title: Formation of filopodia-like bundles in vitro from a dendritic network.
abstract: We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.
authors.affiliation: Northwestern University Medical School, Department of Cell and Molecular Biology, 303 E. Chicago Ave., Ward 8-063, Chicago, IL 60611, USA.
authors.forename: Danijela
authors.initials: D
authors.lastname: Vignjevic
authors.forename: Defne
authors.initials: D
authors.lastname: Yarar
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Welch
authors.forename: John
authors.initials: J
authors.lastname: Peloquin
authors.forename: Tatyana
authors.initials: T
authors.lastname: Svitkina
authors.forename: Gary G
authors.initials: GG
authors.lastname: Borisy
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D051377:Actin-Related Protein 2; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000502:physiology*; D003598:Cytoskeletal Proteins; D003712:Dendrites / Q000378:metabolism* / Q000648:ultrastructure; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D011506:Proteins; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits; D051381:Rats; D014020:Tissue Extracts; D051316:Wiskott-Aldrich Syndrome Protein
chemical_list: D051377:Actin-Related Protein 2; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D011506:Proteins; D014020:Tissue Extracts; C495769:Was protein, rat; D051316:Wiskott-Aldrich Syndrome Protein; C020324:fascin