The role of the Rho family GTPases and their effectors in neurite guidance

The Rho family GTPases play a crucial role in re-arranging the actin cytoskeleton required for neuronal guidance; they mediate their effect through binding to downstream partners - the 'effectors'. In this study, signalling through the Rho GTPases and their effectors was investigated in N1E-115 neuroblastoma cells grown on laminin. The cells were found to respond positively to acetylcholine and insulin-like growth factor which caused morphological differentiation, including neurite outgrowth, as well as acting as attractive guidance cues. The cells responded negatively to a boundary between laminin and chondroitin sulphate proteoglycan (CPSG) or tenascin, which acted as repulsive guidance cues, with most neurites avoiding the boundary. Transient transfection with a dominant negative form of RhoA, RhoA N19 increased the number of neurites crossing onto CSPG or tenascin implicating RhoA in the repulsive guidance response. However, blocking ROK by its inhibitor Y27632 caused only a very small increase in neurite-crossing, suggesting that ROK may only be partially responsible. Furthermore, cells treated with C3 toxin that inactivates RhoA also showed no increase in such crossing. Over-expression of a membrane-targeted form of the Racl/Cdc42 effector αPAK, PAK-CAAX, caused a significant increase in crossing and a kinase-dead form, PAK-CAAX KD, had an even greater effect. The involvement of PAK α, β and γ isoforms was studied by using sequence-specific peptides to inhibit PAK function. There was a significant increase in neurite-crossing at the laminin/CSPG boundary when cells contained a truncated N-terminal γPAK peptide but not the corresponding αPAK or βPAK peptide. The Cdc42 effectors IRS 58 and N-WASP were found not to affect this crossing, when over-expressed. Taken together, these results suggest that guidance away from a repulsive CPSG signal involves both PAK and a balance of signalling through RhoA and Rac1