Fast, SNARE-induced Single-Vesicle Fusion

描述(由申请者提供)：神经疾病的清单很广泛：阿尔茨海默氏症、帕金森氏症、肌萎缩侧索硬化症和多发性硬化症，仅举几例。这项工作的目标是了解神经元在分子水平上是如何工作的。神经元通过质膜(PM)向突触释放神经递质来相互通信，这一过程被称为胞吐作用。神经递质储存在停靠在PM处的小分泌囊泡(直径约50 nm)中当神经元去极化时，钙离子流入PM的胞浆侧，在不到1毫秒的时间内引起钙离子触发的胞吐作用。释放包括囊泡的脂双层与PM的脂双层的融合确定了这种囊泡融合机的许多关键部件的身份。然而，在分子水平上，人们对这些成分如何共同作用进行胞吐作用知之甚少。一个中心成分是反式SNARE复合体，由锚定在小泡中的v-SNARE蛋白Synaptobrevin(Syb)和锚定在PM中的t-SNARE蛋白Synaxin(Syx)和SNAP-25组成突触素(Synaptopagmin，SYT)也定位在囊泡中，可能是钙离子感受器，通过改变蛋白质和脂类之间的结合关系来触发融合。 融合机制是如此复杂，从活体研究中得出的机制推论必然是间接的。我们和其他人一直在努力开发一种重组的模型系统，以忠实地捕获在神经元中观察到的钙离子触发的融合。这样的系统可以通过一个接一个地增加或减少部件来非常直接地研究许多关键的机械问题。我们现在有了一个模型，可以通过宽场荧光显微镜实时直接观察单个v-SNARE囊泡在平面t-SNARE/脂质双层上的对接和融合。到目前为止，我们的体外系统是唯一一个在25毫秒的时间尺度上表现出依赖圈套的融合，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肪成分来确定内在的陷阱驱动融合速度；通过测量1-3ms分辨率的内容释放和脂质混合时间来了解融合孔本身的性质；以及通过将SYT引入检测来重新捕获钙触发。在此过程中，这些实验准备以一种异常犀利的方式回答各种各样的机械论问题。

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.