No abstract

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.

Coordinated regulation of actin cytoskeletal dynamics is critical to growth cone movement. The intracellular molecules calmodulin and profilin actively regulate actin-based motility and participate in the signaling pathways used to steer growth cones. Here we show that in the developing Drosophila embryo, calmodulin and profilin convey complimentary information that is necessary for appropriate growth cone advance. Reducing calmodulin activity by expression of a dominant inhibitor (KA) stalls axon extension of pioneer neurons within the CNS, while a partial loss of profilin function decreases extension of motor axons in the periphery. Yet, surprisingly, when calmodulin and profilin are simultaneously reduced, the ability of both CNS pioneer axons and motor axons to extend beyond the choice points is restored. In the CNS, at the time when growth cones must decide whether to cross or not to cross the midline, a reduction in calmodulin and/or roundabout signaling causes axons to cross the midline inappropriately. These inappropriate crossings are suppressed when profilin activity is simultaneously reduced. Interestingly, the mutual suppression of calmodulin and profilin activity requires a minimal level of profilin. In KA combinations with profilin null alleles, defects in axon extension and midline guidance are synergistically enhanced rather than suppressed. Together, our data indicate that the growth cone must coordinate the activity of both calmodulin and profilin in order to advance past selected choice points, including those dictating midline crossovers.