The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

Abstract

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis

“…It is generally accepted that actin polymerization plays a role in some (but not all, see [52,53]) forms of CME [9,11,18,44,46,47,50,54]. Here, a more accurate measurement of actin dynamics using the ppH assay revealed an ordered sequence of proteins involved in actin dynamics.…”

“…Other proteins showed a similar pattern of biphasic recruitment and thus defined a dynamin module. These included actin-binding proteins such as the actin-and clathrin-binding protein Hip1R [44] as well as the motor protein myosin6, which binds actin and the adaptor protein Dab2 [45]. Other proteins involved in actin dynamics and grouped in the dynamin module included the Arp2/ 3 activator N-WASP [46,47], Eps8, an actin capping protein that forms a complex with Abi1 and binds N-WASP [33] and the motor protein myosin1E [48].…”

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

“…These sorts of intimate and persistent actin/clathrin associations were not seen in control HeLa cells grown without exposure to the "A3" siRNA (unpublished data), nor have they been seen before in any of a wide variety of other cultured cells examined by unroofing over the years (Heuser, 1980;Heuser and Anderson, 1989;Fujimoto et al, 2000;Engqvist-Goldstein et al, 2001). In addition, the collections of F-actin filaments observed in A3 cells have an unusual configuration, not commonly observed on the ventral surfaces of these HeLa (or any other) cultured cells.…”

“…We found that cortactin, the Arp2/3 complex and Abp1 are enriched in these structures (Figure 3 and unpublished data). Similar to Hip1R, cortactin, and Abp1 are actin-binding proteins implicated in both endocytic and actin functions, and they may function at the interface between the actin cytoskeleton and the endocytic machinery (Kessels et al, 2000(Kessels et al, , 2001McNiven et al, 2000;Cao et al, 2003;Mise-Omata et al, 2003). Cortactin and Abp1 both colocalize with the Arp2/3 complex in cells (Kessels et al, 2000;McNiven et al, 2000).…”

“…In the past few years, several proteins including Hip1R, syndapin, cortactin, Abp1, intersectin and myosin VI have been identified as putative linker proteins (Qualmann and Kelly, 2000;Buss et al, 2001;Engqvist-Goldstein et al, 2001;Hussain et al, 2001;Kessels et al, 2001). Currently, in vivo evidence for such linker function is largely lacking.…”