Architecture and control of vesicle fusion in excitable cells

可兴奋细胞中囊泡融合的结构和控制

• 批准号: 8558037
• 负责人: Justin Taraska
• 金额: $ 56.06万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8558037
• 关键词: Accounting; Architecture; Behavior; Biochemistry; Cell membrane; Cell surface; Cells; Clathrin; Color; Complex; Diffuse; Diffusion; Docking; Electron Microscopy; Electrophysiology (science); Endocytic Vesicle; Endocytosis; Exocytosis; Genetic; Goals; Heterogeneity; Image; Individual; Life; Location; Mammalian Cell; Maps; Measures; Membrane; Membrane Fusion; Methods; Microscopy; Modeling; Molecular; Nervous System Physiology; Nervous system structure; Neuroendocrine Cell; Neurosciences; Neurosecretory Systems; Neurotransmitters; PC12 Cells; Pathology; Peptides; Physiology; Protein Dynamics; Proteins; Recycling; Resolution; Role; Running; Signal Pathway; Site; Structure; System; Total Internal Reflection Fluorescent; Transcription Factor AP-2 Alpha; Vesicle; Work; acetylcholine transporter; cell growth regulation; cellular imaging; density; imaging modality; nano; small molecule; three dimensional structure; trafficking; ultra high resolution

Aim 1 Classical neuroscience has proposed two competing models for membrane fusion. In the first, vesicles completely merge with the plasma membrane, dispersing the entirety of their contents. This full fusion model of exocytosis predicts that vesicle contents will spill into the membrane and diffuse away from the site of fusion. In the second, vesicles transiently connect with the plasma membrane and release only a subset of their components. This kiss-and-run model predicts that the vesicle contents will remain within a vesicle cavity and then will be recaptured into the cell mostly intact. To determine which of these two models occurs in neuroendocrine cells, we have imaged single fluorescently-tagged vesicles in living PC12 cells with total internal reflection fluorescent microscopy (TIRF). By watching the diffusive behavior of vesicle components before, during, and after fusion, we will determine if (or which of) the two classical models of fusion fit triggered exocytosis of vesicles in PC12 cells. Through these studies we hope to measure the behavior of individual vesicles to determine the heterogeneity of vesicle fusion behaviors, their topology, relationships, and regulation by cellular signaling pathway and pathologies. Using two-color total internal reflection microscopy we have shown that the dominant mode of fusion for SLMV in PC12 cells is the full fusion model. As such, vesicle transporters, including the vesicular acetylcholine transporter, diffuse into the plasma membrane within seconds. A surprising finding of this work, however, is that the material that exits vesicles is rapidly captured on preformed clusters on the cell's surface. These clusters are composed of the endocytic protein clathrin and AP2 and inhibit the free diffusion of the transporter across the plasma membrane. To further investigate the density and topology of the structures responsible for capturing VAChT on the cell surface after exocytosis, we used three forms of ultra-high resolution imaging methods: 1) photo-activation localization microscopy, 2) ground state depletion (GSD) super-resolution imaging, and 2) electron microscopy. The combinations of these three methods have demonstrated that the density of endocytic clathrin-coated structures in PC12 cells is very high. The density approaches 2 structures per square micron. These structures are randomly distributed across the surface of the cell, and produce a network of endocytic nano-traps capable of rapidly capturing material that escapes from exocytic vesicles. We propose that this system can account for the rapidly recycling of vesicle material in highly excitable cells necessary for the continued function of the nervous and neuroendocrine system. Aim 2 Dozens of proteins control the docking, fusion, and then recapture of vesicles in excitable cells. The identity and functional roles of many of these proteins have been discovered through a combination of genetics, biochemistry, and electrophysiology. However, the architecture, structure, and structural dynamics of these proteins and their complexes have yet to be determined. In this aim we have begun to map the location, architecture, and dynamics of the proteins proposed to act during exocytosis and endocytosis in PC12 cells. To accomplish this, we are using a combination of high-throughput live cell imaging, super-resolution, and electron microscopy. Through this multi-modal approach, the location, and dynamics of individual proteins are being compared to the underlying cellular architecture. This will allow us to map the molecular architecture of the plasma membrane responsible for vesicle trafficking. These studies will determine the complex three dimensional structure of the exocytic and endocytic protein machinery in intact mammalian cells. Our studies are developing a general topographic map of the endocytic and exocytic machinery in living neuroendocrine cells at the plasma membrane. We hope that these studies will provide a network systems level analysis of the machinery reposing for vesicle fusion and recapture in cells of the nervous system.

