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

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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.