An essential role for β-actin mRNA localization and translation in Ca2+-dependent growth cone guidance

Abstract

Axon pathfinding requires directional responses of growth cones to extracellular cues, which have been shown to involve local synthesis of protein. The identity and functions of the locally produced proteins remain, however, unclear. Here we report that Ca2+-dependent bidirectional turning of Xenopus laevis growth cones requires localized distribution and translation of β-actin messenger RNA. Both β-actin mRNA and its zipcode-binding protein, ZBP1, are localized at the growth cone and become asymmetrically distributed upon local exposure to brain-derived neurotrophic factor (BDNF). Inhibition of protein synthesis or antisense interference with β-actin mRNA–ZBP1 binding abolishes both Ca2+-mediated attraction and repulsion. In addition, attraction involves a local increase in β-actin, whereas repulsion is accompanied by a local decrease in β-actin; thus, both produce a synthesis- and ZBP1 binding–dependent β-actin asymmetry but with opposite polarities. Together with a similar asymmetry in Src activity during bidirectional responses, our findings indicate that Ca2+-dependent spatial regulation of β-actin synthesis through Src contributes to the directional motility of growth cones during guidance.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Inhibition of protein synthesis blocks BDNF-induced growth cone turning.
Figure 2: Growth cone turning by FLIP of caged Ca2+ depends on protein synthesis.
Figure 3: Spatial distribution of β-actin mRNA and ZBP1 proteins in X. laevis neurons and growth cones.
Figure 4: Asymmetric distribution of β-actin mRNA and ZBP1 in growth cones during response to local BDNF application.
Figure 5: Ca2+-dependent growth cone turning depends on β-actin mRNA–ZBP1 interactions.
Figure 6: Asymmetric increase in β-actin in growth cones by local BDNF application.
Figure 7: Reverse asymmetry of β-actin distribution in growth cones during repulsion induced by a BDNF gradient under PKA inhibition.
Figure 8: Asymmetric activation of the Src family of kinases by BDNF gradients.

References

  1. 1

    Tessier-Lavigne, M. & Goodman, C.S. The molecular biology of axon guidance. Science 274, 1123–1133 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Dickson, B.J. Molecular mechanisms of axon guidance. Science 298, 1959–1964 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Kalil, K. & Dent, E.W. Touch and go: guidance cues signal to the growth cone cytoskeleton. Curr. Opin. Neurobiol. 15, 521–526 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Dent, E.W. & Gertler, F.B. Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40, 209–227 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Korey, C.A. & Van Vactor, D. From the growth cone surface to the cytoskeleton: one journey, many paths. J. Neurobiol. 44, 184–193 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Campbell, D.S. & Holt, C.E. Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 32, 1013–1026 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Campbell, D.S. & Holt, C.E. Apoptotic pathway and MAPKs differentially regulate chemotropic responses of retinal growth cones. Neuron 37, 939–952 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Ming, G.L. et al. Adaptation in the chemotactic guidance of nerve growth cones. Nature 417, 411–418 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Brittis, P.A., Lu, Q. & Flanagan, J.G. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell 110, 223–235 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Piper, M., Salih, S., Weinl, C., Holt, C.E. & Harris, W.A. Endocytosis-dependent desensitization and protein synthesis-dependent resensitization in retinal growth cone adaptation. Nat. Neurosci. 8, 179–186 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Moccia, R. et al. An unbiased cDNA library prepared from isolated aplysia sensory neuron processes is enriched for cytoskeletal and translational mRNAs. J. Neurosci. 23, 9409–9417 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Willis, D. et al. Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons. J. Neurosci. 25, 778–791 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Piper, M. & Holt, C. RNA translation in axons. Annu. Rev. Cell Dev. Biol. 20, 505–523 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Lee, S.K. & Hollenbeck, P.J. Organization and translation of mRNA in sympathetic axons. J. Cell Sci. 116, 4467–4478 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Ming, G.L., Lohof, A.M. & Zheng, J.Q. Acute morphogenic and chemotropic effects of neurotrophins on cultured embryonic Xenopus spinal neurons. J. Neurosci. 17, 7860–7871 (1997).

