The role of WASP family members and formin proteins in cell protrusion in carcinoma cells

Abstract

Three members of the WASP family, N-WASP, WAVE1 and WAVE2, which promote branched actin filament assembly via Arp2/3, are expressed in MTLn3 carcinoma cells. In this study, the role of N-WASP, WAVE2 and WAVE1 in carcinoma cell motility was investigated. We looked at the localization of WAVE2 and N-WASP after EGF-stimulation and in the case of N-WASP its activation. The role of WAVE2, WAVE1 and N-WASP was also examined following siRNA-mediated knockdown of each protein. The WASP family proteins are recruited to the leading edge at similar times. WAVE1 siRNA knockdown had no effect on lamellipod and/or ruffle formation. In contrast, WAVE2 knockdown suppressed EGF-induced formation of the branched actin network at the leading edge and inhibited lamellipod formation. N-WASP knockdown had no significant effect on lamellipod or filopod formation. However, the simultaneous knockdown of both WAVE2 and N-WASP caused cells to form large jagged protrusions and increased filopod formation. The results suggest that another source of actin nucleation activity is at work in carcinoma cells in response to EGF. Formin homology proteins, in contrast to WASP family proteins that induce Arp2/3-dependent dendritic nucleation, nucleate and processively elongate actin filaments through monomer addition at the barbed end of filaments. A mammalian Diaphanous (mDia) related formin, mDia1 was found to localize at the jagged protrusions formed by the WAVE2/N-WASP double knockdown cells. Constitutively active mDia1 expression recapitulated the phenotype of WAVE2/N-WASP knockdown cells. Conversely, inhibition of mDia1 by siRNA and an interfering dominant negative variant of mDia1 blocked the formation of the protrusions in WAVE2/N-WASP knockdown cells. Increased RhoA activity, which can stimulate mDia1 nucleation activity, was observed in the WAVE2/N-WASP knockdown cells. These data show that both WASP family and mDia proteins control actin assembly and cell protrusions involving the coordinated regulation between these pathways.