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The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity

Nature Neuroscience volume 7, pages 923–929 (2004)Cite this article

Abstract

The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKCλ or PKCζ) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.

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Figure 1: Polarized distribution of Rap1B.
Figure 2: Induction of supernumerary axons by Rap1B and Cdc42.
Figure 3: Distribution of axonal markers after expression of GTPases.
Figure 4: Rap1B and Cdc42 act sequentially.
Figure 5: The induction of multiple axons by actin depolymerization depends on Rap1B.

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References

  1. Da Silva, J.S. & Dotti, C.G. Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat. Rev. Neurosci. 3, 694–704 (2002).

    Article  CAS  Google Scholar 

  2. Horton, A.C. & Ehlers, M.D. Neuronal polarity and trafficking. Neuron 40, 277–295 (2003).

    Article  CAS  Google Scholar 

  3. Bradke, F. & Dotti, C.G. Establishment of neuronal polarity: lessons from cultured hippocampal neurons. Curr. Opin. Neurobiol. 10, 574–581 (2000).

    Article  CAS  Google Scholar 

  4. Dotti, C.G., Sullivan, C.A. & Banker, G.A. The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454–1468 (1988).

    Article  CAS  Google Scholar 

  5. Bradke, F. & Dotti, C.G. The role of local actin instability in axon formation. Science 283, 1931–1934 (1999).

    Article  CAS  Google Scholar 

  6. Macara, I.G. Parsing the polarity code. Nat. Rev. Mol. Cell Biol. 5, 220–231 (2004).

    Article  CAS  Google Scholar 

  7. Nishimura, T. et al. Role of the PAR-3-KIF3 complex in the establishment of neuronal polarity. Nat. Cell Biol. 6, 328–334 (2004).

    Article  CAS  Google Scholar 

  8. Shi, S.H., Jan, L.Y. & Jan, Y.N. Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6 and PI 3-kinase activity. Cell 112, 63–75 (2003).

    Article  CAS  Google Scholar 

  9. Etienne-Manneville, S. & Hall, A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCζ. Cell 106, 489–498 (2001).

    Article  CAS  Google Scholar 

  10. Lin, D. et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat. Cell Biol. 2, 540–547 (2000).

    Article  CAS  Google Scholar 

  11. Yamanaka, T. et al. PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. Genes Cells 6, 721–731 (2001).

    Article  CAS  Google Scholar 

  12. Sohrmann, M. & Peter, M. Polarizing without a c(l)ue. Trends Cell Biol. 13, 526–533 (2003).

    Article  CAS  Google Scholar 

  13. Chuckowree, J.A. & Vickers, J.C. Cytoskeletal and morphological alterations underlying axonal sprouting after localized transection of cortical neuron axons in vitro. J. Neurosci. 23, 3715–3725 (2003).

    Article  CAS  Google Scholar 

  14. Tu, S.S. et al. Antiapoptotic Cdc42 mutants are potent activators of cellular transformation. Biochemistry 41, 12350–12358 (2002).

    Article  CAS  Google Scholar 

  15. Caviston, J.P., Tcheperegine, S.E. & Bi, E. Singularity in budding: a role for the evolutionarily conserved small GTPase Cdc42p. Proc. Natl. Acad. Sci. USA 99, 12185–12190 (2002).

    Article  CAS  Google Scholar 

  16. Irazoqui, J.E., Gladfelter, A.S. & Lew, D.J. Scaffold-mediated symmetry breaking by Cdc42p. Nat. Cell Biol. 5, 1062–1070 (2003).

    Article  CAS  Google Scholar 

  17. Bito, H. et al. A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron 26, 431–441 (2000).

    Article  CAS  Google Scholar 

  18. Luo, L. et al. Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 379, 837–840 (1996).

    Article  CAS  Google Scholar 

  19. Luo, L. Rho GTPases in neuronal morphogenesis. Nat. Rev. Neurosci. 1, 173–180 (2000).

    Article  CAS  Google Scholar 

  20. Ng, J. et al. Rac GTPases control axon growth, guidance and branching. Nature 416, 442–447 (2002).

    Article  CAS  Google Scholar 

  21. Ruchhoeft, M.L., Ohnuma, S., McNeill, L., Holt, C.E. & Harris, W.A. The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPases in vivo. J. Neurosci. 19, 8454–8463 (1999).

