title: Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging.
abstract: Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454-9110, USA.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Smith
authors.forename: Karen
authors.initials: K
authors.lastname: Daugherty-Clarke
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Goode
authors.forename: Jeff
authors.initials: J
authors.lastname: Gelles
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D007700:Kinetics; D036641:Microscopy, Fluorescence, Multiphoton; D008958:Models, Molecular; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D029701:Saccharomyces cerevisiae Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family