New optical approaches to study secretory granule structure and function

描述(由申请人提供)：我们的研究集中在胞吐途径中发生在质膜-细胞质界面高度专门化领域的事件。这一区域由全内反射荧光显微镜(TIRFM)成像，这是我们在研究中广泛使用的核心技术。这一建议是基于这样的假设，即颗粒腔内容物和颗粒膜蛋白的流动性特征塑造了分泌反应。这项提议将提供关于分泌颗粒在胞吐作用中的结构和功能的新的基本见解，并将提供单个颗粒的颗粒、腔和膜蛋白的旋转和平移流动性的第一个定量测量。管腔和颗粒膜蛋白以及单个颗粒的旋转和平移迁移率将通过TIRFM、偏振和FRAP的新组合来测量。该提案有几个相关目标：1)了解颗粒管腔的物理状态，并揭示管腔粘度在决定蛋白质和儿茶酚胺释放速率中的作用；2)确定颗粒膜蛋白的平移迁移率以及这种迁移率是否允许重新聚集到颗粒膜上的融合部位；3)确定单个颗粒的旋转迁移率，以便更好地确定融合前颗粒的拴住和/或笼养状态；以及4)确定融合前颗粒行程的增加是否反映了平移和旋转运动的组合，使颗粒能够与质膜‘滚动’进入融合有效的相互作用。 与公共健康相关：我们的研究重点是激素和神经递质从包括神经元在内的细胞中分泌的机制。这些过程是心血管、内分泌和神经系统功能的基础。这一过程(胞吐作用)是由储存的囊泡或颗粒与细胞(质膜)刺激融合而发生的。这项提议将建立新的、强大的光学技术来探索活细胞和分泌细胞中分泌颗粒的结构和功能。这些发现将有助于我们了解颗粒膜与细胞膜融合的机制以及控制颗粒内容物释放的因素。这些研究直接适用于健康所需的过程和疾病中的故障。

DESCRIPTION (provided by applicant): Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a core technique that we use extensively in our studies. This proposal is based upon the hypothesis that the mobility characteristics of granule lumenal contents and of granule membrane proteins shape the secretory response. The proposal will provide fundamental new insights concerning secretory granule structure and function in exocytosis and will provide the first quantitative measures of the rotational and translational mobility of granule lumenal and membrane proteins of individual granules. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization and FRAP. There are several related goals in the proposal: 1) to understand the physical state of the granule lumen and reveal the role of lumenal viscosity in determining the rates of protein and catecholamine release, 2) to determine the translational mobility of granule membrane proteins and whether the mobility permits recruitment to the fusion site on the granule membrane, 3) to determine the rotational mobility of individual granules in order to better define the tethered and/or caged state of the granules before fusion, and 4) and to determine whether the increase in granule travel immediately before fusion reflects a combination of translational and rotational motion that permits the granule to 'roll' into a fusion competent interaction with the plasma membrane. PUBLIC HEALTH RELEVANCE: Our research focuses on the mechanisms by which hormones and neurotransmitters are secreted from cells including neurons. These processes underlie the function of the cardiovascular, endocrine and nervous systems. The process (exocytosis) occurs by the stimulated fusion of a storage vesicle or granule with the cell (plasma) membrane. This proposal will establish new, powerful optical techniques to probe the structure and function of secretory granules in living and secreting cells. The findings will help us understand the mechanism of granule membrane fusion with the cell membrane and the factors which control release of granule contents. These studies have direct application to processes that are required for health and malfunction in disease.