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

• Published in: The Journal of biological chemistry 2007-09, Vol.282 (36), p.26528-26541
• Main Authors: George, Sudeep P., Wang, Yaohong, Mathew, Sijo, Srinivasan, Kamalakkannan, Khurana, Seema
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Actins - genetics; Actins - metabolism; Amino Acid Sequence; Animals; Binding Sites - genetics; Caco-2 Cells; Cell Line; Dimerization; Dogs; Epithelial Cells - chemistry; Epithelial Cells - metabolism; Epithelial Cells - ultrastructure; Fluorescence Resonance Energy Transfer; Humans; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Microvilli - chemistry; Microvilli - genetics; Microvilli - metabolism; Microvilli - ultrastructure; Protein Binding - genetics; Protein Structure, Tertiary - genetics; Pseudopodia - chemistry; Pseudopodia - genetics; Pseudopodia - metabolism; Pseudopodia - ultrastructure; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Deletion; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M703617200

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.