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Mol Biol Cell. 2014 Jul 1;25(13):2084-93. doi: 10.1091/mbc.E14-02-0771. Epub 2014 May 7.

Systematic spatial mapping of proteins at exocytic and endocytic structures.

Author information

1
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
2
Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 justin.taraska@nih.gov.

Abstract

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.

PMID:
24807904
PMCID:
PMC4072581
DOI:
10.1091/mbc.E14-02-0771
[Indexed for MEDLINE]
Free PMC Article
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