The Plasma Membrane-Granule Interface in Exocytosis

这项拟议的研究的目标是了解对胞吐作用重要的晚期事件，这一过程 是内分泌、外分泌和神经系统细胞间通讯的基础。研究将集中在 围绕质膜和紧邻的分泌颗粒的体积。这个 潜在的假设是，颗粒的行为和相互作用紧邻血浆 膜是高度调节的，在决定分泌反应中很重要。它的确切性质 颗粒向质膜的运动和质膜对接步骤 猜测，但尚未直接调查。我们在填补这一空白方面处于独特的地位。在上一次 授权期内，我们应用了强大的光学技术--全内反射荧光显微镜 (TIRFM)显示活细胞中紧邻质膜的颗粒。我们描述了 首次从可移动颗粒膜蛋白的角度进行融合，发现 颗粒运动受胞吐作用的两种调节剂--三磷酸腺苷和微摩尔钙离子的调节。我们开发了 强大的分析和生化工具来研究邻近的颗粒和质膜运动 质膜用TIRFM。我们还测量了静息细胞的质膜运动。 使用TIRFM，增加与单个胞吐事件相关的质膜动力学 共聚焦显微镜，并开发了一种分子内FRET探针，可以直接检测胞吐 诱捕活细胞中的相互作用。我们将把这些进步与几部小说结合起来，但很好 基于TIRFM的嗜铬细胞光学技术研究：a)颗粒的调节 运动，b)颗粒与质膜相互作用，c)胞吐过程中的质膜动力学， 以及d)在胞吐之前和期间在胞吐部位形成圈套复合体。这些研究将进一步 我们对细胞间通讯的关键功能的了解--荷尔蒙或 神经递质。这些结果将对理解神经学， 健康和疾病中的内分泌和心血管系统。

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.