Axon extension and guidance require a coordinated assembly of F-actin and microtubules as well as regulated translation. The molecular basis of how the translation of mRNAs encoding guidance proteins could be closely tied to the pace of cytoskeletal assembly is poorly understood. Previous studies have shown that the F-actin-microtubule crosslinker Short stop (Shot) is required for motor and sensory axon extension in the Drosophila embryo. Here, we provide biochemical and genetic evidence that Shot functions with a novel translation inhibitor, Krasavietz (Kra, Exba), to steer longitudinally directed CNS axons away from the midline. Kra binds directly to the C-terminus of Shot, and this interaction is required for the activity of Shot to support midline axon repulsion. shot and kra mutations lead to weak robo-like phenotypes, and synergistically affect midline avoidance of CNS axons. We also show that shot and kra dominantly enhance the frequency of midline crossovers in embryos heterozygous for slit or robo, and that in kra mutant embryos, some Robo-positive axons ectopically cross the midline that normally expresses the repellent Slit. Finally, we demonstrate that Kra also interacts with the translation initiation factor eIF2␤ and inhibits translation in vitro. Together, these data suggest that Kra-mediated translational regulation plays important roles in midline axon repulsion and that Shot functions as a direct physical link between translational regulation and cytoskeleton reorganization.

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Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. Here, we present evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, our results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline.

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