Molecular mechanisms of exocytotic vesicle fusion and release.

胞吐囊泡融合和释放的分子机制。

• 批准号: 10529686
• 负责人: Manfred LINDAU
• 金额: $ 28.58万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10529686
• 关键词: Molecular; mechanisms; exocytotic; vesicle; fusion

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. .

膜结合囊泡与靶膜的融合对细胞功能无处不在 从细胞内贩运到各种不同细胞类型的各种介体的胞胞胞吐释放。 胞吞作用及其调节的机制是我的研究计划的核心主题 实验室。胞吐释放发生在分泌囊泡的内部到细胞的外部。 融合孔的形成。网罗（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蛋白质触发蛋白-2，syntaxin-1和snap-25， 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，这是先天免疫系统的一部分并释放 抗菌蛋白；在嗜酸性粒细胞中，将细胞毒性蛋白释放为对抗寄生虫。目前我们 主要集中于铬蛋白细胞中胞吐融合机制的研究，我们已经有 选择其针对实验方法的特定优势和由于其神经元分子融合 机械比其他细胞类型的机械更为知名。在接下来的5年中，神经元的功能 SNARE蛋白stynaptobrevin2，语法1，SNAP25和辅助蛋白突触蛋白突触蛋白，复合素， 囊泡融合和启动中的munc18和munc13将作为模型系统研究，以了解 一般机制囊泡对接，启动和融合。对于这些研究，我们将结合高度创新的 在我的实验室开发的实验和计算方法以阐明纳米力学 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 微生物的电化学检测器阵列将用于实验询问特定的分子 与细胞和重构系统中融合孔形成相关的相互作用和构象变化。 分子动力学模拟将补充实验研究以解释 实验数据并指导实验设计。机械部分的各种晶体结构 已经确定，可以提供很高的原子分辨率，但它们代表静态结构。到 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，启动，融合和释放。我们最终的愿景是获得现实的分子电影 融合机的作用，深入了解囊泡启动和融合孔的机制 形成。这项研究还将提高我们对相关细胞内贩运融合的理解 事件以及使用密切相关的融合机制的病毒进入。 。