title: Mutagenetic and electron microscopy analysis of actin filament severing by Cordon-Bleu, a WH2 domain protein.
abstract: Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer » of actin assembly, inducing fast turnover of actin filaments and oscillatory polymerization regime via nucleation, severing, and rapid depolymerization activities. Cobl is the most efficient severer of actin filaments characterized so far. To understand which primary sequence elements determine the filament severing activity of the WH2 repeats, here we combine a mutagenetic/domain swapping approach of the minimal fully active Cobl-KAB construct, which comprises the lysine rich region K preceding the two first WH2 domains A and B. The mutated Cobl constructs display variable loss of the original filament nucleating activities of native Cobl-KAB, without any strict correlation with a loss in actin binding, which emphasizes the functional importance of the electrostatic environment of WH2 domains. Filament severing displayed the greatest stringency and was abolished in all mutated forms of Cobl-KAB. Filament severing and re-annealing by Cobl-KAB, which is key in its rapid remodeling of a population of actin filaments, and most likely responsible for its function in ciliogenesis, was analyzed by electron microscopy in comparison with Spire and ADF.
authors.affiliation: Cytoskeleton Dynamics and Cell Motility Team, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, 91198, Gif-sur-Yvette, France.
authors.forename: Yue
authors.initials: Y
authors.lastname: Jiao
authors.forename: Matt
authors.initials: M
authors.lastname: Walker
authors.forename: John
authors.initials: J
authors.lastname: Trinick
authors.forename: Julien
authors.initials: J
authors.lastname: Pernier
authors.forename: Pierre
authors.initials: P
authors.lastname: Montaville
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Carlier
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D006801:Humans; D057446:Hydrodynamics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D050505:Mutant Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D016415:Sequence Alignment; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; C561633:Cobl protein, human; D008840:Microfilament Proteins; D050505:Mutant Proteins; D051316:Wiskott-Aldrich Syndrome Protein
keywords: Cordon-Bleu; WASP homology 2 domain; actin; electron microscopy; intrinsically disordered proteins; mutagenesis; protein complexes