Fast, SNARE-induced Single-Vesicle Fusion

描述（由申请人提供）：神经系统疾病的列表很广泛：阿尔茨海默氏症，帕金森症，ALS和MS，仅举几例。这项工作的目标是了解神经元是如何在分子水平上工作的。神经元通过质膜（PM）向突触释放神经递质，这一过程被称为胞吐（exocytosis）。神经递质储存在停靠在PM的小分泌囊泡（直径约50 nm）中。当神经元去极化时，Ca2+流入PM的细胞质侧，在不到1ms的时间内引起Ca2+触发的胞外分泌。释放涉及囊泡的脂质双分子层与PM的脂质双分子层融合。建立了这种囊泡融合机制的许多关键部件的身份。然而，在分子水平上，对这些成分如何协同工作以进行胞吐作用知之甚少。一个核心成分是trans-SNARE复合物，包括锚定在囊泡中的v-SNARE蛋白synaptobrevin （Syb）和锚定在PM中的t-SNARE蛋白syntaxin （Syx）和SNAP-25。Synaptotagmin （Syt）也锚定在囊泡中，可能是Ca2+传感器，通过改变蛋白质和脂质之间的结合关系来触发融合。

DESCRIPTION (provided by applicant): The list of neurological diseases is extensive: Alzheimer's, Parkinson's, ALS, and MS, to name just a few. The goal of this work is to understand how neurons work at the molecular level. Neurons communicate with each other by releasing neurotransmitters through the plasma membrane (PM) into the synapse in a process called exocytosis. The neurotransmitter is stored in small secretory vesicles (~50 nm diameter) docked at the PM. When a neuron depolarizes, an influx of Ca2+ to the cytosolic side of the PM causes Ca2+- triggered exocytosis in less than 1 ms. Release involves fusion of the lipid bilayer of the vesicle with that of the PM. The identity of many key components of this vesicle fusion machinery is established. However, little is known at the molecular level about how the components work together to carry out exocytosis. A central component is the trans-SNARE complex, comprising the v-SNARE protein synaptobrevin (Syb) anchored in the vesicle and the t-SNARE proteins syntaxin (Syx) and SNAP-25 anchored in the PM. Synaptotagmin (Syt), also anchored in the vesicle, is probably the Ca2+ sensor that evidently triggers fusion by altering binding relationships among proteins and lipids. The fusion machinery is so complex that mechanistic inferences drawn from in vivo studies are necessarily indirect. We and others have been working to develop a reconstituted model system that faithfully captures the Ca2+-triggered fusion observed in neurons. Such a system would enable very direct study of many key mechanistic questions by adding or subtracting components one by one. We now have a model that allows direct observation of single v-SNARE vesicle docking and fusion on a planar t-SNARE/lipid bilayer in real time by widefield fluorescence microscopy. Ours is the only in vitro system thus far that exhibits SNARE-dependent fusion on a 25-ms time scale, approaching that in nature. The goals of this work are: to determine the intrinsic SNARE-driven rate of fusion by optimizing protein and lipid components; to learn about the nature of the fusion pore itself by measuring the contents release and lipid mixing time scales with 1-3 ms resolution; and to recapture Ca2+ triggering by introducing Syt into the assay. Along the way, these experiments are poised to answer a wide variety of mechanistic questions in an unusually incisive manner.