Molecular mechanisms of exocytotic vesicle fusion and release.

膜结合囊泡与靶膜的融合对于细胞功能具有普遍的重要性 从细胞内运输到各种不同细胞类型的各种介质的胞吐释放。 胞吐作用的机制及其调节是我的研究计划的中心主题 实验室。胞吐释放通过以下方式从分泌囊泡内部到细胞外部发生： 融合孔的形成。 SNARE（可溶性 NSF 附着受体）复合物，在哺乳动物中 神经元和神经内分泌细胞由 synaptobrevin-2、syntaxin-1 和 SNAP-25 蛋白组成， 在囊泡融合中起关键作用。我的实验室研究了肥大细胞中的囊泡融合机制， 释放组胺和肝素；在中性粒细胞中，它是先天免疫系统的一部分并释放 抗菌蛋白；嗜酸性粒细胞释放细胞毒性蛋白来对抗寄生虫。目前我们 主要集中在嗜铬细胞胞吐融合机制的研究上，我们有 因其实验方法的特定优势以及其神经元分子融合而被选中 机器比其他细胞类型更广为人知。在接下来的5年里，神经元的功能 SNARE 蛋白 synaptobrevin2、syntaxin 1、SNAP25 和辅助蛋白 synaptotagmin、complexin、 Munc18 和 Munc13 在囊泡融合和启动中的作用将作为模型系统进行研究，以了解 囊泡对接、启动和融合的一般机制。对于这些研究，我们将结合高度创新的 我的实验室开发的实验和计算方法来阐明纳米力学 导致囊泡融合和胞吐释放的运动和相互作用。事件相关显微镜使用 微制造的电化学检测器阵列将用于通过实验询问特定的分子 与细胞和重建系统中融合孔形成相关的相互作用和构象变化。 实验研究将得到分子动力学模拟的补充，以解释 实验数据并指导实验设计。机械零件的各种晶体结构 已经确定，它们提供非常高的原子分辨率，但它们代表静态结构。至 为了了解这些蛋白质复合物的功能，需要阐明它们的动态结构变化。 我研究的长期目标是真正理解纳米力学机制 囊泡融合对接、启动、融合和释放。我们的最终愿景是获得逼真的分子电影 融合机的作用，深入了解囊泡启动和融合孔的机制 形成。这项研究还将增进我们对相关细胞内运输融合的理解 事件以及病毒进入，它们采用密切相关的融合机制。 。

Fusion of membrane bound vesicles with a target membrane is of ubiquitous importance for cellular function from intracellular trafficking to exocytotic release of various mediators from a wide range of different cell types. The mechanisms of exocytosis and their regulation are the central topic of the research program in my laboratory. Exocytotic release occurs from the interior of secretory vesicles to the outside of the cell via formation of a fusion pore. The SNARE (Soluble NSF Attachment REceptor) complex, which in mammalian neurons and neuroendocrine cells is composed of the proteins synaptobrevin-2, syntaxin-1, and SNAP-25, plays a key role in vesicle fusion. My laboratory has investigated vesicle fusion mechanisms in mast cells, which release histamine and heparin; in neutrophils, which are part of the innate immune system and release antimicrobial proteins; and in eosinophils, which release cytotoxic proteins to combat parasites. At present we are mostly focused on the investigation of the exocytotic fusion mechanism in chromaffin cells, which we have chosen for their specific advantages for experimental approaches and because their neuronal molecular fusion machinery is better known than those of other cell types. Over the next 5 years, the function of the neuronal SNARE proteins synaptobrevin2, syntaxin 1, SNAP25, and the accessory proteins synaptotagmin, complexin, Munc18 and Munc13 in vesicle fusion and priming will be investigated as a model system to understand the general mechanisms vesicle docking, priming and fusion. For these studies we will combine highly innovative experimental and computational approaches developed in my laboratory to elucidate the nanomechanical motions and interactions that lead to vesicle fusion and exocytotic release. Event Correlation Microscopy using microfabricated Electrochemical Detector Arrays will be used to experimentally interrogate specific molecular interactions and conformational changes related to fusion pore formation in cells and reconstituted systems. The experimental research will be complemented by molecular dynamics simulations to interpret the experimental data and to guide the experimental design. Various crystal structures of parts of the machinery have been determined, which provide very high atomistic resolution, but they represent static structures. To understand the functions of these protein complexes, their dynamic structural changes need to be elucidated. The long term goal of my research is to achieve a true understanding of the nanomechanical mechanisms of vesicle fusion docking, priming, fusion, and release. Our ultimately vision is to obtain realistic molecular movies of the actions of fusion machine, providing deep insight into the mechanisms of vesicle priming and fusion pore formation. This research will also advance our understanding of the related intracellular trafficking fusion events as well as viral entry, which employ closely related fusion mechanisms. .