Nucleation and dynamics of exocytotic fusion pores

描述（由申请人提供）：胞吐作用是神经递质和激素释放的基础。在神经元中，包装有神经递质的突触囊泡（SV）与质膜融合以释放它们的内容物，这些内容物通过突触间隙被感觉到。这一过程受到严格调控：在动作电位到达后，游离钙浓度的局部增加刺激释放。激素通过含有激素的分泌颗粒（SG）与质膜融合，以类似的方式使用一些相同的蛋白质机制释放。SV或SG与质膜之间的初始连接是一个小孔（约1 nm宽），可以在永久关闭（瞬时或吻和跑融合）或完全扩张之前连续打开和关闭。细胞类型之间（孔开放时间跨度约为100 μs至10 μ s）和同一细胞内（一些孔闪烁，一些突然扩张）的行为存在很大差异。孔闪烁是由生理输入，如刺激强度，与重要的后果是什么释放（只有小货物可以通过小孔逃逸），在什么时间过程中，以及如何胞吐耦合到内吞。尽管融合孔在调节神经递质和激素释放方面具有根本的重要性，但关于控制孔成核和动力学的机制知之甚少。这主要是由于难以研究具有明确定义的蛋白质和膜组分的重构系统中的融合孔，所述蛋白质和膜组分将允许分离每个组分的作用。在过去的15年里，由胞吐SNARE蛋白及其调节因子介导的融合已经被重建和研究。然而，现有的方法不能解析单个重构融合孔，并且不能以足够的时间分辨率跟踪孔动力学。我们的目标是（1）设计新的实验方法，使探测机制的成核和闪烁的胞吐融合孔。结合电生理学方法、单粒子荧光、微加工装置和人工 双层技术我们将开发体外检测方法，可以直接，同时监测单孔闪烁和脂质混合;计数蛋白质数量和/或探测蛋白质-蛋白质相互作用;控制膜曲率和张力。使用这些测定，我们将（2）确定控制SNARE介导的融合孔的成核和动力学的因素。我们将解决膜力学和融合蛋白的动力学如何共同决定融合所需的SNARE复合物的数量。此外，我们将量化膜张力的作用，模仿细胞骨架，曲率和突变孔闪烁和扩张的约束。这些基础研究将促进我们对神经递质和激素释放如何调节的理解，并对人类健康产生长期的潜在影响。

DESCRIPTION (provided by applicant): Exocytosis underlies neurotransmitter and hormone release. In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. This process is tightly regulated: release is stimulated by a local increase in the free calcium concentration following the arrival of an action potential. Hormones are released in a similar fashion using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial connection between a SV or SG and the plasma membrane is a small pore (~1 nm wide) that can open and close in succession before either closing permanently (transient, or kiss-and-run fusion) or dilating fully. There is large variabiliy in behavior 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 fundamental importance of fusion pores in regulating neurotransmitter and hormone 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 component. Fusion mediated by exocytotic SNARE proteins and their regulators has been reconstituted and studied for the past 15 years. However, existing methods are not able to resolve single reconstituted fusion pores and follow pore dynamics with sufficient time resolution. We aim (1) to engineer novel experimental approaches to enable probing mechanisms of nucleation and flickering of exocytotic fusion pores. Combining electrophysiological methods, single-particle fluorescence, microfabricated devices, and artificial bilayer technologies we will develop in vitro assays that allow direct, simultaneous monitoring of single pore flickering and lipid mixing; counting protein numbers and/or probing protein-protein interactions; and controlling membrane curvature and tension. Using these assays, we will then (2) determine factors that govern nucleation and dynamics of SNARE- mediated fusion pores. We will resolve how membrane mechanics and the dynamics of fusion proteins together determine the number of SNARE complexes required for fusion. Further, we will quantify the roles of membrane tension, constraints mimicking the cytoskeleton, curvature, and mutations on pore flickering and expansion. 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.