素和钙离子如何释放这个钳制。我们的研究将揭示陷阱是如何 突变可能导致神经病变，以及复合素和突触素如何调节钙依赖的释放。 我们的研究将提供具体的分子机制，作为治疗神经疾病的新药物靶点。

Project Summary – Aleksander A. Rebane Imbalances in Ca2+-triggered exocytosis of hormones and neurotransmitters cause severe diseases, including type 2 diabetes and various neurological disorders such as Parkinson’s disease, schizophrenia, and epilepsy. Yet it remains poorly understood how Ca2+ triggers exocytosis. Of particular interest is synaptic exocytosis of neurotransmitters, which occurs in response to the local influx of Ca2+, triggered by the arrival of an action potential at the axonal terminal. The core machinery responsible for membrane fusion in regulated exocytosis consists of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), complexin, and synaptotagmin. SNAREs form a broad class of molecular fusion machines that associate by forcefully zippering into a coiled-coil four-helix structure, thus drawing opposing membranes into close proximity for fusion. Additional components are required to turn fusion on and off to regulate synaptic transmission. This task is achieved by complexin and synaptotagmin, which suppress unwanted spontaneous synaptic exocytosis by suspending SNARE zippering halfway as a clamp. Controlled release occurs when calcium removes the clamp to resume SNARE zippering and induce neurotransmitter release. However, the exact constituents of the clamp and the mechanism of clamping and de-clamping are poorly understood. It has been difficult to observe the molecular events along the regulated SNARE assembly pathway using traditional ensemble-based methods. These events are inherently transient and occur solely in the presence of the membrane’s repulsive force. We address these difficulties by using optical tweezers to apply precisely known pulling forces on single SNARE complex molecules to mimic membrane repulsion and to stabilize the partially assembled intermediates, while using molecular extension measurements to determine the structures on millisecond timescale and at nanometer resolution. We analyze these measurements with state-of-the-art methods to derive the conformations, energies, and kinetics of SNARE complex assembly intermediates. In Aim 1, we will analyze the effect of two mutations in the SNARE complex, SNAP-25 I67T and I67N, which cause severe neuropathy, including ataxia and intellectual disability. We will use optical tweezers to measure how these mutations change the energetics, kinetics, and pathways of SNARE zippering. We will then employ reconstituted membrane-fusion assays to compare how the observed changes in SNARE zippering affect SNARE-mediated membrane fusion. In Aim 2, we will directly observe the effect of complexin, synaptotagmin, and calcium on SNARE assembly. We will investigate by what mechanism SNARE assembly is clamped by complexin, and how synaptotagmin and Ca2+ release this clamp. Our research will reveal how SNARE mutations may cause neuropathy and how complexin and synaptotagmin regulate Ca2+-dependent release. Our research will provide concrete molecular mechanisms to act as new drug targets for neurological disease.