Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders.
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.
George
Sudeep P
SP
Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Wang
Yaohong
Y
Mathew
Sijo
S
Srinivasan
Kamalakkannan
K
Khurana
Seema
S
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D018938:Caco-2 Cells; D002460:Cell Line; D019281:Dimerization; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008871:Microvilli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011554:Pseudopodia / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000199:Actins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C026615:villin