title: Villin severing activity enhances actin-based motility in vivo.
abstract: Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.
authors.affiliation: Laboratoire de Morphogenèse et Signalisation Cellulaires, Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique/Institut Curie, 75248 Paris Cedex 05, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Revenu
authors.forename: Matthieu
authors.initials: M
authors.lastname: Courtois
authors.forename: Alphée
authors.initials: A
authors.lastname: Michelot
authors.forename: Cécile
authors.initials: C
authors.lastname: Sykes
authors.forename: Daniel
authors.initials: D
authors.lastname: Louvard
authors.forename: Sylvie
authors.initials: S
authors.lastname: Robine
mesh_terms: D051344:Actin Capping Proteins / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D004285:Dogs; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008863:Microspheres; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics; D011817:Rabbits; D012763:Shigella flexneri / Q000166:cytology; D013552:Swine
chemical_list: D051344:Actin Capping Proteins; D000199:Actins; D008840:Microfilament Proteins; D050505:Mutant Proteins; C026615:villin