A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.
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.
Yarar
Defne
D
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Waterman-Storer
Clare M
CM
Schmid
Sandra L
SL
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism* / Q000648:ultrastructure; D022163:Clathrin-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D047630:Depsipeptides / Q000494:pharmacology; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D000097573:Red Fluorescent Protein
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002966:Clathrin; D047630:Depsipeptides; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D013844:Thiazoles; D053778:Thiazolidines; D000097573:Red Fluorescent Protein; C057531:jasplakinolide; C037067:latrunculin A