Larson Ben T, Sochacki Kem A, Kindem Jonathan M, Taraska Justin W
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
Mol Biol Cell. 2014 Jul 1;25(13):2084-93. doi: 10.1091/mbc.E14-02-0771. Epub 2014 May 7.
Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here we develop a high-resolution and high-throughput fluorescence imaging-based approach for the unbiased mapping of 78 proteins at single exocytic vesicles and endocytic structures in neuroendocrine PC12 cells. This analysis uses two-color single-frame images to provide a systems-level map of the steady-state distributions of proteins at individual exocytic and endocytic structures in the cell. Along with this quantitative map, we find that both calcium-regulated exocytic vesicles (dense core vesicles) and endocytic structures (clathrin-coated structures) and the proteins associated with these structures exhibit a random spatial distribution in unstimulated neuroendocrine PC12 cells. This approach is broadly applicable for quantitatively mapping the molecular composition and spatial organization of discrete cellular processes with central molecular hubs.
囊泡分泌（胞吐作用）涉及囊泡成分通过内吞作用的释放及随后的补偿性回收利用。这一基本细胞过程受数十种蛋白质在质膜上的协同组装和相互作用所控制。了解单个胞吐和内吞结构的分子组成及其在质膜上的组织方式对于理解这两个细胞过程的行为和调控至关重要。在此，我们开发了一种基于高分辨率和高通量荧光成像的方法，用于在神经内分泌PC12细胞的单个胞吐囊泡和内吞结构上对78种蛋白质进行无偏差定位。该分析使用双色单帧图像来提供细胞中单个胞吐和内吞结构上蛋白质稳态分布的系统级图谱。连同这一定量图谱，我们发现钙调节的胞吐囊泡（致密核心囊泡）和内吞结构（网格蛋白包被结构）以及与这些结构相关的蛋白质在未受刺激的神经内分泌PC12细胞中呈现随机空间分布。这种方法广泛适用于对具有核心分子枢纽的离散细胞过程的分子组成和空间组织进行定量定位。
