Dimerization and Actin-bundling Properties of Villin and Its Role in the Assembly of Epithelial Cell Brush Borders

Abstract

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factorstimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actinbundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis

“…Cerulean-tagged villin was cloned as described previously (34). HeLa GFP-cytochrome c cells were transiently transfected with cerulean-tagged villin, treated with 1 M staurosporine for 0 -5 h, and live cell images captured at different time points (0 -5 h) using confocal laser scanning microscope and a 63ϫ oil objective (LSM 5 PASCAL; Carl Zeiss, Thornwood, NY).…”

Actin bundling in plants

“…1B, model b). In contradiction with these data George et al [2007] demonstrated that villin can form dimers both in vitro and in vivo [George et al, 2007]. In their model, bundling activity implicates the parallel arrangement of villin dimers mediated by an N-terminal domain, and involves exclusively one ABD in the HP (Fig.…”

“…Lily ABP-135 contains six gelsolin-like domains (G1-G6) and a C-terminal headpiece (HP). The asterisk indicates the position of a potential dimerization site [George et al, 2007]. An alternative to a single ABD located between G1 and G2 (ABD1a), is two ABDs located in G1 (ABD1b) and G2 (ABD1c), respectively [Hampton et al, 2008].…”

“…In this case, ABD2 only holds villin on the first AF. (3) AF bundling by ABD2 following villin dimerization [George et al, 2007]. Fimbrins.…”

The two domains of cotton WLIM1a protein are functionally divergent

“…It is known that the plant actin-binding proteins such as villin and formin require dimerization for their actin bundling activity (George et al, 2007;Xue et al, 2011). Concerning LIM proteins, one assumption is that LIM domains could dimerize to fulfill their actin-bundling function (Thomas et al, 2007).…”