Methods Enzymol 2006 Jan 01;406:727-39. doi: 10.1016/S0076-6879(06)06057-5.

In vitro assembly of filopodia-like bundles.

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A breakthrough in understanding the mechanism of lamellipodial protrusion came from development of an in vitro model system, namely the rocketing movement of microbes and activated beads driven by actin comet tails (Cameron et al., 1999, 2000; Loisel et al., 1999; Theriot et al., 1994). As a model for investigation of the other major protrusive organelle, the filopodium, we developed in vitro systems for producing filopodia-like bundles (Vignjevic et al., 2003), one of which uses cytoplasmic extracts and another that reconstitutes like-like bundles from purified proteins. Beads coated with 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 star 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. Similar to cells, the transition from a dendritic (lamellipodial) to a bundled (filopodial) organization is induced by depletion of capping protein, and add-back of this protein restores the dendritic mode. By use of purified proteins, a small number of components are sufficient for the assembly of filopodia-like bundles: WASP-coated beads, actin, Arp2/3 complex, and fascin. On the basis of analysis of this system, we proposed 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.

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Species referenced: Xenopus
Genes referenced: actl6a fscn1 tbx2 was