Nucleation and dynamics of exocytotic fusion pores

项目概要： 在神经元中，包裹有神经递质的突触囊泡（SV）与质膜融合， 它们的内容通过突触间隙被感知。释放是由局部钙的增加引发的 去极化后的浓度。释放动力学包括同步阶段（钙释放后0.1-5 ms）。 仰角），以及慢得多的异步阶段（~100 ms）。膜融合是如何被触发的 如何快速地调节动力学还没有很好地理解。荷尔蒙也以类似的方式释放， 具有多个动力学阶段，使用一些相同的蛋白质机制，通过融合含有激素的 分泌颗粒（SG）与质膜。融合之间的初始~1-3 nm宽的连接 被称为融合孔的分隔间，在永久关闭之前， （短暂融合）或完全扩张。细胞类型之间存在很大的差异（孔开放时间跨度约为100 µs 到10秒）和在同一细胞内（一些孔闪烁，一些突然扩张）。孔隙闪烁由以下因素调节 生理输入，例如刺激强度，具有关于释放的重要后果（仅 小货物可以通过小孔逃逸），在什么时间过程中，以及胞吐作用如何与 内吞作用尽管融合孔在调节释放中的重要性，但关于融合孔的作用， 控制孔隙成核和动力学的机制。这主要是由于研究聚变的困难 具有明确蛋白质和膜组分的重构系统中的孔， 每个人的角色。由胞吐SNARE蛋白及其调节剂介导的融合已经被重建， 研究了20年。然而，可以监测单个重构融合孔与亚细胞的方法， MS的分辨率有所欠缺。在上一个周期，我们首次开发了这种方法， 探索了调节单独由SNARE诱导的融合孔的机制。在下一个周期，我们建议使用 这些方法（1）确定SNARE相互作用蛋白质在成核和动力学中的作用， 熔融孔和小孔的选择性。为了描述胞吐作用的钙传感器 和释放机制的其他基本成分有助于融合孔特性，我们将使用 电生理学，纳米盘，工程细胞，单粒子荧光显微镜，微加工 装置和人造双层。我们还将表征小融合孔对离子的选择性， 与确定瞬时聚变事件期间释放的物质高度相关。然后我们将（2）解剖 有助于钙触发胞吐的动力学机制。方法将是 增强以允许使用微灌注或紫外线闪光光解快速（~1 ms）[Ca 2 +]升高。这些 将能够定义不同的传感器和释放复合物如何调节释放动力学，以及是什么决定了 高钙协同释放。这些基础研究将促进我们理解 神经递质和激素的释放受到调节，对人类健康有长期的潜在影响。

PROJECT SUMMARY: In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. Release is triggered by a local increase in the calcium concentration following depolarization. Release kinetics comprise a synchronous phase (0.1-5 ms after calcium elevation), and a much slower asynchronous phase (~100 ms). How membrane fusion can be triggered so rapidly and how the kinetics are regulated are not well understood. Hormones are released in a similar fashion, with multiple kinetic phases, using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial ~1-3 nm wide connection between the fusing compartments, called the fusion pore, can flicker open-closed in succession before either closing permanently (transient fusion) or dilating fully. There is large variability between cell types (pore open times span ~100 µs to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the importance of fusion pores in regulating release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each. Fusion mediated by exocytic SNARE proteins and their regulators has been reconstituted and studied for the past 20 years. However, methods that can monitor single reconstituted fusion pores with sub- ms resolution have been lacking. During the last cycle, we developed such methods for the first time, and explored mechanisms regulating fusion pores induced by SNAREs alone. In the next cycle, we propose to use those methods to (1) define the role of SNARE-interacting proteins in nucleation and dynamics of fusion pores and the selectivity of small pores. To characterize how the calcium sensors for exocytosis and other essential components of the release machinery contribute to fusion pore properties, we will use electrophysiology, nanodiscs, engineered cells, single-particle fluorescence microscopy, microfabricated devices, and artificial bilayers. We will also characterize selectivity of small fusion pores for ions, which is highly relevant for determining what is released during transient fusion events. We will then (2) dissect mechanisms contributing to kinetics of calcium-triggered exocytosis. The approaches will be augmented to allow rapid (~1 ms) [Ca2+] elevation using microperfusion or ultraviolet flash photolysis. These will enable defining how different sensors and release complexes regulate release kinetics and what determines the high calcium-cooperativity of release. These fundamental studies will advance our understanding of how neurotransmitter and hormone release are regulated, with potential impact on human health in the long term.