目标1 经典神经科学提出了两种相互竞争的膜融合模型。在第一种情况下，囊泡与质膜完全融合，分散了全部内容物。这种胞吐作用的完全融合模型预测，囊泡内容物将溢出到膜中，并从融合部位扩散开来。在第二种情况下，囊泡瞬间与质膜连接，只释放其部分成分。该模型预测囊泡内容物将保留在囊泡腔内，然后被重新捕获到细胞内，基本完好无损。 为了确定这两种模式中的哪一种发生在神经内分泌细胞中，我们用全内反射荧光显微镜(TIRF)对活的PC12细胞中的单个荧光标记的小泡进行了成像。通过观察融合前、融合中和融合后囊泡成分的扩散行为，我们将确定两种经典融合模型是否(或哪一种)适合在PC12细胞中触发囊泡的胞吐。通过这些研究，我们希望通过测量单个小泡的行为来确定小泡融合行为的异质性、它们的拓扑结构、关系以及细胞信号通路和病理学的调节。 利用双色全内反射显微镜，我们证明了SLMV在PC12细胞中的主要融合模式是全融合模式。因此，囊泡转运体，包括囊泡乙酰胆碱转运体，在几秒钟内扩散到质膜。然而，这项工作的一个令人惊讶的发现是，退出囊泡的物质很快就会被细胞表面的预成簇捕获。这些簇由内吞蛋白clathrin和AP2组成，抑制转运蛋白在质膜上的自由扩散。 为了进一步研究胞吐后负责捕获细胞表面Vacht的结构的密度和拓扑结构，我们使用了三种形式的超高分辨率成像方法：1)光激活定位显微镜，2)基态耗竭(GSD)超分辨率成像，2)电子显微镜。这三种方法的结合表明，PC12细胞内的胞内笼状蛋白包被结构的密度很高。密度接近每平方微米2个结构。这些结构随机分布在细胞表面，并产生一个由胞内纳米陷阱组成的网络，能够快速捕获从胞外小泡中逸出的物质。我们认为，这个系统可以解释高度兴奋的细胞中囊泡物质的快速循环，这是神经和神经内分泌系统继续发挥功能所必需的。 目标2 数十种蛋白质控制着可兴奋细胞中囊泡的对接、融合和重新捕获。通过遗传学、生物化学和电生理学的结合，人们已经发现了许多这些蛋白质的身份和功能作用。然而，这些蛋白质及其复合体的结构、结构和结构动力学尚未确定。 在这个目标中，我们已经开始绘制在PC12细胞的胞吐和内吞过程中可能起作用的蛋白质的位置、结构和动态。为了实现这一点，我们使用了高通量活细胞成像、超分辨率和电子显微镜的组合。通过这种多模式方法，可以将单个蛋白质的位置和动态与潜在的细胞结构进行比较。这将使我们能够绘制负责囊泡运输的质膜的分子结构。这些研究将确定完整哺乳动物细胞胞外和胞内蛋白质结构的复杂三维结构。 我们的研究正在开发质膜上活的神经内分泌细胞的内分泌和外分泌机制的总体地形图。我们希望这些研究将为神经系统细胞中小泡融合和重新捕获的机制提供网络系统水平的分析。