Filopodia are dynamic actin-rich cell surface protrusions involved in cell 
migration, axon guidance, and wound healing. The RhoGTPase Cdc42 generates 
filopodia via IRSp53, a multidomain protein that links the processes of plasma 
membrane deformation and actin dynamics required for their formation in 
mammalian cells. The Src homology 3 domain of IRSp53 binds to the actin 
regulators Mena, Eps8, WAVE1, WAVE2, mDia1, and mDia2. We show that mDia1 and 
WAVE2 synergize with IRSp53 to form filopodia. IRSp53 also interacts directly 
with these two proteins within filopodia, as observed in acceptor photobleaching 
FRET studies. Measurement of filopodium formation by time-lapse imaging of live 
cells also revealed that depleting neuronal cells of either mDia1 or WAVE2 
protein decreases the ability of IRSp53 to induce filopodia. In contrast, IRSp53 
does not appear to partner WAVE1 or mDia2 to give rise to these structures. In 
addition, although all three isoforms of mDia are capable of inducing filopodia, 
IRSp53 requires only mDia1 to do so. These findings suggest that mDia1 and WAVE2 
are important Src homology 3 domain partners of IRSp53 in forming filopodia.