The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.
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We thank L. Cabanié for technical support and members of the Louvard laboratory and M. Beckerle for valuable discussions. We also thank P. Chavrier, P. Cossart, E. Gouin, L. Hoffman, C. Koehler and L. Machesky for generously providing reagents. This work was supported by a Curie Fellowship (to R.M.G. and J.P.), a MENRT fellowship (to J.F. and V.N.), and grants from the Association pour la Recherche sur le Cancer and the Centre National de la Recherche Scientifique (France).
Figure S1 Effect of cytochalasin D on the ATPase activity of actin during polymerization. (PDF 26 kb)
Figure S2 Inhibition of actin polymerization on the surface of WA or ActA-Pro-beads by increasing concentrations of latrunculin.
Figure S3 Quantification of fluorescence associated with WA or ActA-Pro beads immunostained for VASP or Arp3 in the absence of latrunculin A.
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