    CAS  Article  Google Scholar 

  16. 16

    Song, H.J., Ming, G.L. & Poo, M.M. cAMP-induced switching in turning direction of nerve growth cones. Nature 388, 275–279 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Guirland, C., Suzuki, S., Kojima, M., Lu, B. & Zheng, J.Q. Lipid rafts mediate chemotropic guidance of nerve growth cones. Neuron 42, 51–62 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Li, Y. et al. Essential role of TRPC channels in the guidance of nerve growth cones by brain-derived neurotrophic factor. Nature 434, 894–898 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Zheng, J.Q. Turning of nerve growth cones induced by localized increases in intracellular calcium ions. Nature 403, 89–93 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Wen, Z., Guirland, C., Ming, G.L. & Zheng, J.Q.A. CaMKII/calcineurin switch controls the direction of Ca2+-dependent growth cone guidance. Neuron 43, 835–846 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Zhang, H.L. et al. Neurotrophin-induced transport of a β-actin mRNP complex increases β-actin levels and stimulates growth cone motility. Neuron 31, 261–275 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Yisraeli, J.K. VICKZ proteins: a multi-talented family of regulatory RNA-binding proteins. Biol. Cell. 97, 87–96 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Yaniv, K., Fainsod, A., Kalcheim, C. & Yisraeli, J.K. The RNA-binding protein Vg1 RBP is required for cell migration during early neural development. Development 130, 5649–5661 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Farina, K.L., Huttelmaier, S., Musunuru, K., Darnell, R. & Singer, R.H. Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment. J. Cell Biol. 160, 77–87 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Huttelmaier, S. et al. Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512–515 (2005).

    Article  Google Scholar 

  26. 26

    Robles, E., Woo, S. & Gomez, T.M. Src-dependent tyrosine phosphorylation at the tips of growth cone filopodia promotes extension. J. Neurosci. 25, 7669–7681 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Bassell, G.J. & Kelic, S. Binding proteins for mRNA localization and local translation, and their dysfunction in genetic neurological disease. Curr. Opin. Neurobiol. 14, 574–581 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Steward, O. Translating axon guidance cues. Cell 110, 537–540 (2002).

    Article  Google Scholar 

  29. 29

    Martin, K.C. Local protein synthesis during axon guidance and synaptic plasticity. Curr. Opin. Neurobiol. 14, 305–310 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Willis, D.E. & Twiss, J.L. The evolving roles of axonally synthesized proteins in regeneration. Curr. Opin. Neurobiol. 16, 111–118 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Bassell, G.J. et al. Sorting of β-actin mRNA and protein to neurites and growth cones in culture. J. Neurosci. 18, 251–265 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Zhang, H.L., Singer, R.H. & Bassell, G.J. Neurotrophin regulation of β-actin mRNA and protein localization within growth cones. J. Cell Biol. 147, 59–70 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Leung, K.-M. et al. Asymmetrical β-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat. Neurosci. 9, 1247–1256 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Kislauskis, E.H., Zhu, X. & Singer, R.H. β-Actin messenger RNA localization and protein synthesis augment cell motility. J. Cell Biol. 136, 1263–1270 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Oleynikov, Y. & Singer, R.H. Real-time visualization of ZBP1 association with β-actin mRNA during transcription and localization. Curr. Biol. 13, 199–207 (2003).

    CAS  Article  Google Scholar 

  36. 36

    Hong, K., Nishiyama, M., Henley, J., Tessier-Lavigne, M. & Poo, M. Calcium signalling in the guidance of nerve growth by netrin-1. Nature 403, 93–98 (2000).

    CAS  Article  Google Scholar 

  37. 37

    Henley, J.R., Huang, K.H., Wang, D. & Poo, M.M. Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein. Neuron 44, 909–916 (2004).

