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. This results in polarized distribution to the leading edge of the growth cone. 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). We used EBP-2::GFP expression in VD neurons to monitor MT + ends in VD growth cones. We found that mutations in components of the UNC-5, UNC-33/CRMP pathway result in excess EBP-2::GFP puncta in VD growth cones, suggesting that this pathway might normally restrict MT + end entry into the growth cones to inhibit growth cone protrusion. Interestingly, a mutation affecting specifically the Rac GEF domain of
unc-73 did not affect EBP-2::GFP distribution in growth cones despite having excessively protrusive growth cones. F-actin distribution was affected, indicating that inhibition of protrusion can act through both actin and MTs. We find that the MT-dependent mechanism involves RHO-1 and its upstream activator RHGF-1/PDZ RhoGEF.
rhgf-1 mutants show large protrusive growth cones similar to those seen in
unc-5 and
unc-33 mutants and displayed greatly increased numbers of EBP-2::GFP puncta in VD growth cones and protrusions. The dominant negative form of RHO-1,
rho-1(T19N) also resembles
unc-5, with excess lamellipodial and filopodial protrusions and show increased EBP-2::GFP puncta. In sum, these studies indicate that growth cone protrusion can be inhibited by UNC-6/Netrin signaling by restricting MT + ends from growth cones, and that ectopic MT + ends in growth cones drive ectopic protrusion.