A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner.

Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms--l-afadin, having the F-actin-binding domain, and s-afadin, lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching.

R-Ras controls axon branching through afadin in cortical neurons.

Regulation of axon growth, guidance, and branching is essential for constructing a correct neuronal network. R-Ras, a Ras-family small GTPase, has essential roles in axon formation and guidance. During axon formation, R-Ras activates a series of phosphatidylinositol 3-kinase signaling, inducing activation of a microtubule-assembly promoter-collapsin response mediator protein-2. However, signaling molecules linking R-Ras to actin cytoskeleton-regulating axonal morphology remain obscure. Here we identify afadin, an actin-binding protein harboring Ras association (RA) domains, as an effector of R-Ras inducing axon branching through F-actin reorganization. We observe endogenous interaction of afadin with R-Ras in cortical neurons during the stage of axonal development. Ectopic expression of afadin increases axon branch number, and the RA domains and the carboxyl-terminal F-actin binding domain are required for this action. RNA interference knockdown experiments reveal that knockdown of endogenous afadin suppressed both basal and R-Ras-mediated axon branching in cultured cortical neurons. Subcellular localization analysis shows that active R-Ras-induced translocation of afadin and its RA domains is responsible for afadin localizing to the membrane and inducing neurite development in Neuro2a cells. Overall, our findings demonstrate a novel signaling pathway downstream of R-Ras that controls axon branching.