Supramolecular Dynamics of Secretory Granules Studied by New Optical Techniques

描述（由申请人提供）：尽管约10%的人类基因组由分泌蛋白组成，但对融合前和融合期间颗粒内腔和膜蛋白的物理状态知之甚少。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。我们的研究集中在胞吐途径中发生在质膜-细胞质界面的高度专业化领域的事件。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。该提议得到了强有力的初步结果的支持，表明不同的内腔蛋白，NPY-Cerulean和tPA-Cerulean的不同的迁移率。具有低得多的迁移率的tPA-Cerulean在单个颗粒融合时释放得慢得多，并且融合与慢得多的融合孔扩张相关。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，偏振，光漂白后荧光恢复（FRAP）和荧光相关光谱（FCS）的新组合进行测量。提案中有几个相关的目标：1）了解颗粒腔中蛋白质的迁移特性，并揭示它们在确定蛋白质和儿茶酚胺释放速率中的影响，2）确定颗粒膜蛋白质的翻译迁移率，以及迁移率是否允许它们通过扩散到颗粒膜上的融合位点而被募集，3）为了确定整个颗粒的旋转运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，四、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，允许颗粒“滚动”进入融合与质膜的有效相互作用，和5）首次使用超临界角发射测量颗粒与质膜之间的绝对距离，从而确定融合前颗粒与质膜接合的时间。此外，预计新技术将不仅适用于分泌细胞生物学，而且更普遍地适用于质膜-胞质溶胶界面处的众多细胞生物学问题。

DESCRIPTION (provided by applicant): Despite ~10% of the human genome being comprised of secretory proteins, little is known about the physical states of granule lumenal and membrane proteins before and during fusion. 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. 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. The proposal is supported by strong preliminary results demonstrating distinct mobilities of different lumenal proteins, NPY-Cerulean and tPA-Cerulean. tPA-Cerulean, which has a much lower mobility, is released much more slowly upon fusion of individual granules and the fusion is associated with much slower fusion pore expansion. The rotational and translational mobility of lumenal and granule membrane proteins and of individual granules will be measured by novel combinations of TIRFM, polarization, fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). There are several related goals in the proposal: 1) To understand the MOBILITY CHARACTERISTICS OF PROTEINS IN THE GRANULE LUMEN and reveal their influence in determining the rates of protein and catecholamine release, 2) To determine the translational MOBILITY OF GRANULE MEMBRANE PROTEINS and whether the mobility permits their recruitment by diffusion to the fusion site on the granule membrane, 3) To determine the ROTATIONAL MOBILITY OF ENTIRE GRANULES in order to better define the tethered and/or caged state of the granules before fusion, 4) 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, and 5) To measure for the first time the ABSOLUTE DISTANCE BETWEEN THE GRANULE AND THE PLASMA MEMBRANE using supercritical angle emission, thereby determining the timing of engagement of the granule with the plasma membrane before fusion. In addition, it is anticipated that the new techniques will not only be applicable to secretory cell biology, but more generally to the multitude of cell biological issues at the plasma membrane- cytosol interface.