The Rho GTPase Rif signals through IRTKS, Eps8 and WAVE2 to generate dorsal membrane ruffles and filopodia

Rif induces dorsal filopodia but the signalling pathway responsible for this has not been identified. We show here that Rif interacts with the I-BAR family protein IRTKS via its I-BAR domain. Rif also interacts with Pinkbar in N1E-115 mouse neuroblastoma cells. IRTKS and Rif induce dorsal membrane ruffles and filopodia. Dominant negative Rif inhibits the formation of IRTKS-induced morphological structures and Rif activity is blocked in IRTKS KO cells. To further define the Rif-IRTKS signalling pathway, we identify Eps8 and WAVE2 as IRTKS interactors. We find that Eps8 regulates the size and number of dorsal filopodia and membrane ruffles downstream of Rif-IRTKS, while WAVE2 modulates dorsal membrane ruffling. Furthermore, our data suggests that Tir, a protein essential for enterohemorrhagic E.coli infection, may compete for Rif for interaction with the I-BAR domain of IRKS. Based on these evidences we propose a model in which Rho family GTPases use the I-BAR proteins, IRSp53, IRTKS and Pinkbar, as a central mechanism to modulate cell morphology.