Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 16, Issue 2, Pages 964-975Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E04-09-0774
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Funding
- NHLBI NIH HHS [5T32HL07695, T32 HL007695] Funding Source: Medline
- NIGMS NIH HHS [R01 GM067230, GM67230, U01 GM067230] Funding Source: Medline
- NIMH NIH HHS [R37 MH061345, R01 MH061345, MH61345] Funding Source: Medline
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Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.
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