Abstract
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.
Highlights
Arp2/3 complex is an important cytoskeletal regulator that nucleates actin filament networks important in a broad range of cellular processes, including cell motility, differentiation, endocytosis, meiotic spindle positioning, and DNA repair (Goley and Welch, 2006; Hurst et al, 2019; Rotty et al, 2013; Yi et al, 2011)
Through single molecule total internal reflection fluorescence (TIRF) microscopy along with kinetic assays and modeling, we find that the role of Wsp1 in initiation is likely due to its ability to synergize with Dip1 to activate Arp2/3 complex
Deletion of the WASP CA segment causes a decrease in the patch initiation rate To test their relative importance in the initiation versus propagation of endocytic actin networks, we measured the influence of Dip1 and Wsp1 mutations on actin dynamics in fission yeast using the endocytic actin patch marker Fim1 labeled with green fluorescent protein (GFP) (Berro and Pollard, 2014)
Summary
Arp2/3 complex is an important cytoskeletal regulator that nucleates actin filament networks important in a broad range of cellular processes, including cell motility, differentiation, endocytosis, meiotic spindle positioning, and DNA repair (Goley and Welch, 2006; Hurst et al, 2019; Rotty et al, 2013; Yi et al, 2011). Analysis of mutant or knockout strains of Wsp and Dip suggests that activation of Arp2/3 complex by both of these NPFs is required for normal endocytic actin assembly (Basu and Chang, 2011; Sirotkin et al, 2005; Wagner et al, 2013). It is currently unknown how Wsp and Dip cooperate to assemble functional endocytic actin networks in S. pombe, but key biochemical differences between these NPFs have
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