The Plasma Membrane-Granule Interface in Exocytosis

描述（由申请人提供）：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、外分泌和神经系统中细胞间通讯的基础过程。研究将集中在包围质膜和紧邻的分泌颗粒的体积上。基本假设是紧邻质膜的颗粒行为和相互作用受到高度调节，并且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精确性质已经被推测，但尚未直接研究。我们处于填补这一空白的独特地位。在上一个资助期，我们应用了强大的光学技术，全内反射荧光显微镜（TIRFM），以可视化颗粒紧邻质膜在活细胞。我们首次从移动的颗粒膜蛋白的角度描述了融合事件，并发现颗粒运动受两种胞吐调节剂ATP和微摩尔Ca 2+的调节。我们开发了强大的分析和生化工具，研究颗粒和质膜运动与TIRFM质膜附近。我们还测量了质膜运动在静息细胞中使用TIRFM，增加质膜动态与个人胞吐事件使用共聚焦显微镜，并已开发出一种分子内FRET探针直接检测胞吐陷阱在活细胞中的相互作用。我们将应用这些进展与几个新的，但有充分依据的TIRFM为基础的光学技术，在嗜铬细胞中调查：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，和d）SNARE复合物形成在胞吐前和胞吐过程中的胞吐位点。这些研究将进一步加深我们对细胞间通讯的关键功能激素或神经递质的释放的认识。这些结果将对了解健康和疾病中的神经系统、内分泌系统和心血管系统具有根本的重要性。

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.