Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex

Abstract

Summary When activated by WASP family proteins, Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2–4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, that unlike WASP, activate Arp2/3 complex without a preformed filament [4]. While this property may allow WDS proteins to serve as seed filament generators, it is unknown if actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here we use two color single molecule TIRF microscopy to show that Dip1, the S. pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.

Single-Turnover Activation of Arp2/3 Complex by Dip1 May Balance Nucleation of Linear versus Branched Actin Filaments

“…Monitoring the dynamics of Dip1 at comet tail sites revealed that Dip1 treadmills inward from the cortex at the same rate as Fim1-marked actin (Figure 3B). We previously showed that Dip1 does not bind actin filaments directly and only binds strongly to Arp2/3 complex on an actin filament pointed end if it has cooperated with the complex to nucleate that filament [8]. Therefore, our observation that Dip1 treadmills is consistent with a model in which it remains bound to Arp2/3 complex after triggering nucleation.…”

“…To determine whether ''programmed'' Dip1 release is a feature of its activation mechanism, we labeled it with Alexa Fluor 568 (Alexa568-Dip1) and visualized its action on Arp2/3 complex in reactions containing Oregon-Green-labeled actin using TIRF microcopy. As we reported previously, these reactions produced multiple events in which a new linear actin filament grew from an Alexa568-Dip1 molecule non-specifically adsorbed to the surface [8] (Figure 2A). Dip1 does not nucleate filaments on its own and does not bind actin filaments in the absence of Arp2/3 complex, so we interpret these events as Dip1 and Arp2/3 complex nucleating a linear filament [7,8].…”

“…As we reported previously, these reactions produced multiple events in which a new linear actin filament grew from an Alexa568-Dip1 molecule non-specifically adsorbed to the surface [8] (Figure 2A). Dip1 does not nucleate filaments on its own and does not bind actin filaments in the absence of Arp2/3 complex, so we interpret these events as Dip1 and Arp2/3 complex nucleating a linear filament [7,8]. Importantly, Dip1 did not release from filament ends upon nucleation, and the Alexa Fluor 568 signal was visible at the filament end for many seconds as the filament elongated (Figure 2A).…”

“…The SE is calculated from the measurements of the full field of view from 2 or 3 reactions and is shown in a lighter shade around the means at each time point (dark lines). The increased polymer accumulation rate is due to faster nucleation in the Dip1-containing reactions, because Dip1 does not influence filament barbed end elongation rates [7,8]. (F) Plot of the branching rate versus the concentration of Dip1 added to reactions as shown in (A).…”

Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

“…Therefore, the polarity of the mother filament defines the polarity of the resultant branched actin network. Our study uses diffusing linear actin filament nucleating proteins (Balzer et al, 2018;Basu and Chang, 2011;Wagner et al, 2013) to seed a defined number of randomly oriented mother filaments near the endocytic site. Alternatively, simulations using a pool of cytoplasmic linear actin filaments (Aroush et al, 2017) allowed for similar internalization, but with less reliable timing of initiation (data not shown).…”