Supramolecular Dynamics of Secretory Granules Studied by New Optical Techniques

描述(申请人提供)：尽管约10%的人类基因组由分泌蛋白组成，但对融合前和融合过程中颗粒腔蛋白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。我们的研究集中在胞吐途径中的事件，这些事件发生在质膜-细胞质界面的高度专门化领域。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。这一提议得到了强有力的初步结果的支持，这些结果表明不同的管腔蛋白NPY-Cerulean和tPA-Cerulean具有不同的迁移性。TPA-Cerulean的迁移率要低得多，在单个颗粒融合时释放慢得多，融合与慢得多的融合孔扩张有关。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，将测量管腔和颗粒膜蛋白的旋转和平移迁移率。在该提案中有几个相关的目标：1)了解蛋白质在颗粒管腔中的运动特性并揭示它们在确定蛋白质和儿茶酚胺释放速率方面的影响，2)确定颗粒膜蛋白的平移迁移率，以及该迁移率是否允许它们通过扩散到颗粒膜上的融合部位而被招募，3)确定整个颗粒的旋转迁移率，以便更好地确定融合前颗粒的系留和/或笼养状态，4)确定融合前颗粒运动的增加是否反映了平移和旋转运动的组合，该平移和旋转运动允许颗粒‘滚动’进入与质膜的融合适宜的相互作用，5)首次利用超临界角度发射测量颗粒与质膜的绝对距离，从而确定融合前颗粒与质膜结合的时间。此外，预计新技术不仅适用于分泌细胞生物学，而且更广泛地适用于质膜-胞浆界面的多种细胞生物学问题。

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.