The Plasma Membrane-Granule Interface in Exocytosis

胞吐作用中的质膜-颗粒界面

• 批准号: 7574406
• 负责人: RONALD W HOLZ
• 金额: $ 33.21万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-15 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7574406
• 关键词: Acute; Behavior; Biochemical; Cardiovascular system; Cell membrane; Cells; Chromaffin Cells; Chromaffin granule; Complex; Confocal Microscopy; Cytoplasmic Granules; Disease; Docking; Endocrine; Event; Exocytosis; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Grant; Health; Hormones; Image; Individual; Interphase Cell; Kinetics; Knowledge; Life; Measures; Membrane; Membrane Proteins; Motion; Movement; Nature; Nervous system structure; Neurologic; Neurotransmitters; Optics; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Physiological; Positioning Attribute; Process; Progress Reports; Regulation; Regulation of Exocytosis; Relative (related person); Research; SNAP receptor; Secretory Vesicles; Site; Techniques; Time; Vesicle; base; intercellular communication; novel; response; syntaxin; tool

The goal of the proposed research is to understand the late events important for exocytosis, a process that underlies intercellular communication in the endocrine, exocrine and nervous systems. Studies will focus on the volume encompassing the plasma membrane and immediately adjacent secretory granules. The underlying hypothesis is that granule behavior and interactions immediately adjacent to the plasma membrane are highly regulated and important in determining the secretory response. The precise nature of the movement of granules to the plasma membrane and the plasma membrane docking step have been surmised, but have not been directly investigated. We are in a unique position to fill this gap. In the previous grant period we applied the powerful optical technique, total internal reflection fluorescence microscopy (TIRFM) to visualize granules immediately adjacentto the plasma membrane in living cells. We described for the first time fusion events from the point of view of a mobile granule membrane protein and discovered that granule motion is regulated by two regulators of exocytosis, ATP and micromolar Ca2+. We developed powerful analytical and biochemical tools to investigate granule and plasma membrane motion adjacent to the plasma membrane with TIRFM. We have also measured plasma membrane motions in resting cells using TIRFM, increased plasma membrane dynamics associated with individual exocytotic events using confocal microscopy, and have developed an intramolecular FRET probe to directly detect exocytotic SNARE interactions in living cells. We will apply these advances together with several novel, but well founded, TIRFM-based optical techniques to investigate in chromaffin cells: a) the regulation of granule motion, b) granule interaction with the plasma membrane, c) plasma membrane dynamics during exocytosis, and d) SNARE complex formation at exocytotic sites before and during exocytosis. These studies will further our knowledge of the key function in intercellular communication-the release of hormones or neurotransmitters. The results will be of fundamental importance in the understanding of neurological, endocrine and cardiovascular systems in health and disease.

拟议研究的目的是了解对胞吐作用很重要的后期事件，这一过程 内分泌，外分泌和神经系统中细胞间交流的基础。研究将重点 包含质膜和紧邻分泌颗粒的体积。这 潜在的假设是颗粒行为和相互作用紧邻等离子体 膜受到高度调节，对于确定分泌反应很重要。的确切本质 颗粒向质膜和质膜对接步骤的运动已经 推测，但尚未直接调查。我们处于填补这一空白的独特位置。在上一个 授予期我们应用了强大的光学技术，总内反射荧光显微镜 （TIRFM）可视化颗粒紧邻活细胞中的质膜。我们描述了 从移动颗粒膜蛋白的角度，第一次融合事件，发现 颗粒运动由两个胞吐作用，ATP和微摩尔Ca2+的调节剂调节。我们开发了 强大的分析和生化工具研究颗粒和质膜运动 带有TIRFM的质膜。我们还测量了静息细胞中的质膜运动 使用TIRFM，使用与单个胞吐事件相关的质膜动力学增加了 共聚焦显微镜，并开发了分子内的FRET探针直接检测胞吐 活细胞中的圈圈相互作用。我们将与几本小说一起运用这些进步，但是 建立的基于TIRFM的光学技术可在铬蛋白细胞中进行研究：a）颗粒的调节 运动，b）与质膜的颗粒相互作用，c）胞吐作用期间的质膜动力学， d）在胞吐作用前和期间，在胞吐部位的圈型复合物形成。这些研究将进一步 我们对细胞间交流中关键功能的了解 - 激素的发布或 神经递质。结果将在理解神经系统方面具有至关重要的重要性 健康和疾病的内分泌和心血管系统。