Nucleation and dynamics of exocytotic fusion pores

胞吐融合孔的成核和动力学

• 批准号: 10376228
• 负责人: ERDEM KARATEKIN
• 金额: $ 36.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376228
• 关键词: Address; Affect; Affinity; Artificial Membranes; Binding; Biochemical; Biological Assay; Calcium; Cell fusion; Cell membrane; Cells; Charge; Communication; Complex; Coupled; Dependence; Docking; Electrophysiology (science); Endocrine; Endocytosis; Engineering; Event; Evolution; Exocytosis; Fluorescence Microscopy; Health; Hormones; Human; Individual; Ions; Kinetics; Lipids; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Methods; Molecular; Monitor; Neuroendocrine Cell; Neurons; Neurotransmitters; Phase; Physiological; Probability; Process; Property; Protein Isoforms; Proteins; Recycling; Regulation; Resolution; Role; SNAP receptor; Secretory Vesicles; Shapes; Speed; Synaptic Cleft; Synaptic Vesicles; System; Technology; Testing; Time; Vesicle; cell type; experience; experimental study; flash photolysis; microdevice; nanodisk; neurotransmitter release; particle; reconstitution; response; sensor; synaptotagmin I; target SNARE proteins; ultraviolet; vesicular SNARE proteins

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.

项目摘要： 在神经元中，用神经递质与质膜融合的突触蔬菜（SV）释放 他们在突触裂缝中感应的内容。释放是由钙的局部增加触发的 去极化后的浓度。释放动力学包括同步相（钙后0.1-5毫秒 海拔），异步相（〜100 ms）较慢。如何触发膜融合 迅速地以及如何调节动力学尚未得到充分理解。激素以类似的方式发布 使用多个动力学相，使用一些相同的蛋白质机制，通过融合含有荷酮的蛋白质机械 分泌颗粒（SG）和质膜。融合之间的初始〜1-3 nm宽连接 隔室称为融合孔，可以连续闪烁，然后永久关闭之前 （瞬态融合）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〜100 µs 到S）和同一细胞内的10s（有些毛孔闪烁，有些突然扩张）。孔闪烁是由 物理输入（例如模拟强度），对发布的内容产生重要后果（只有 小货物可以通过小毛孔逃脱），在什么时间过程中，以及胞吞作用的耦合 内吞作用。尽管融合孔在法规发布中很重要，但关于 控制孔成核和动力学的机制。这主要是由于研究融合的困难 具有明确定义的蛋白质和膜成分的重构系统中的孔，可以隔离 每个的作用。由外囊泡蛋白及其调节剂介导的融合已重组，并且 在过去的20年中进行了研究。但是，可以监视单个重构融合孔的方法 MS决议缺乏。在最后一个周期中，我们首次开发了此类方法， 探索了单独由鼻子引起的融合孔的机制。在下一个周期中，我们建议使用 那些（1）定义了核断相互作用蛋白在成核和动力学中的作用的方法 融合孔和小孔的选择性。为了表征胞吐作用的钙传感器 发行机械的其他基本组件有助于融合孔的性能，我们将使用 电生理学，纳米盘，工程细胞，单粒子荧光显微镜，微生物 设备和人造双层。我们还将表征离子小融合孔的选择性，即 与确定瞬态融合事件中发布的内容高度相关。然后我们将（2）解剖 促进钙触发胞吐作用动力学的机制。方法将是 增强以允许使用微灌注或紫外线闪光光解的快速（〜1 ms）[Ca2+]升高。这些 将启用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什么决定 释放的高钙合并性。这些基本研究将提高我们对 神经递质和马释释放受到调节，从长远来看对人类健康有潜在的影响。