Abstract
Arp2/3 complex nucleates branched actin filaments important for cellular motility, endocytosis, meiosis, and cellular differentiation [1-4]. Wiskott-Aldrich syndrome proteins (WASPs), the prototypical Arp2/3 complex activators, activate Arp2/3 complex only once it is bound to the side of an actin filament [5, 6]. This ensures WASP-activated Arp2/3 complex only nucleates branched actin filaments but means branched actin networks must be seeded with an initial preformed filament. Dip1 and other WISH/DIP/SPIN90 family proteins activate Arp2/3 complex without preformed filaments [7], creating seed filaments that activate WASP-bound Arp2/3 complex [8]. Importantly, Dip1-mediated activation of Arp2/3 complex creates linear filaments instead of branches [7]. Cells may therefore need to limit Dip1 activity relative to WASP to preserve the dendritic nature of actin networks, although it is unclear whether such regulatory mechanisms exist. Here, we use total internal reflection fluorescence (TIRF) microscopy to show that Dip1 causes actin assembled with WASP and Arp2/3 complex to form disconnected networks with many linear filaments rather than highly branched arrays. We discover a key biochemical difference between Dip1 and WASP that may limit linear filament nucleation in cells; although WASP must be released for nucleation, Dip1 stays associated with Arp2/3 complex on the pointed ends of nucleated actin filaments, so Dip1 is consumed in the reaction. Using live-cell imaging of fission yeast, we provide evidence that Dip1 is a single-turnover activator of Arp2/3 complex in vivo, revealing a mechanism by which Dip1 can initiate branched actin networks at endocytic sites without disrupting their branched architectures.