Analysis of Wnt/Planar Cell Polarity Pathway in Cultured Cells

  • Mitsuharu Endo
  • Michiru Nishita
  • Yasuhiro MinamiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 839)


Planar cell polarity (PCP) pathway of Wnt signaling plays a crucial role to establish the polarization of cells during tissue development. Our recent findings using in vitro analyses have revealed that Ror2, a member of the Ror-family receptor tyrosine kinases, acts as a receptor or co-receptor for Wnt5a and plays a crucial role for Wnt5a-induced polarized cell migration through activating PCP pathway. Indeed, analyses of both Wnt5a and Ror2 mutant mice have shown that Wnt5a-Ror2 signaling is involved in establishing the PCP in epithelial tissues in vivo, indicating that in vitro analyses of polarized cell migration and PCP signaling induced by Wnt5a can be useful tools to explore putative regulators involved in Wnt/PCP pathway. Here, we introduce in vitro methods using cultured cells to monitor polarized cell migration and PCP signaling induced by Wnt5a.

Key words

PCP Wnt5a Ror2 JNK Non-canonical Wnt signaling Cell polarity Wound-healing 


  1. 1.
    Gubb, D., and Garcia-Bellido, A. (1982) A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster. J Embryol Exp Morphol 68, 37–57.PubMedGoogle Scholar
  2. 2.
    Adler, P. N. (2002) Planar signaling and morphogenesis in Drosophila. Dev Cell 2, 525–535.PubMedCrossRefGoogle Scholar
  3. 3.
    Klein, T. J., and Mlodzik, M. (2005) Planar cell polarization: an emerging model points in the right direction. Annu Rev Cell Dev Biol 21, 155–176.PubMedCrossRefGoogle Scholar
  4. 4.
    Mlodzik, M. (2002) Planar cell polarization: do the same mechanisms regulate Drosophila tissue polarity and vertebrate gastrulation? Trends Genet 18, 564–571.PubMedCrossRefGoogle Scholar
  5. 5.
    Fanto, M., and McNeill, H. (2004) Planar polarity from flies to vertebrates. J Cell Sci 117, 527–533.PubMedCrossRefGoogle Scholar
  6. 6.
    Cadigan, K. M., and Nusse, R. (1997) Wnt signaling: a common theme in animal development. Genes Dev 11, 3286–3305.PubMedCrossRefGoogle Scholar
  7. 7.
    Sokol, S. Y. (1999) Wnt signaling and dorso-ventral axis specification in vertebrates. Curr Opin Genet Dev 9, 405–410.PubMedCrossRefGoogle Scholar
  8. 8.
    Moon, R. T., Campbell, R. M., Christian, J. L., McGrew, L. L., Shih, J., and Fraser, S. (1993) Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development 119, 97–111.PubMedGoogle Scholar
  9. 9.
    Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000) Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76–81.PubMedCrossRefGoogle Scholar
  10. 10.
    Sokol, S. (2000) A role for Wnts in morpho-genesis and tissue polarity. Nat Cell Biol 2, E124–125.PubMedCrossRefGoogle Scholar
  11. 11.
    Tada, M., and Smith, J. C. (2000) Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127, 2227–2238.PubMedGoogle Scholar
  12. 12.
    Oishi, I., Suzuki, H., Onishi, N., Takada, R., Kani, S., Ohkawara, B., Koshida, I., Suzuki, K., Yamada, G., Schwabe, G. C., Mundlos, S., Shibuya, H., Takada, S., and Minami, Y. (2003) The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway. Genes Cells 8, 645–654.PubMedCrossRefGoogle Scholar
  13. 13.
    Qian, D., Jones, C., Rzadzinska, A., Mark, S., Zhang, X., Steel, K. P., Dai, X., and Chen, P. (2007) Wnt5a functions in planar cell polarity regulation in mice. Dev Biol 306, 121–133.PubMedCrossRefGoogle Scholar
  14. 14.
    Nomachi, A., Nishita, M., Inaba, D., Enomoto, M., Hamasaki, M., and Minami, Y. (2008) Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A. J Biol Chem 283, 27973–27981.PubMedCrossRefGoogle Scholar
  15. 15.
    Boutros, M., Paricio, N., Strutt, D. I., and Mlodzik, M. (1998) Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118.PubMedCrossRefGoogle Scholar
  16. 16.
    Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M., Hanafusa, H., Takada, R., Takada, S., and Nishida, E. (2002) JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. EMBO Rep 3, 69–75.PubMedCrossRefGoogle Scholar
  17. 17.
    Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025–1037.PubMedCrossRefGoogle Scholar
  18. 18.
    Cheyette, B. N., Waxman, J. S., Miller, J. R., Takemaru, K., Sheldahl, L. C., Khlebtsova, N., Fox, E. P., Earnest, T., and Moon, R. T. (2002) Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Dev Cell 2, 449–461.PubMedCrossRefGoogle Scholar
  19. 19.
    Pukrop, T., Klemm, F., Hagemann, T., Gradl, D., Schulz, M., Siemes, S., Trumper, L., and Binder, C. (2006) Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines. Proc Natl Acad Sci USA 103, 5454–5459.PubMedCrossRefGoogle Scholar
  20. 20.
    Nishita, M., Itsukushima, S., Nomachi, A., Endo, M., Wang, Z., Inaba, D., Qiao, S., Takada, S., Kikuchi, A., and Minami, Y. (2010) Ror2/Frizzled complex mediates Wnt5a-induced AP-1 activation by regulating Dishevelled polymerization. Mol Cell Biol. 30, 3610–3619.Google Scholar
  21. 21.
    Yamamoto, S., Nishimura, O., Misaki, K., Nishita, M., Minami, Y., Yonemura, S., Tarui, H., and Sasaki, H. (2008) Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell 15, 23–36.PubMedCrossRefGoogle Scholar
  22. 22.
    Kani, S., Oishi, I., Yamamoto, H., Yoda, A., Suzuki, H., Nomachi, A., Iozumi, K., Nishita, M., Kikuchi, A., Takumi, T., and Minami, Y. (2004) The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon. J Biol Chem 279, 50102–50109.PubMedCrossRefGoogle Scholar
  23. 23.
    Scliwa, M., and Honer, B. (1993) Microtubules, centrosomes and intermediate filaments in directed cell movement. Trends Cell Biol 3, 377–380.PubMedCrossRefGoogle Scholar
  24. 24.
    Nobes, C. D., and Hall, A. (1999) Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol 144, 1235–1244.PubMedCrossRefGoogle Scholar
  25. 25.
    Lallemand, D., Ham, J., Garbay, S., Bakiri, L., Traincard, F., Jeannequin, O., Pfarr, C. M., and Yaniv, M. (1998) Stress-activated protein kinases are negatively regulated by cell density. Embo J 17, 5615–5626.PubMedCrossRefGoogle Scholar
  26. 26.
    Takada, R., Hijikata, H., Kondoh, H., and Takada, S. (2005) Analysis of combinatorial effects of Wnts and Frizzleds on beta-catenin/armadillo stabilization and Dishevelled phosphorylation. Genes Cells 10, 919–928.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Mitsuharu Endo
    • 1
  • Michiru Nishita
    • 1
  • Yasuhiro Minami
    • 2
    Email author
  1. 1.Department of Physiology and Cell Biology, Graduate School of MedicineKobe UniversityKobeJapan
  2. 2.Department of Physiology and Cell Biology, School of MedicineKobe UniversityKobeJapan

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