Villin function in the organization of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro.
Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca(2+)-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca(2+) as well as its bundling and severing activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.
Friederich
E
E
Laboratoire de Morphogenèse et Signalisation Cellulaire, Centre National de la Recherche Scientifique, UMR 144, Institut Curie, 26, rue d'Ulm, Paris 75248 Cedex 05 France. Evelyne.Friederich@curie.fr
Vancompernolle
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K
Louvard
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D
Vandekerckhove
J
J
D000199:Actins / Q000502:physiology* / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000502:physiology* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; C026615:villin