Credit: Image courtesy of D. Vignjevic et al., Northwestern Medical school, Chicago, USA.

Actin-rich finger-like filopodia let cells explore their environment, but little is known about how filopodial formation is initiated. The lack of an in vitro system for producing filopodia-like bundles has certainly been a handicap, so the development of such a system by Vignjevic et al., reported in the Journal of Cell Biology, is a welcome step. Using this system, the authors propose that filopodia form by the elongation of a pre-existing actin filament network by inhibition of capping and the subsequent bundling of these filaments.

The authors coated beads with Wiskott–Aldrich syndrome protein (WASP), a protein that facilitates filopodia formation, introduced them with actin into a rat brain extract, and visualized the resultant actin structures. Depending on their position under the coverslip, the structures were very different. 'Stars' of straight actin bundles emanating from the beads formed at the edge, whereas 'clouds' of actin filaments, or 'comets', formed in the centre. Vignjevic et al. reasoned that one or more proteins became depleted during sample spreading by adsorption to the glass coverslip. And indeed, reducing the concentration of some factor(s) in the extract — by glass depletion or fivefold dilution — resulted in uniform star formation.

The stars' radial actin bundles resembled filopodia, so Vignjevic et al. looked for other similarities. Kinetic and structural analysis of star formation showed that close to the bead was a dendritic network (resembling lamellipodia), from which long, unbranched filaments emanated and merged into bundles (resembling filopodia) further away. There were also differences in the localization of proteins associated with lamellipodia or filopodia, such as the actin-related protein 2/3 (Arp2/3) complex. Arp2/3 was present in the dendritic network, but not in bundles.

WASP-family members activate the Arp 2/3 complex, which nucleates actin filaments. Depleting Arp2/3 abolished actin assembly around beads, but star formation was restored when Arp2/3 was added back, indicating that Arp2/3 mediates star formation. Furthermore, the adsorption/dilution experiments had hinted that a decreased amount of factor(s) in the extract were crucial in star formation. Increased levels of a candidate factor, capping protein, antagonized star formation. Decreased levels (for example, at the edge of the coverslip) probably mediate actin-filament elongation.

On the basis of their results, Vignjevic et al. attempted reconstitution experiments using WASP-coated beads, actin and Arp2/3, but found that they had to add an actin-bundling protein, fascin, to promote star formation. So the authors propose three steps — nucleation (by Arp2/3), elongation (by decreased capping protein) and bundling (by fascin) — for star, or filopodia, formation.