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The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments

Abstract

Regulated assembly of actin-filament networks provides the mechanical force that pushes forward the leading edge of motile eukaryotic cells1 and intracellular pathogenic bacteria2 and viruses3. When activated by binding to actin filaments and to the WA domain of Wiskott–Aldrich-syndrome protein (WASP)/Scar proteins, the Arp2/3 complex nucleates new filaments that grow from their barbed ends4,5,6,7,8. The Arp2/3 complex binds to the sides9 and pointed ends10,11 of actin filaments, localizes to distinctive 70° actin-filament branches present in lamellae12, and forms similar branches in vitro6,8,10. These observations have given rise to the dendritic nucleation model for actin-network assembly10,13, in which the Arp2/3 complex initiates branches on the sides of older filaments. Recently, however, an alternative mechanism for branch formation has been proposed8. In the `barbed-end nucleation' model, the Arp2/3 complex binds to the free barbed end of a filament and two filaments subsequently grow from the branch. Here we report the use of kinetic and microscopic experiments to distinguish between these models. Our results indicate that the activated Arp2/3 complex preferentially nucleates filament branches directly on the sides of pre-existing filaments.

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Figure 1: Length distribution of actin filament branches mediated by the Arp2/3 complex.
Figure 2: Effect of capped and uncapped filaments on actin polymerization in the presence of activated Arp2/3 complex.
Figure 3: Fluorescence micrographs of actin filaments nucleated by uncapped green mother filaments and the Arp2/3 complex.
Figure 4: Fluorescence micrographs of actin filaments nucleated by capped green mother filaments and the Arp2/3 complex.

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Acknowledgements

This work was supported by research grants from the National Institutes of Health. We thank D. Kaiser for amoeba profilin and Scar-WA, H. Higgs for bovine Arp2/3 complex, L. Blanchoin for amoeba capping protein, V. Sirotkin and K. M. Roh for measuring filaments, and members of our laboratory for discussions. We also thank D. Pantaloni and M-F. Carlier for discussion of their work on barbed-end nucleation and for providing us with a copy of their Nature Cell Biology paper before publication, and M-F. Carlier in particular for suggesting the mother/daughter nomenclature for branches.

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Correspondence to Thomas D. Pollard.

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Amann, K., Pollard, T. The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments. Nat Cell Biol 3, 306–310 (2001). https://doi.org/10.1038/35060104

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