A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Molecular Biology of the Cell (Impact Factor: 4.55). 03/2005; 16(2):964-75. DOI: 10.1091/mbc.E04-09-0774
Source: PubMed

ABSTRACT 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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Available from: Clare M Waterman-Storer, Aug 25, 2014
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    • "However, by a number of tests, foci of polymerization were not closely associated with clathrin puncta within the spine, and the velocity of actin monomers on filaments near the EZ was not different than the surrounding spine milieu. This is particularly surprising given that the endocytic zone contains numerous actin-binding molecules which likely regulate endocytosis (Engqvist-Goldstein and Drubin, 2003; Rocca et al., 2008; Yarar et al., 2005). However, consistent with a limited tonic role of actin polymerization at the spine EZ, clathrin puncta are not disassembled or disrupted during latrunculin application (Blanpied et al., 2002), whereas AMPA receptors and PSD scaffold proteins are quickly lost (Kuriu et al., 2006; Zhou et al., 2001). "
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    • "Coupled with the above studies on the various dystonin-a isoforms was the finding that the plakin domain of dystonin-a interacts with the protein clathrin in brain tissues (Bhanot et al., 2011). Clathrin is a protein involved in the coating of newly developed vesicles during both endocytosis and Golgi-exocytosis, and utilizes the actin cytoskeleton in these processes (Galletta et al., 2010; Yarar et al., 2005). This suggests that dystonin may play a role in linking clathrin-coated vesicles with the cytoskeleton. "
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    • "We previously found that inactive forms of PAK1 locally sequester these GTPases and prevent their action in catalysing actin polymerization (Bisson et al., 2007). Since, regulation of the actin cytoskeleton has been implicated in endocytosis (Boulant et al., 2011; Liu et al., 2010; Taylor et al., 2011; Yarar et al., 2005). Our data suggest a possible role for E- Syt2 binding of PAK1 in this process. "
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