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Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Nature volume 404, pages 1007–1011 (2000)Cite this article

Abstract

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane1,2. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward3. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed4 in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott–Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

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Figure 1: Comparison of the kinetics of actin polymerization in the presence of Arp2/3 complex and WASP WA domain with the structure of the products observed by light microscopy.
Figure 2: Regulation of branch length and observation of branch point rigidity.
Figure 3: Model for the assembly and disassembly of the dendritic actin filament network at the leading edge of a cell.

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Acknowledgements

This work was supported by an NIH research grant to T.D.P., fellowships from National Research Service Award to K.J.A. and H.N.H. and from the Association pour la Recherche contre le Cancer to J.B.M. We thank J. Howard for advice on calculating the spring constant.

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Author notes

  1. Laurent Blanchoin and Kurt J. Amann: These authors contributed equally to this work

  2. Thomas D. Pollard: Correspondence and requests for materials should be addressed to T.D.P.

Authors and Affiliations

  1. Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Laurent Blanchoin, Kurt J. Amann, Henry N. Higgs, Jean-Baptiste Marchand, Donald A. Kaiser & Thomas D. Pollard

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Blanchoin, L., Amann, K., Higgs, H. et al. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature 404, 1007–1011 (2000). https://doi.org/10.1038/35010008

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