Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

Abstract

<jats:title>Abstract</jats:title><jats:p>The actin filament nucleator Arp2/3 complex is activated at cortical sites in <jats:italic>S. pombe</jats:italic> 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 pre-existing filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from pre-existing filaments. Here we show that Wsp1 is important not only for propagation, but also for initiation of endocytic actin networks. Using single molecule TIRF microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic coactivation does not require pre-existing actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.</jats:p>

Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly

“…In contrast, the Y275A mutant that showed little activity on its own but that synergized with WASP in vitro was far less defective in vivo . These observations support a previously proposed model in which Wsp1 and Dip1 synergize in cells to initiate the assembly of endocytic actin networks ( 12 ). Whether synergy between Wsp1 and Dip1 might be required for initiation of endocytic actin networks or if Dip1 can also initiate new actin patch assembly on its own is an important open question.…”

“…In this way, branched actin network initiation and propagation would be coordinated through a single signaling pathway. However, our previous cell biological data show that deletion of the Wsp1 CA segment, which likely blocks Wsp1 from synergizing with Dip1 ( 12 ), decreases the rate of initiation of new actin patches, but not to the same extent as deletion of DIP1. Whether this means Dip1 has some ability to initiate new actin patches on its own or whether Dip1 synergizes with one of the other NPFs at endocytic sites ( e.g.…”

“…While there are multiple possible explanations for this behavior, one possibility is that cellular factors might rescue the activity of some Dip1 mutants so that their in vivo activity is closer to WT Dip1. Importantly, we recently showed that Dip1 activity is increased by Wsp1 in vitro and provided evidence that the two NPFs might synergize in vivo ( 12 ). Therefore, we asked if Wsp1 could rescue Arp2/3 complex activation by mutant Dip1 proteins.…”

“…Whether synergy between Wsp1 and Dip1 might be required for initiation of endocytic actin networks or if Dip1 can also initiate new actin patch assembly on its own is an important open question. A requirement for synergy with Wsp1 could be advantageous in that it would provide a simple signaling pathway to trigger Dip1 activity; factors that activate the NPF activity of Wsp1 would—through synergy—activate Dip1, without the requirement to activate a separate Dip1-specific activation pathway ( 12 ). In this way, branched actin network initiation and propagation would be coordinated through a single signaling pathway.…”

“…Therefore, WDS proteins are thought to provide the initial (seed) filaments to stimulate WASP-mediated activation of Arp2/3 complex during branched actin assembly in cells. While early models postulated a clear demarcation of Dip1 as the initiator of branched networks and WASP as the propagator, recent data show that the two NPFs potently synergize to coactivate Arp2/3 complex without a pre-existing actin filament, suggesting that WASP could play a role in both propagation and initiation of branched actin networks ( 12 ).…”