Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.

BACKGROUND: Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. RESULTS: Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. CONCLUSIONS: Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

A cytoskeletal platform for local translation in axons.

Recent data indicate that local translation in growth cones is critical for axon guidance. Evidence from the Drosophila midline axon guidance system suggests that the F-actin-microtubule cross-linker Short stop (Shot) might link the translation machinery to the cytoskeleton in the growth cone. The identification of a complex of translation factors attached to the actin and microtubule networks points to a mechanism by which cytoskeletal dynamics regulate translation in axons and vice versa.

A role for S1P signalling in axon guidance in the Xenopus visual system.

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.

Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1.

Local protein synthesis regulates the turning of growth cones to guidance cues, yet little is known about which proteins are synthesized or how they contribute to directional steering. Here we show that beta-actin mRNA resides in Xenopus laevis retinal growth cones where it binds to the RNA-binding protein Vg1RBP. Netrin-1 induces the movement of Vg1RBP granules into filopodia, suggesting that it may direct the localization and translation of mRNAs in growth cones. Indeed, a gradient of netrin-1 activates a translation initiation regulator, eIF-4E-binding protein 1 (4EBP), asymmetrically and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. Inhibition of beta-actin translation abolishes both the asymmetric rise in beta-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized beta-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus.