Fast, SNARE-induced Single-Vesicle Fusion

神经系统疾病的名单是广泛的：阿尔茨海默氏症，帕金森氏症，ALS和MS，仅举一个例子。 几个这项工作的目标是了解神经元如何在分子水平上工作。神经元交流 通过释放神经递质通过质膜（PM）进入突触， 这个过程叫做胞吐作用。神经递质储存在小的分泌囊泡（直径约50 nm）中 停靠在PM。当神经元去极化时，Ca 2+流入PM的胞质侧，导致Ca 2 +-ATP酶活性降低。 释放涉及囊泡的脂质双层与囊泡的脂质双层的融合。 首相这种囊泡融合机制的许多关键组成部分的身份建立。不过小 在分子水平上已知这些成分如何协同工作以进行胞吐作用。中央 该组分是反式SNARE复合物，其包含锚定在细胞中的v-SNARE蛋白小突触蛋白（Syb）。 囊泡和锚定在PM上的t-SNARE蛋白syntaxin（Syx）和SNAP-25。Synaptotagmin (Syt)也锚定在囊泡中，可能是Ca 2+传感器，其明显地通过改变 蛋白质和脂质之间的结合关系。 融合机制是如此复杂，以至于从体内研究中得出的机制推断是不可靠的。 必然是间接的。我们和其他人一直在努力开发一个重建的模型系统， 忠实地捕捉在神经元中观察到的Ca 2+触发的融合。这样的系统可以非常直接地 通过一个接一个地增加或减少组件来研究许多关键的机械问题。我们现在有一个 允许直接观察单个v-SNARE囊泡在平面t-SNARE/脂质上的对接和融合的模型 双分子层的真实的时间通过宽场荧光显微镜。我们的是迄今为止唯一一个 在25毫秒的时间尺度上表现出依赖于SNARE的融合，接近自然界中的融合。这项工作的目标 是：通过优化蛋白质和脂质组分来确定固有的SNARE驱动的融合速率; 通过测量内容物释放和脂质混合时间尺度来了解融合孔本身的性质 以1-3 ms的分辨率;并通过将Syt引入到测定中来重新捕获Ca 2+触发。沿着， 这些实验准备回答各种各样的机械问题， 方式

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 frans-SNARE complex, comprising the v-SNARE protein synaptobrevin (Syb) anchored in the vesicle and the t-SNARE proteins syntaxin (Syx) and SNAP-25anchored 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 systemthat 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 theway, these experiments are poised to answer a wide variety of mechanistic questions in an unusually incisive manner.