The small GTPase Rif is an alternative trigger for the formation of actin stress fibers in epithelial cells.

Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation--the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.