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J. Biol. Chem., Vol. 282, Issue 36, 26528-26541, September 7, 2007

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

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

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 factor-stimulated 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 actin-bundling 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.

Received for publication, May 1, 2007 , and in revised form, June 11, 2007.

^* This work was supported by NIDDK, National Institutes of Health Grants DK-65006 and DK-54755 (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1-S3, and Videos 1 and 2.

¹ To whom correspondence should be addressed: University of Tennessee Health Science Center, 894 Union Ave., Nash 402, Memphis TN 38163. Tel.: 901-448-3410; Fax: 901-448-3505; E-mail: skhurana{at}utmem.edu.

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				Y. Wang, K. Srinivasan, M. R. Siddiqui, S. P. George, A. Tomar, and S. Khurana A Novel Role for Villin in Intestinal Epithelial Cell Survival and Homeostasis J. Biol. Chem., April 4, 2008; 283(14): 9454 - 9464. [Abstract] [Full Text] [PDF]