Significance Actin filament nucleation by Arp2/3 complex must be triggered by activators like WASP family proteins. Understanding how WASP proteins activate Arp2/3 complex has been a major challenge due to a lack of high-resolution structures of the complex in an activated state. We determined a high-resolution (∼3.9 Å) structure of the WASP-activated Arp2/3 complex at a branch junction and used biochemical, cell biological, and molecular dynamic simulations to understand the mechanism of WASP-mediated activation. This work shows in detail the contacts between the fully activated Arp2/3 complex, the nucleated daughter actin filament, and the mother actin filament and provides important insights into how conformational rearrangements in the Arp2/3 complex are stimulated during activation.

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Highlights d Actin filaments stimulate the short pitch conformational change weakly or not at all d Actin filaments stimulate an activating step distinct from WASP d The WASP-and actin filament-mediated Arp2/3 activation steps are unconcerted d Both steps are required to create sustained force-producing branched actin networks

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate ‘seed’ filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

Cytidine triphosphate synthase catalyzes the synthesis of cytidine 5′-triphosphate (CTP) from uridine 5′-triphosphate (UTP), the final step in the production of cytidine nucleotides. CTP synthases also form filamentous structures of different morphologies known as cytoophidia, whose functions in most organisms are unknown. Here, we identified and characterized a novel CTP synthase (TgCTPS) from Toxoplasma gondii. We show that TgCTPS is capable of substituting for its counterparts in the otherwise lethal double mutant (ura7Δ ura8Δ) of Saccharomyces cerevisiae. Equally, recombinant TgCTPS purified from Escherichia coli encodes for a functional protein in enzyme assays. The epitope-tagged TgCTPS under the control of its endogenous promoter displays a punctate cytosolic distribution, which undergoes spatial reorganization to form foci or filament-like structures when the parasite switches from a nutrient-replete (intracellular) to a nutrient-scarce (extracellular) condition. An analogous phenotype is observed upon nutrient stress or after treatment with a glutamine analog, 6-diazo-5-oxo-L-norleucine (DON). The exposure of parasites to DON disrupts the lytic cycle, and the TgCTPS is refractory to a genetic deletion, suggesting an essential requirement of this enzyme for T. gondii. Not least, this study, together with previous studies, supports that CTP synthase can serve as a potent drug target, because the parasite, unlike human host cells, cannot compensate for the lack of CTP synthase activity.