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Published 17 March 2003. doi:10.1083/jcb.200208059
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© The Rockefeller University Press, 0021-9525/2003/3/951 $5.00
The Journal of Cell Biology, Volume 160, Number 6, 951-962

Article

 Formation of filopodia-like bundles in vitro from a dendritic network

Danijela Vignjevic1, Defne Yarar2, Matthew D. Welch2, John Peloquin1, Tatyana Svitkina1 and Gary G. Borisy1

1 Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611
2 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

Address correspondence to Danijela Vignjevic, Northwestern University Medical School, Department of Cell and Molecular Biology, 303 E. Chicago Ave., Ward 8-063, Chicago, IL 60611. Tel.: (312) 503-2854. Fax: (312) 501-7912. E-mail: nele{at}northwestern.edu

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.

Key Words: filopodia; actin; Arp2/3; capping protein; fascin

* Abbreviations used in this paper: Arp2/3, actin-related protein 2/3; BB, brain buffer; Ena/VASP, enabled/vasodilator-stimulated phosphoprotein; REF, rat embryo fibroblast; VCA, verprolin-homology/connecting/acidic domain of WASP; WASP, Wiskott-Aldrich syndrome protein.


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