Negative Guidance Factor-Induced Macropinocytosis in the Growth Cone Plays a Critical Role in Repulsive Axon Turning

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.