Nucleation and dynamics of exocytotic fusion pores

项目总结： 在神经元中，包裹着神经递质的突触小泡(SV)与质膜融合释放 它们的内容通过突触缝隙被感觉到。释放是由局部钙的增加触发的 去极化后的浓度。释放动力学包括一个同步阶段(钙化后0.1-5ms 高度)，以及慢得多的异步阶段(~100毫秒)。膜融合是如何被触发的 快速以及如何调控动力学还不是很清楚。荷尔蒙以类似的方式释放， 通过融合含有以下成分的激素，使用一些相同的蛋白质机制，具有多个动力学相 分泌颗粒(SG)与质膜。熔断器之间的初始~1-3 nm宽连接 被称为融合孔的隔室可以在永久关闭之前连续闪烁开启和关闭 (瞬时融合)或完全扩张。不同细胞类型之间有很大的差异(孔道开放时间跨度约为100uM S 到S的十分之一)和同一细胞内(有些毛孔闪烁，有些突然扩张)。毛孔闪烁由以下因素调制 生理输入，如刺激强度，对释放的内容有重要影响(仅 小货物可以通过小孔逃逸)，在什么时间进程中，以及胞吐作用是如何耦合的 内吞作用。尽管融合孔在调节释放方面很重要，但人们对其了解甚少。 控制气孔成核和动力学的机制。这主要是由于研究核聚变的困难。 重组系统中的孔，具有明确定义的蛋白质和膜组件，可以分离 每个人的角色。胞外SNARE蛋白及其调控因子介导的融合已重组并 在过去的20年里一直研究。然而，可以监测单个重组融合孔的方法 微软的分辨率一直欠缺。在上一个周期中，我们第一次开发了这样的方法，并且 探索了单独由圈套诱导的融合孔的调节机制。在下一个周期中，我们建议使用 这些方法(1)确定SNARE相互作用蛋白在核化和动力学中的作用 融合毛孔和小毛孔的选择性。为了描述细胞吐出的钙感受器 和其他有助于释放机械关键部件的熔融气孔特性，我们将使用 电生理学、纳米盘、工程细胞、单粒子荧光显微镜、微型制造 设备和人造双层。我们还将表征小孔对离子的选择性，这是 与确定在瞬变聚变事件期间释放什么高度相关。然后我们将(2)解剖 钙离子引发胞吐作用的动力学机制。这些方法将是 通过微灌流或紫外线闪光光解，使钙离子迅速升高(~1ms)。这些 将能够定义不同的传感器和释放复合体如何调节释放动力学，以及什么决定 高钙协同释放性。这些基础性研究将促进我们对 神经递质和激素的释放受到调节，从长远来看，可能会对人类健康产生影响。

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.