R-Ras controls axon branching through afadin in cortical neurons

• Published in: Molecular biology of the cell 2012-07, Vol.23 (14), p.2793-2804
• Main Authors: Iwasawa, Nariaki, Negishi, Manabu, Oinuma, Izumi
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - metabolism; Animals; Axons - metabolism; Axons - physiology; Axons - ultrastructure; Cell Differentiation; Cell Line; Cell Proliferation; Cerebral Cortex - metabolism; HEK293 Cells; Humans; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Mice; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Microtubules - genetics; Microtubules - metabolism; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Neurogenesis; Phosphatidylinositol 3-Kinase - metabolism; Promoter Regions, Genetic; ras Proteins - metabolism; RNA Interference; RNA, Small Interfering; Signal Transduction
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.e12-02-0103

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.