CHROMAFFIN GRANULE MOVEMENT NEAR THE PLASMA MEMBRANE

描述(改编自申请者的摘要)：尽管知道的很多 关于嗜铬细胞的胞吐作用，对分泌物知之甚少。 最后融合前靠近质膜的颗粒运动 事件。该提案的一个关键方面是我们能够可视化染色质 实时的颗粒。心钠素与心钠素的融合蛋白 瞬时转基因表达的绿色荧光蛋白是 分类为嗜铬粒。利用TOTAL的光学技术 内反射，我们可以看到荧光嗜铬颗粒在 分布在1-2个胞浆颗粒直径内的活细胞 薄膜。我假设嗜铬颗粒的移动和放置 靠近质膜的是高度受调控的过程，有助于定义 颗粒可用于进行胞吐作用。这件事的重点是 建议是确定辅因子和蛋白质是否会影响 最终的分泌反应至少部分是通过控制这些 再融合事件。我的具体目标是：(1)确定 靠近质膜的嗜铬颗粒的位置和运动 在基础和分泌条件下，(2)确定关系 已知的ATP和Ca~(2+)对颗粒启动的影响 与质膜相邻的分泌和颗粒运动以及(3) 确定SNARES和Rab3a这两种分泌物特异性蛋白的作用 颗粒在质膜附近运动和放置的途径。

DESCRIPTION (Adapted from applicant's abstract): Although much is known about exocytosis in chromaffin cells, little is known about secretory granule movement close to the plasma membrane preceding the final fusion event. A key aspect of the proposal is our ability to visualize chromaffin granules in real time. A fusion protein of atrial natriuretic peptide and green fluorescent protein that is expressed by transient transfection is sorted to chromaffin granules. Using the optical technique of total internal reflection, we can visualize fluorescent chromaffin granules in living cells that are localized within 1-2 granule diameters of the plasma membrane. I hypothesize that movement and placement of chromaffin granules close to the plasma membrane are highly regulated processes that help define the availability of granules to undergo exocytosis. The focus of this proposal is to determine whether cofactors and proteins known to influence the final secretory response act at least in part by controlling these prefusion events. My specific aims are the following: (1) to determine the location and movement of chromaffin granules close to the plasma membrane under basal and secretory conditions, (2) to determine the relationship between the known effects of ATP and Ca2+ on granule priming before secretion and granule movement adjacent to the plasma membrane and (3) to determine the role of SNAREs and Rab3a, specific proteins of the secretory pathway in granule movement and placement adjacent to the plasma membrane.