    Article  CAS  Google Scholar 

  22. Threadgill, R., Bobb, K. & Ghosh, A. Regulation of dendritic growth and remodeling by Rho, Rac, and Cdc42. Neuron 19, 625–634 (1997).

    Article  CAS  Google Scholar 

  23. Da Silva, J.S. et al. RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability. J. Cell. Biol. 162, 1267–1279 (2003).

    Article  CAS  Google Scholar 

  24. Tsygankova, O.M., Saavedra, A., Rebhun, J.F., Quilliam, L.A. & Meinkoth, J.L. Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A. Mol. Cell. Biol. 21, 1921–1929 (2001).

    Article  CAS  Google Scholar 

  25. Weiner, O.D. et al. A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity. Nat. Cell Biol. 4, 509–513 (2002).

    Article  CAS  Google Scholar 

  26. Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).

    Article  CAS  Google Scholar 

  27. Etienne-Manneville, S. Cdc42—the centre of polarity. J. Cell Sci. 117, 1291–1300 (2004).

    Article  CAS  Google Scholar 

  28. Bos, J.L., de Rooij, J. & Reedquist, K.A. Rap1 signalling: adhering to new models. Nat. Rev. Mol. Cell Biol. 2, 369–377 (2001).

    Article  CAS  Google Scholar 

  29. Caron, E. Cellular functions of the Rap1 GTP-binding protein: a pattern emerges. J. Cell Sci. 116, 435–440 (2003).

    Article  CAS  Google Scholar 

  30. Chant, J. Cell polarity in yeast. Annu. Rev. Cell Dev. Biol. 15, 365–391 (1999).

    Article  CAS  Google Scholar 

  31. Park, H.O., Kang, P.J. & Rachfal, A.W. Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast. J. Biol. Chem. 277, 26721–26724 (2002).

    Article  CAS  Google Scholar 

  32. Pruyne, D. & Bretscher, A. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J. Cell Sci. 113, 365–375 (2000).

    CAS  PubMed  Google Scholar 

  33. Etienne-Manneville, S. & Hall, A. Rho GTPases in cell biology. Nature 420, 629–35 (2002).

    Article  CAS  Google Scholar 

  34. Rolls, M.M. & Doe, C.Q. Cell polarity: from embryo to axon. Nature 421, 905–906 (2003).

    Article  CAS  Google Scholar 

  35. Brewer, G.J., Torricelli, J.R., Evege, E.K. & Price, P.J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576 (1993).

    Article  CAS  Google Scholar 

  36. Washbourne, P. & McAllister, A.K. Techniques for gene transfer into neurons. Curr. Opin. Neurobiol. 12, 566–573 (2002).

    Article  CAS  Google Scholar 

  37. Bradke, F. & Dotti, C.G. Differentiated neurons retain the capacity to generate axons fro m dendrites. Curr. Biol. 10, 1467–1470 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to R. Fiore, V. Gerke, C. Klämbt, and M. Müller for comments on the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP1111) and Fonds der Chemischen Industrie (A.W.P.) and a fellowship from the Boehringer Ingelheim Fonds (J.C.S.).

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  1. Abteilung Molekularbiologie, Institut für Allgemeine Zoologie und Genetik, Westfälische Wilhelms-Universität Münster, Schloßplatz 5, Münster, D-48149, Germany

    Jens C Schwamborn & Andreas W Püschel

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  1. Jens C Schwamborn
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Correspondence to Andreas W Püschel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Distribution of Rap1B, Par3, P-Akt, and Cdc42. (PDF 832 kb)

Supplementary Fig. 2

Rap1B localization after inhibition of neurite extension. (PDF 120 kb)

Supplementary Fig. 3

Effects of different GTPases on the extension of axons and dendrites. (PDF 638 kb)

Supplementary Fig. 4

Stage-dependent effects of Rap1B. (PDF 22 kb)

Supplementary Fig. 5

Effects of mPar3 and mPar6 expression. (PDF 113 kb)

Supplementary Fig. 6

The establishment of polarity in hippocampal neurons. (PDF 757 kb)

Supplementary Table 1

Number of axons and minor neurites. (PDF 17 kb)

Supplementary Table 2

Length of minor neurites. (PDF 18 kb)

Supplementary Methods (PDF 17 kb)

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Schwamborn, J., Püschel, A. The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity. Nat Neurosci 7, 923–929 (2004). https://doi.org/10.1038/nn1295

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