Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

Abstract

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 clathrincoated 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.

RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

“…We will refer to these as clathrin-coated structures (CCSs). As shown in Figure 4A, F-actin does not colocalize with CCSs in control cells, although in some cells coated-pits appear to align along stress fibers as previously reported (Bennett et al, 2001). It should be noted that because HeLa cells are Strikingly, in A1/A3 cells F-actin colocalized with CCSs and appeared enriched in these structures ( Figure 4A, see arrows in the enlargement, and unpublished data).…”

Alix regulates cortical actin and the spatial distribution of endosomes

“…Clathrin hub expression leads to dissociation of the actin-binding protein Hip1R from coated pits, and disrupts the alignment with the actin cytoskeleton. This suggests a role for the clathrin-Hip1R interaction in mediating the spatial relationship between endosomes and the cortical actin cytoskeleton (Bennett et al, 2001a). Even though overexpression of the clathrin hub domain inhibits Tf endocytosis, it does not significantly affect endocytic recycling (Bennett et al, 2001b).…”

Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

“…BDM has been utilized as an uncompetitive inhibitor of myosin ATPase activity (Higuchi and Takemori, 1989;Herrmann et al, 1992). Exposure of cells to BDM dissociates myosin from actin filaments, impairing myosin involvement in events as varied as muscle contraction (Herrman et al, 1992) and myosin-based vesicle transport (Bennett et al, 2001;Duran et al, 2003). The ability of BDM to inhibit myosins other than myosin II family members has recently been questioned (Ostap, 2002), and other work suggests that it may affect actin dynamics through myosinindependent mechanisms (Yarrow et al, 2003).…”