Molecular mechanisms of exocytotic vesicle fusion and release.

膜结合的囊泡与靶膜的融合对细胞功能具有普遍的重要性 从细胞内转运到多种不同细胞类型的各种介质的胞外释放。 胞吐作用的机制及其调控是我的研究项目的中心议题 实验室。胞吐释放是从分泌小泡的内部通过 形成融合孔。SNARE(可溶性NSF附着受体)复合体，在哺乳动物中 神经元和神经内分泌细胞由突触素-2、突触素-1和SNAP-25蛋白组成， 在囊泡融合中起着关键作用。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，中性粒细胞是先天免疫系统的一部分，并释放 抗微生物蛋白；以及嗜酸性粒细胞，嗜酸性粒细胞释放细胞毒蛋白来对抗寄生虫。目前我们 主要集中在研究嗜铬细胞的胞吐融合机制，我们有 选择它们是因为它们在实验方法上的特殊优势，以及它们的神经元分子融合 机械比其他类型的细胞更为人所知。在接下来的5年里，神经元的功能 SNARE蛋白Synaptobrevin2、Synaxin 1、SNAP25以及辅助蛋白synaptopagmin、Complex in、 Munc18和Munc13在囊泡融合和启动中的作用将作为一个模型系统进行研究，以了解 一般机制囊泡对接、引爆和融合。对于这些研究，我们将结合高度创新 我的实验室开发了实验和计算方法来阐明纳米机械 导致囊泡融合和胞吐释放的运动和相互作用。事件关联显微镜使用 微型制造的电化学探测器阵列将用于实验询问特定分子 细胞和重组系统中与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 并对实验数据进行分析，指导实验设计。机械零件的各种晶体结构 已经确定，它们提供了非常高的原子分辨率，但它们代表静态结构。至 要了解这些蛋白质复合体的功能，还需要阐明它们的动态结构变化。 我的研究的长期目标是实现对纳米力学机制的真正理解 囊泡融合对接、引爆、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，提供了对囊泡启动和融合孔的机制的深入了解 队形。这项研究也将促进我们对相关的细胞内运输融合的理解 事件以及病毒进入，这采用了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. .