Actin-crosslinking proteins organize actin into highly dynamic and 
architecturally diverse subcellular scaffolds that orchestrate a variety of 
mechanical processes, including lamellipodial and filopodial protrusions in 
motile cells. How signalling pathways control and coordinate the activity of 
these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can 
associate with the Rho-GTPases Rac and Cdc42, participates in these processes 
mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD 
has actin-bundling activity in vitro and is sufficient to induce filopodia in 
vivo. However, the manner of regulation of this activity in the full-length 
protein remains largely unknown. Eps8 is involved in actin dynamics through its 
actin barbed-ends capping activity and its ability to modulate Rac activity. 
Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking 
activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in 
mediating actin bundling in vitro, enhancing IRSp53-dependent membrane 
extensions in vivo. Cdc42 binds to and controls the cellular distribution of the 
IRSp53-Eps8 complex, supporting the existence of a Cdc42-IRSp53-Eps8 signalling 
pathway. Consistently, Cdc42-induced filopodia are inhibited following 
individual removal of either IRSp53 or Eps8. Collectively, these results support 
a model whereby the synergic bundling activity of the IRSp53-Eps8 complex, 
regulated by Cdc42, contributes to the generation of actin bundles, thus 
promoting filopodial protrusions.