title: Integration of linear and dendritic actin nucleation in Nck-induced actin comets.
abstract: The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.
authors.affiliation: R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030.
authors.forename: Sofya
authors.initials: S
authors.lastname: Borinskaya
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269.
authors.forename: Katrina B
authors.initials: KB
authors.lastname: Velle
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269.
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Campellone
authors.affiliation: Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT 06519.
authors.forename: Arthur
authors.initials: A
authors.lastname: Talman
authors.affiliation: Biotechnology Research Institute, University of San Martin, 1650 San Martin, Argentina.
authors.forename: Diego
authors.initials: D
authors.lastname: Alvarez
authors.affiliation: Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT 06519.
authors.forename: Hervé
authors.initials: H
authors.lastname: Agaisse
authors.affiliation: R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030 Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT 06030.
authors.forename: Yi I
authors.initials: YI
authors.lastname: Wu
authors.affiliation: R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030 bmayer@uchc.edu les@uchc.edu.
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Loew
authors.affiliation: R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030 Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT 06030 bmayer@uchc.edu les@uchc.edu.
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Mayer
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D003712:Dendrites / Q000378:metabolism; D005326:Fetal Proteins / Q000378:metabolism; D000080865:Formins; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D005326:Fetal Proteins; D000080865:Formins; D008840:Microfilament Proteins; C078332:Nck protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal