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胞吐作用的重要晚期事件，这是内分泌，外分泌和神经系统中细胞间通讯的基础过程。研究将集中在围绕质膜和邻近分泌颗粒的体积上。潜在的假设是，紧邻质膜的颗粒行为和相互作用受到高度调节，在决定分泌反应中很重要。颗粒向质膜运动和质膜对接步骤的精确性质已被推测，但尚未直接研究。我们处于一个独特的位置来填补这一空白。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）来观察活细胞中紧邻质膜的颗粒。我们首次从移动颗粒膜蛋白的角度描述了融合事件，并发现颗粒运动受胞外分泌的两种调节剂，ATP和微摩尔Ca2+的调节。我们开发了强大的分析和生化工具来研究颗粒和质膜在质膜附近的运动。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外活动相关的质膜动力学，并开发了分子内FRET探针来直接检测活细胞中胞外活动的SNARE相互作用。我们将把这些进展与一些新的，但建立良好的基于tirfm的光学技术一起应用于研究染色质细胞：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，d)胞吐前和胞吐过程中胞吐部位的SNARE复合物形成。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。研究结果将对理解神经系统、内分泌系统和心血管系统在健康和疾病中的作用具有重要意义。

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.