Clathrin hub expression dissociates the actin-binding protein Hip1R from coated pits and disrupts their alignment with the actin cytoskeleton.
The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin-Hip1R interaction is involved in the cytoskeletal organization of coated pits.
Bennett
E M
EM
The G. W. Hooper Foundation, Department of Microbiology and Immunology, University of California, San Francisco 94143-0552, USA.
Chen
C Y
CY
Engqvist-Goldstein
A E
AE
Drubin
D G
DG
Brodsky
F M
FM
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*
D000199:Actins; D002966:Clathrin; D008840:Microfilament Proteins