Cyclic Nucleotide Control of Microtubule Dynamics for Axon Guidance.

unlabelledGraded distribution of intracellular second messengers, such as Ca(2+) and cyclic nucleotides, mediates directional cell migration, including axon navigational responses to extracellular guidance cues, in the developing nervous system. Elevated concentrations of cAMP or cGMP on one side of the neuronal growth cone induce its attractive or repulsive turning, respectively. Although effector processes downstream of Ca(2+) have been extensively studied, very little is known about the mechanisms that enable cyclic nucleotides to steer migrating cells. Here, we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance via modulating microtubule dynamics and membrane organelle transport. In embryonic chick dorsal root ganglion neurons in culture, contact of an extending microtubule with the growth cone leading edge induces localized membrane protrusion at the site of microtubule contact. Such a contact-induced protrusion requires exocytosis of vesicle-associated membrane protein 7 (VAMP7)-positive vesicles that have been transported centrifugally along the microtubule. We found that the two cyclic nucleotides counteractively regulate the frequency of microtubule contacts and targeted delivery of VAMP7 vesicles: cAMP stimulates and cGMP inhibits these events, thereby steering the growth cone in the opposite directions. By contrast, Ca(2+) signals elicit no detectable change in either microtubule contacts or VAMP7 vesicle delivery during Ca(2+)-induced growth cone turning. Our findings clearly demonstrate growth cone steering machinery downstream of cyclic nucleotide signaling and highlight a crucial role of dynamic microtubules in leading-edge protrusion for cell chemotaxis. SIGNIFICANCE STATEMENT: Developing neurons can extend long axons toward their postsynaptic targets. The tip of each axon, called the growth cone, recognizes extracellular guidance cues and navigates the axon along the correct path. Here we show that asymmetric cyclic nucleotide signaling across the growth cone mediates axon guidance through localized regulation of microtubule dynamics and resulting recruitment of specific populations of membrane vesicles to the growth cone's leading edge. Remarkably, cAMP stimulates microtubule growth and membrane protrusion, whereas cGMP promotes microtubule retraction and membrane senescence, explaining the opposite directional polarities of growth cone turning induced by these cyclic nucleotides. This study reveals a novel microtubule-based mechanism through which cyclic nucleotides polarize the growth cone steering machinery for bidirectional axon guidance.