New optical approaches to study secretory granule structure and function

描述（由申请人提供）：我们的研究重点是发生在质膜-细胞质界面高度专业化领域的胞吐途径中的事件。这个区域是极好的成像全内反射荧光显微镜（TIRFM），我们在我们的研究中广泛使用的核心技术。这一建议是基于这样的假设，即颗粒内腔内容物和颗粒膜蛋白质的流动性特征形成分泌反应。该建议将提供有关分泌颗粒的结构和功能的胞吐作用的基本新的见解，并将提供第一个定量措施的颗粒内腔和膜蛋白的单个颗粒的旋转和平移的流动性。内腔和颗粒膜蛋白和单个颗粒的旋转和平移流动性将通过TIRFM，极化和FRAP的新组合来测量。提案中有几个相关的目标：1）了解颗粒腔的物理状态并揭示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移动性以及该移动性是否允许募集到颗粒膜上的融合位点，3）确定单个颗粒的旋转移动性以便更好地限定颗粒在融合之前的束缚和/或笼状状态，和4）并确定在融合之前颗粒移动的增加是否反映了平移和旋转运动的组合，颗粒“滚动”成与质膜的融合能力相互作用。

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.