DYNAMICS OF CHROMAFFIN GRANULE SECRETION

描述（来自申请人摘要） 这个项目研究了这个过程中所涉及的生物物理过程 嗜铬细胞分泌含肾上腺素的颗粒。的 总体假设是嗜铬颗粒向血浆运动 膜对于持续的分泌反应至关重要，并且 这种运动的调节为细胞提供了一种控制 分泌物除了测试这个假设，这个项目还 一个总体的主要目标，介绍和展示新的光学 显微镜技术，应该找到细胞生物学的普遍效用。 研究结果不仅对理解人类如何 肾上腺髓质分泌一种主要的激素， 身体，而且（a）了解分泌机制， 其它具有化学递质的细胞（例如，神经细胞 系统）和（B）证明新型荧光显微镜的实用性 选择性探测近膜突起的技术。这 一个项目建议使用几种新的技术变体， PI实验室：全内反射荧光（TIRF） 显微镜-同时提供真实的时间成像和 足够薄的光学切片。TIRF的专业知识是 结合合作研究者和合作者的专业知识， 通过瞬时转染荧光标记颗粒以表达 一种颗粒特异性绿色荧光蛋白（GFP）融合蛋白。的 具体目标分为五个相关领域。(a)颗粒运动 (on几个纳米的尺度）靠近质膜（内 几十纳米）将以速度为特征， 距离、单向性、横向运动和人口类别 在颗粒之间。(b)这些运动参数将在 细胞的基础状态和分泌状态。特别是， 颗粒中分泌途径的特异性蛋白（SNARE和Rab 3a） 运动和对接将在预先确定的ELL中检查。（c）第（1）款 细胞膜的亚显微形态学变化 将通过新的和灵敏的光学技术检查分泌物。（d）其他事项 将检查颗粒内部的有效粘度 通过偏振TIR荧光光漂白技术。如果颗粒 内部呈凝胶状，则整个颗粒的翻滚速率将 作为区分游离颗粒和停靠颗粒的手段进行测量。（五） 丝状蛋白和局部Ca 2+瞬变的变化一致 将在初步研究中检查颗粒融合。

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.