Pathfinding by nerve growth cones depends on attractive and repulsive turning in response to a variety of guidance cues. Here we present direct evidence to demonstrate an essential and instructive role for microtubules (MTs) in growth cone steering. First, both growth cone attraction and repulsion induced by diffusible cues in culture can be completely blocked by low concentrations of drugs that specifically inhibit dynamic microtubule ends in the growth cone. Second, direct focal photoactivated release of the microtubule-stabilizing drug taxol on one side of the growth cone consistently induces attraction (turning toward the site of application). Using the focal pipette application method, we also show that local MT stabilization by taxol induces growth cone attraction, whereas local MT destabilization by the microtubule-disrupting drug nocodazole induces repulsion (turning away). Finally, the microtubule-initiated attractive turning requires the participation of the actin cytoskeleton: local microtubule stabilization induces preferential protrusion of lamellipodia before the attractive turning, and the attraction can be abolished by inhibition of either actin polymerization or the Rho family GTPases. Together, these results demonstrate a novel steering mechanism for growth cones in which local and selective modification of dynamic microtubules can initiate and instruct directional steering. With the subsequent concerted activity of the actin cytoskeleton, this microtubule-initiated mechanism provides the growth cone with the additional means to efficiently navigate through its environment.

Developing axons are guided to their appropriate targets by environmental cues through the activation of specific receptors and intracellular signaling pathways. Here we report that gradients of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide widely expressed in the developing nervous system, induce marked attraction of Xenopus growth cones in vitro. PACAP exerted its chemoattractive effects through PAC1, a PACAP-selective G-protein-coupled receptor (GPRC) expressed at the growth cone. Furthermore, the attraction depended on localized cAMP signaling because it was completely blocked either by global elevation of intracellular cAMP levels using forskolin or by inhibition of protein kinase A using specific inhibitors. Moreover, local direct elevation of intracellular cAMP by focal photolysis of caged cAMP compounds was sufficient to induce growth cone attraction. On the other hand, blockade of Ca 2ϩ , phospholipase C, or phosphatidyl inositol-3 kinase signaling pathways did not affect PACAP-induced growth cone attraction. Finally, PACAP-induced attraction also involved the Rho family of small GTPases and required local protein synthesis. Taken together, our results establish cAMP signaling as an independent pathway capable of mediating growth cone attraction induced by a physiologically relevant peptide acting through GPCRs. Such a direct cAMP pathway could potentially operate in other guidance systems for the accurate wiring of the nervous system.

Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

In growth cones, local Arp 2/3-dependent actin assembly mechanically buffers apCAM adhesions from retrograde flow–associated traction forces. The resulting propulsive forces drive the exploratory motility of inductopodia. Increasing the stiffness of apCAM targets induces an extensive 3D actin cup to form at the adhesion during evoked growth responses.