DYNAMICS OF CHROMAFFIN GRANULE SECRETION

描述(摘自申请人的摘要) 这个项目考察了这一过程中涉及的生物物理过程。 嗜铬细胞分泌的含肾上腺素颗粒。这个 总体假设是嗜铬颗粒向血浆运动 膜对于持续的分泌反应是必不可少的，而且 对这一运动的调节为细胞提供了一种控制 分泌物。除了检验这一假设，该项目还拥有 推出和展示新光纤的总体主要目标 应该在细胞生物学中找到普遍用途的显微技术。 这一结果不仅应该对理解 肾上腺髓质分泌一种影响全身的主要激素 身体，但也为了(A)了解分泌的机制 具有化学递质的其他细胞(例如，神经性细胞 系统)和(B)证明新型荧光显微镜的实用性 选择性探查膜-近端突起的技术。这 该项目提议使用几种开创性技术的新变种 在PI的实验室：全内反射荧光(TIRF) 显微镜-同时提供实时成像和 足够薄的光学切片。TIRF的专业知识是 结合合作调查员和合作者的专业知识 瞬时转染法荧光标记颗粒表达 颗粒特异性绿色荧光蛋白(GFP)融合蛋白。这个 具体目标分为五个相关领域。(A)颗粒运动 (在几纳米尺度上)靠近质膜(在 几十纳米)将在速度方面进行表征， 距离、单向性、横向运动和人口类别 在颗粒物中。(B)这些运动参数将与 细胞的基本和分泌状态。尤其是 颗粒中分泌途径的特异性蛋白(SNARES和Rab3a) 运动和对接将在预稳定化的ELLS中进行检查。(C) 细胞质膜的亚显微形态变化 分泌物将通过新的灵敏的光学技术进行检查。(D) 将检测颗粒内部的有效粘度 通过偏振的TIR荧光光漂白技术。如果颗粒剂 内部是凝胶状的，那么整个颗粒的翻滚速度就会 作为区分自由颗粒和对接颗粒的一种手段进行测量。(E) 丝状蛋白和局部钙瞬变的变化一致 颗粒融合将在初步研究中进行检查。

DESCRIPTION (from applicant's abstract) This project examines the biophysical process involved in the process of secretion of adrenalin-containing granules from chromaffin cells. The overall hypothesis is that chromaffin granule motion toward the plasma membrane is essential for a sustained secretory response, and that regulation of this motion provides the cell with a means to control secretion. In addition to testing that hypothesis, this project also has an overall major goal of introducing and demonstrating new optical microscopy techniques that should find general utility in cell biology. The results should have significance not only to understanding how the adrenal medulla gland secretes a major hormone that affects the whole body, but also for (a) understanding the mechanisms of secretion in other cells with chemical transmitters (e.g., cells in the neurological system) and (b) proving the utility of novel fluorescence microscope techniques that selectively probe membrane-proximal processes. This project proposes to use several new variations of a technique pioneered in the PI's lab: total internal reflection fluorescence (TIRF) microscopy - to simultaneously provide both real time imaging and sufficiently thin optical sectioning. The expertise with TIRF is combined with the co-investigator's and collaborator's expertise in fluorescence-marking the granules by transient transfection to express a granule-specific green fluorescent protein (GFP) fusion protein. The specific aims are grouped into five related areas. (a) Granule motions (on the scale of several nanometer) near the plasma membrane (within tens of nanometers) will be characterized with respect to speed, distance, unidirectionality, lateral motion, and population classes among granules. (b) These motion parameters will be compared between the basal and the secretory state of the cells. In particular the role of specific proteins of the secretory pathway (SNAREs and Rab3a) in granule motion and docking will be examined in premeabilized ells. (c) Submicroscopic morphological changes in the plasma membrane during secretion will be examined by new and sensitive optical techniques. (d) The effective viscosity of the inside of the granule will be examined by a polarized TIR fluorescence photobleaching technique. If the granule is internally gel-like, then tumbling rates of the whole granule will be measured as a means to distinguish free from docked granules. (e) Changes in filamentous proteins and in local Ca2+ transients coincident with granule fusion will be examined in a preliminary study.