Role of rho-family guanosine triphosphatase effectors in filopodia dynamics

Filopodia play sensory roles by acting like 'antennae' to sense the cell's surroundings. In nerve growth cone, they promote motility towards an attractive cue or away from a repulsive one. Filopodia have been reported to be involved in wound healing, adhesion to the extracellular matrix and embryonic development. A number of cytoskeletal regulatory proteins have been implicated in regulating initiation, formation, maintenance and extension/retraction cycle of such protrusions. Some of these proteins either bind to the Rho family GTPases, Cdc42 and Rac, as effectors or function downstream of such effectors to regulate signaling pathways involved in cytoskeletal reorganization.;The purpose of this study was to determine whether certain proteins, which had well-defined binding interfaces with proteins downstream of GTPase-regulated proteins, were implicated in filopodial dynamics. Synthetic binding peptides (BPs) were used to impede the interaction of Cdc42 and Rac with the effectors and to interfere at protein-protein binding interfaces downstream of the GTPases. Single cell peripheries were analyzed to determine the levels of positive outgrowths of filopodia. This assay allowed for rapid determination of whether the BPs had an effect on filopodia formation. BPs, namely IQGAP (132--140), ACK and Par6 showed a negative effect on filopodia, whereas IQGAP (84--93), PAK and WASP elevated filopodia formation or had no effect compared to control. When interactions of PAK2 with Abl and PAK4 with integrin-beta5 were inhibited, filopodia prevalence increased.;Based on several previous findings, the tyrosine kinase ACK is thought to mediate internalization, processing and trafficking pathways of cell surface receptors, especially epidermal growth factor receptors (EGFRs). ACK is an oncogene and its overexpression or mutation is implicated in several human cancers. During this study, when the E3 ubiquitin ligase, neural precursor cell expressed developmentally downregulated protein (Nedd4) was inhibited from binding to ACK, both cell periphery coverage with filopodia and number of cells showing filopodia, increased significantly compared to control. Since earlier findings have suggested that ACK and RTK (especially EGFR) are co-processed, this study suggests that, ACK-mediated RTK processing may play a role in regulating filopodial dynamics.