    CAS  Article  Google Scholar 

  38. 38

    Gomez, T.M. & Zheng, J.Q. The molecular basis for calcium-dependent axon pathfinding. Nat. Rev. Neurosci. 7, 115–125 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Shuster, C.B. & Herman, I.M. Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity. J. Cell Biol. 128, 837–848 (1995).

    CAS  Article  Google Scholar 

  40. 40

    Shestakova, E.A., Singer, R.H. & Condeelis, J. The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility. Proc. Natl. Acad. Sci. USA 98, 7045–7050 (2001).

    CAS  Article  Google Scholar 

  41. 41

    Wang, J. et al. Reversible glutathionylation regulates actin polymerization in A431 cells. J. Biol. Chem. 276, 47763–47766 (2001).

    CAS  Article  Google Scholar 

  42. 42

    Zheng, J.Q., Wan, J.J. & Poo, M.M. Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient. J. Neurosci. 16, 1140–1149 (1996).

    CAS  Article  Google Scholar 

  43. 43

    Rehder, V. & Kater, S.B. Regulation of neuronal growth cone filopodia by intracellular calcium. J. Neurosci. 12, 3175–3186 (1992).

    CAS  Article  Google Scholar 

  44. 44

    Piper, M. et al. Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones. Neuron 49, 215–228 (2006).

    CAS  Article  Google Scholar 

  45. 45

    Wu, K.Y. et al. Local translation of RhoA regulates growth cone collapse. Nature 436, 1020–1024 (2005).

    CAS  Article  Google Scholar 

  46. 46

    Guirland, C., Buck, K.B., Gibney, J.A., DiCicco-Bloom, E. & Zheng, J.Q. Direct cAMP signaling through G-protein-coupled receptors mediates growth cone attraction induced by pituitary adenylate cyclase-activating polypeptide. J. Neurosci. 23, 2274–2283 (2003).

    CAS  Article  Google Scholar 

  47. 47

    Peng, H.B., Baker, L.P. & Chen, Q. Tissue culture of Xenopus neurons and muscle cells as a model for studying synaptic induction. Methods Cell Biol. 36, 511–526 (1991).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank C.E. Holt and colleagues (University of Cambridge) for sharing unpublished work and R.H. Singer (Albert Einstein College of Medicine) for the antibody to ZBP1. This work was supported by grants from the US National Institutes of Health (NS36241 to J.Q.Z. and HD46368 to G.J.B.) and a research grant from New Jersey Commission on Spinal Cord Research (04-3029-SCR to J.Q.Z.).

Author information

Affiliations

Authors

Contributions

J.Y. performed most of the experiments and analyses in this study. Y.S. designed zipcode antisense and did FISH and immunostaining on β-actin mRNA and ZBP1. Z.W. assisted with the turning assay. G.J.B. contributed to the development of the project and directed the antisense and FISH experiments. J.Q.Z. designed and oversaw the research project, and directed most of the experiments, analyses and writing.

Corresponding author

Correspondence to James Q Zheng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

The schematic diagram showing the design of antisense oligonucleotides to the Xenopus β-actin zipcode sequences. (PDF 23 kb)

Supplementary Fig. 2

BDNF induced co-localization of β-actin mRNA and ZBP1 in growth cone filopodia. (PDF 25 kb)

Supplementary Fig. 3

Analysis of β-actin asymmetry in Xenopus growth cones exposed to a BDNF gradient. (PDF 35 kb)

Supplementary Fig. 4

The hypothesized model on spatial regulation of β-actin local synthesis in the growth cone. (PDF 73 kb)

Supplementary Fig. 5

Filopodia elicitation by KCl depolarization depends on protein synthesis and β-actin translocation. (PDF 317 kb)

Supplementary Methods (PDF 99 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yao, J., Sasaki, Y., Wen, Z. et al. An essential role for β-actin mRNA localization and translation in Ca2+-dependent growth cone guidance. Nat Neurosci 9, 1265–1273 (2006). https://doi.org/10.1038/nn1773

Download citation

Further reading