Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts

We investigated the effect of actin filament barbed end uncapping on Arp2/3 complex function both in vivo and in vitro. Arp2/3 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp2/3 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp2/3 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp2/3 complex nucleation. We conclude that Arp2/3 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.