The Plasma Membrane-Granule Interface in Exocytosis

描述(申请人提供)：拟议研究的目标是了解对胞吐作用重要的晚期事件，胞吐作用是内分泌、外分泌和神经系统细胞间通讯的基础过程。研究将集中在围绕质膜和紧邻的分泌颗粒的体积。潜在的假设是，颗粒行为和紧邻质膜的相互作用受到高度调控，在决定分泌反应方面很重要。颗粒运动到质膜和质膜对接步骤的确切性质已经被推测，但尚未被直接调查。我们在填补这一空白方面处于独特的地位。在之前的资助期间，我们应用了强大的光学技术-全内反射荧光显微镜(TIRFM)来显示活细胞中紧邻质膜的颗粒。我们首次从可移动的颗粒膜蛋白的角度描述了融合事件，并发现颗粒运动受到胞吐作用的两个调节因子--ATP和微摩尔钙的调节。我们开发了强大的分析和生化工具，用TIRFM研究质膜附近的颗粒和质膜运动。我们还使用TIRFM测量了静息细胞中的质膜运动，使用共聚焦显微镜增加了与单个胞吐事件相关的质膜动力学，并开发了一种分子内FRET探针来直接检测活细胞中的胞吐陷阱相互作用。我们将应用这些新技术和基于TIRFM的光学技术来研究嗜铬细胞：a)颗粒运动的调节，b)颗粒与质膜的相互作用，c)胞吐过程中的质膜动力学，以及d)在胞吐之前和期间胞吐部位的SNARE复合体的形成。这些研究将进一步加深我们对细胞间通讯中的关键功能--激素或神经递质的释放的了解。这一结果将对了解健康和疾病中的神经、内分泌和心血管系统具有重要意义。

DESCRIPTION (provided by applicant): 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 adjacent to 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.