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

Nature Neuroscience volume 9, pages 1247–1256 (2006)Cite this article

Abstract

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 β-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 β-actin translation on the near side of the growth cone before growth cone turning. Inhibition of β-actin translation abolishes both the asymmetric rise in β-actin and attractive, but not repulsive, turning. Our data suggest that newly synthesized β-actin, concentrated near sites of signal reception, provides the directional bias for polymerizing actin in the direction of an attractive stimulus.

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Figure 1: Vg1RBP is expressed in retinal growth cones and interacts with β-actin mRNA.
Figure 2: Netrin-1 or cell-contact induce translocation of Vg1RBP-eGFP into filopodia.
Figure 3: Netrin-1 induces β-actin translation driven by its 3′ UTR.
Figure 4: Vg1RBP granules move into filopodia closest to a netrin-1 source.
Figure 5: Netrin-1 gradient causes asymmetric activation of translation regulator.
Figure 6: Netrin-1 gradient elicits asymmetric increase of β-actin across the growth cone.
Figure 7: β-actin morpholinos block netrin-1–induced attractive turning.

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Acknowledgements

We thank M. Spira for suggesting use of Kaede–β-actin 3′ UTR; R. Adams for suggesting use of the Zenon labeling kit; J. Ireland and B. Kvinlaug for preliminary data on β-actin translation; J. Zheng, D. Campbell and L. Strochlic for sharing unpublished data; A. Dwivedy, I. Pradel and K. Zivraj for technical assistance; L. Poggi and J. Falk for help with image analysis; and W. Harris, M. Piper and D. Campbell for comments on the manuscript. This work was supported by a Croucher Scholarship (K.-M.L.), an EMBO Long Term Fellowship (F.P.G.v.H.), an NSF Graduate Research Fellowship (A.C.L.), a BBSRC Studentship (R.A.) and a Wellcome Trust Programme Grant (C.E.H.).

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Author notes

  1. Kin-Mei Leung and Francisca PG van Horck: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK

    Kin-Mei Leung, Francisca PG van Horck, Andrew C Lin & Christine E Holt

  2. Departments of Biochemistry, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK

    Rachel Allison & Nancy Standart

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Contributions

K.-M.L. did the experiments on β-actin synthesis and function in Figures 3, 6, 7 and Supplementary Figure 2. F.P.G.v.H. did the experiments on Vg1RBP in Figures 1, 2, 4, Supplementary Figures 1 and 2k–o and Supplementary Videos 1,2,3,4. A.C.L. did the gradient assays on 4EBP and β-actin in Figures 5 and 6e. R.A. and N.S. provided Vg1RBP reagents and constructs and discussed experiments. K.-M.L., F.P.G.v.H., A.C.L. and C.E.H. wrote the manuscript and discussed experiments.

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Correspondence to Christine E Holt.

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Supplementary information

Supplementary Fig. 1

Netrin-1 induces transport of endogenous Vg1RBP and β-actin mRNA into filopodia. (PDF 2415 kb)

Supplementary Fig. 2

Differential β-actin translation in response to attractive and repulsive cues. (PDF 2725 kb)

Supplementary Fig. 3

Model for polarized synthesis of β-actin. (PDF 696 kb)

Supplementary Video 1

Movement of Vg1RBP-eGFP granules in retinal growth cones. Retinal explants taken from stage 33/34 embryos injected with Vg1RBP-eGFP mRNA were cultured for 24 hours and analyzed in real time. Images were taken every 12 seconds. Vg1RBP-eGFP granules show bidirectional movement in the axon shaft, growth cone central domain and filopodia. (MOV 1151 kb)

Supplementary Video 2

The effect of cytochalasin D on Vg1RBP-eGFP granules movement. Live imaging of Vg1RBP-eGFP positive growth cones. Bath application of cytochalasin D (0.1 μM) results in retraction of Vg1RBP-eGFP granules from the growth cone. Images were taken every 12 seconds. (MOV 598 kb)

Supplementary Video 3

Movement of Vg1RBP-eGFP granules into filopodial contact sites. Live imaging of Vg1RBP-eGFP positive growth cones. The arrows indicate anterograde transport of a Vg1RBP-eGFP granule moving to the contact site, followed by a pause and retrograde movement to the base of the filopodium. Images were taken every 12 seconds. (MOV 1297 kb)

Supplementary Video 4

Netrin-1 induces transport of Vg1RBP-eGFP granules into filopodia. Vg1RBP-eGFP expressing retinal growth cones were stimulated with netrin-1 after 5 minutes and followed in real time. Images were taken every 12 seconds for 15 minutes. The arrows indicate filopodia in which the increased transport of Vg1RBP-eGFP granules is very prominent. (MOV 1675 kb)

Supplementary Methods (PDF 131 kb)

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Leung, KM., van Horck, F., Lin, A. et al. Asymmetrical β-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat Neurosci 9, 1247–1256 (2006). https://doi.org/10.1038/nn1775

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