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Vol. 16, Issue 2, 964-975, February 2005
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Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
Submitted September 3, 2004;
Revised November 3, 2004;
Accepted December 1, 2004
Monitoring Editor: David Drubin
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.
Abbreviations used: CME, clathrin-mediated endocytosis; CCP, clathrin-coated pit; CCS, clathrin-coated structure; CCV, clathrincoated vesicle; TIR-FM, total internal reflection fluorescence microscopy; WF-EFM, wide-field epi-fluorescence microscopy; CLC-DsRed, clathrin light-chain DsRed; Pll, poly-L-lysine; latA, latrunculin A; jasp, jasplakinolide.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Sandra L. Schmid (slschmid{at}scripps.edu) or Clare M. Waterman-Storer (waterman{at}scripps.edu).
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