Previous studies showed that UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5 control axon guidance by regulating protrusion of growth cone lamellipodia and filopodia (Norris et al., 2011; Norris et al., 2014). UNC-6 stimulates protrusion in growth cones attracted to UNC-6 via the UNC-40 homodimeric receptor, and inhibits protrusion in growth cones repelled from UNC-6 via the UNC-5/UNC-40 heterodimeric receptor. Protrusion via UNC-40 requires CDC-42, the Rac GTPases MIG-2 and CED-10, the Rac GEF TIAM-1, and the actin modulating molecules Arp2/3 and UNC-115/abLIM. We delineated a pathway downstream of UNC-5/UNC-40 required to inhibit protrusion that also involves the Rac GTPases and a distinct GEF, UNC-73/Trio, as well as the microtubule-interacting protein UNC-33, similar to Collapsin response mediating protein (CRMP). In repelled VD growth cones, it is likely that both of these complexes are active, as unc-40 mutants suppress the excess growth cone protrusion of unc-5 mutants (Norris et al., 2011). UNC-5 and UNC-6 restrict F-actin accumulation to the dorsal, protrusive region of the VD growth cone (Norris et al., 2011).
unc-33 mutant VD growth cones resembled unc-5, with excess lamellipodial and filopodial protrusion. We used EBP-2::GFP expression in VD neurons to monitor microtubule + ends in VD growth cones. We found that unc-33 mutants displayed greatly increased numbers of EBP-2::GFP puncta in VD growth cones and protrusions, suggesting that UNC-33 normally prevents MT + end accumulation in growth cones. unc-44/ankyrin mutants showed the same phenotype. Importantly, unc-5 mutants also showed increased numbers of EBP-2::GFP puncta in growth cones, consistent with UNC-33 acting in the UNC-5 pathway. Interestingly, unc-73(rh40), which specifically affects the Rac GEF domain, did not cause increased EBP-2::GFP puncta despite having excessively protrusive growth cones similar to UNC-33, indicating that increased numbers of EBP-2::GFP puncta were not a secondary consequence of an enlarged growth cone. unc-73(e936), which affects both the Rac and Rho GEF domains, did show increased EBP-2::GFP puncta similar to unc-33. These experiments suggest MT-dependent and independent mechanisms, and possibly that the MT-dependent mechanism involves Rho and the independent mechanism involves Rac and possibly actin. In sum, these studies indicate that growth cone protrusion can be inhibited by restricting MT + ends from growth cones, and that UNC-33 is involved in this process. Asymmetric regulation of UNC-33 across the growth cone might result in directed migration away from UNC-6 in axon repulsion.