The Plasma Membrane-Granule Interface in Exocytosis

胞吐作用中的质膜-颗粒界面

• 批准号: 7363607
• 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/7363607
• 关键词: Acute; Behavior; Biochemical; Cardiovascular system; Cell membrane; Cells; Chromaffin Cells; Chromaffin granule; Complex; Confocal Microscopy; Cytoplasmic Granules; Disease; Disruption; 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; Numbers; 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.

拟议研究的目标是了解对胞吐作用重要的晚期事件，该过程 是内分泌、外分泌和神经系统中细胞间通讯的基础。研究将集中在 包围质膜和紧邻的分泌颗粒的体积。的 基本假设是紧邻等离子体的颗粒行为和相互作用 膜是高度调节的，在决定分泌反应中很重要。的确切性质 颗粒向质膜的移动和质膜对接步骤已经被 猜测，但没有直接调查。我们处于填补这一空白的独特地位。上一 授权期间，我们应用了强大的光学技术--全内反射荧光显微镜 （TIRFM），以显示活细胞中紧邻质膜的颗粒。我们描述了 第一次从移动的颗粒膜蛋白的角度融合事件，并发现， 颗粒运动受胞吐作用的两种调节剂ATP和微摩尔Ca 2+调节。我们开发 强大的分析和生物化学工具，以研究颗粒和质膜运动相邻 用TIRFM对质膜进行了观察。我们还测量了静息细胞的质膜运动 使用TIRFM，增加与个体胞吐事件相关的质膜动力学， 共聚焦显微镜，并已开发出一种分子内FRET探针，直接检测胞吐 活细胞中的陷阱相互作用。我们将把这些进展与几个新颖的，但很好地 基于TIRFM的光学技术在嗜铬细胞中研究：a）颗粒的调节 运动，B）颗粒与质膜的相互作用，c）胞吐期间的质膜动力学， 和d）在胞吐作用之前和期间在胞吐作用位点处形成SNARE复合物。这些研究将进一步 我们对细胞间通讯的关键功能-激素的释放或 神经传递素这些结果将对理解神经学， 内分泌和心血管系统的健康和疾病。