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

Regulated assembly of actin-filament networks provides the mechanical force that pushes forward the leading edge of motile eukaryotic cells(1) and int racellular pathogenic bacteria(2) and viruses(3). When activated by binding to actin filaments and to the WA domain of Wiskott-Aldrich syndrome protei n (WASP)/Scar proteins, the Arp2/3 complex nucleates new filaments that gro w from their barbed ends(4-8). The Arp2/3 complex binds to the sides(9) and pointed ends(10,11) of actin filaments, localizes to distinctive 70 degree s actin-filament branches present in lamellae(12), and forms similar branch es in vitro(6,8,10). These observations have given rise to the dendritic nu cleation model for actin-network assembly(10,13) in which the Arp2/3 comple x initiates branches on the sides of older filaments. Recently, however, an alternative mechanism for branch formation has been proposed(8). In the 'b arbed-end nucleation' model, the Arp2/3 complex binds to the free barbed en d of a filament and two filaments subsequently grow from the branch. Here w e report the use of kinetic and microscopic experiments to distinguish betw een these models. Our results indicate that the activated Arp2/3 complex pr eferentially nucleates filament branches directly on the sides of pre-exist ing filaments.