Phosphorylation of Arp2 is necessary for lamellipodia formation. (A) Control, luciferase RNAi-treated D. melanogaster S2 cells on concanavalin A–coated coverslips spread symmetrical, round lamellipodia. S2 cells depleted of the Arp2 subunit by siRNA (Arp2 RNAi) exhibited a stellate morphology with membrane protrusions radiating from the cell body. A wild-type morphology of circular lamellipodia was restored in Arp2-depleted S2 cells expressing wild-type Arp2-GFP (Arp2-WT-GFP), or mutated Arp2-T237/238A-GFP or Arp2-Y202A-GFP. Arp2-depleted S2 cells expressing the triple mutant Arp2-T237/238A-Y202A-GFP remained stellate. Bar, 5 μM. (B) The number of cells with wild-type or stellate morphology were scored for the indicated conditions and expressed as the mean ± SD of three separate cell preparations, with >500 cells scored for each condition per cell preparation. (C) Model for activation of the Arp2/3 complex by phosphorylation of the Arp2 subunit. In the “inactive” form of the Arp2/3 complex, the Arp2 and Arp3 subunits are splayed open. Phosphorylated T237/T238 and Y202 residues in Arp2 interact with arginine residues on adjacent subunits to maintain a conformation that stabilizes the complex in the “active” form. This conformation aligns the Arp2 and Arp3 subunits to form a protonucleus for actin nucleation.