Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization

Nature Cell Biology volume 11, pages 286–294 (2009)Cite this article

An Erratum to this article was published on 01 April 2009

This article has been updated

Abstract

Dishevelled (Dsh) is a cytoplasmic multidomain protein that is required for all known branches of the Wnt signalling pathway1,2,3. The Frizzled/planar cell polarity (Fz/PCP) signalling branch requires an asymmetric cortical localization of Dsh, but this process remains poorly understood. Using a genome-wide RNA interference (RNAi) screen in Drosophila melanogaster cells, we show that Dsh membrane localization is dependent on the Na+/H+ exchange activity of the plasma membrane exchanger Nhe2. Manipulating Nhe2 expression levels in the eye causes PCP defects, and Nhe2 interacts genetically with Fz. Our data show that the binding and surface recruitment of Dsh by Fz is pH- and charge-dependent. We identify a polybasic stretch within the Dsh DEP domain that binds to negatively charged phospholipids and appears to be mechanistically important. Dsh recruitment by Fz can be abolished by converting these basic amino-acid residues into acidic ones, as in the mutant, DshKR/E. In vivo, the DshKR/E(2×) mutant with two substituted residues fails to associate with the membrane during active PCP signalling but rescues canonical Wnt signalling defects in a dsh-background. These results suggest that direct interaction between Fz and Dsh is stabilized by a pH and charge-dependent interaction of the DEP domain with phospholipids. This stabilization is particularly important for the PCP signalling branch and, thus, promotes specific pathway selection in Wnt signalling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nhe2/NHE3 activity is required for Fz-mediated Dsh recruitment.
Figure 2: Nhe2 shows gain- and loss-of-function PCP phenotypes in D. melanogaster eye, and interacts genetically with Fz.
Figure 3: The Dsh DEP domain interacts with acidic phospholipids.
Figure 4: Fz-mediated Dsh membrane recruitment is charge-dependent.
Figure 5: The polybasic stretch in the Dsh DEP domain is essential for PCP signalling in vivo.

Similar content being viewed by others

Change history

  • 24 February 2009

    In the version of this article initially published online, Fig. 5s had an erroneous 8th transmembrane domain on Frizzled. Supplementary Methods were also missing from the Supplementary Information. These errors have been corrected for the HTML and PDF versions of the article.

References

  1. Boutros, M., Paricio, N., Strutt, D. I. & Mlodzik, M. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118 (1998).

    Article  CAS  Google Scholar 

  2. Seifert, J. R. & Mlodzik, M. Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility. Nature Rev. Genet. 8, 126–138 (2007).

    Article  CAS  Google Scholar 

  3. Veeman, M. T., Axelrod, J. D. & Moon, R. T. A second canon. Functions and mechanisms of β-catenin-independent Wnt signaling. Dev. Cell 5, 367–377 (2003).

    Article  CAS  Google Scholar 

  4. Axelrod, J. D. Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling. Genes Dev. 15, 1182–1187 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Bilic, J. et al. Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. Science 316, 1619–1622 (2007).

    Article  CAS  Google Scholar 

  6. Wong, H-C. et al. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled. Mol. Cell 12, 1251–1260 (2003).

    Article  CAS  Google Scholar 

  7. Rothbacher, U. et al. Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis. EMBO J. 19, 1010–1022 (2000).

    Article  CAS  Google Scholar 

  8. Wong, H. C. et al. Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway. Nature Struct. Biol. 7, 1178–1184 (2000).

    Article  CAS  Google Scholar 

  9. Boutros, M., Mihaly, J., Bouwmeester, T. & Mlodzik, M. Signaling specificity by Frizzled receptors in Drosophila. Science 288, 1825–1828 (2000).

    Article  CAS  Google Scholar 

  10. Boutros, M. et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science 303, 832–835 (2004).

    Article  CAS  Google Scholar 

  11. Ossipova, O., Dhawan, S., Sokol, S. & Green, J. B. Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development. Dev. Cell 8, 829–841 (2005).

    Article  CAS  Google Scholar 

  12. Putney, L. K., Denker, S. P. & Barber, D. L. The changing face of the Na+/H+ exchanger, NHE1: structure, regulation, and cellular actions. Annu. Rev. Pharmacol. Toxicol. 42, 527–552 (2002).

    Article  CAS  Google Scholar 

  13. Donowitz, M. & Li, X. Regulatory binding partners and complexes of NHE3. Physiol. Rev. 87, 825–872 (2007).

    Article  CAS  Google Scholar 

  14. Ro, H. A. & Carson, J. H. pH microdomains in oligodendrocytes. J. Biol. Chem. 279, 37115–37123 (2004).

    Article  CAS  Google Scholar 

  15. Frantz, C., Karydis, A., Nalbant, P., Hahn, K. M. & Barber, D. L. Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells. J. Cell Biol. 179, 403–410 (2007).

    Article  CAS  Google Scholar 

  16. Wang, D., King, S. M., Quill, T. A., Doolittle, L. K. & Garbers, D. L. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nature Cell Biol. 5, 1117–1122 (2003).

    Article  CAS  Google Scholar 

  17. Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008).

    Article  CAS  Google Scholar 

  18. Giannakou, M. E. & Dow, J. A. Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family. J. Exp. Biol. 204, 3703–3716 (2001).

    CAS  PubMed  Google Scholar 

  19. Mustonen, P., Lehtonen, J., Koiv, A. & Kinnunen, P. K. Effects of sphingosine on peripheral membrane interactions: comparison of adriamycin, cytochrome c, and phospholipase A2. Biochemistry 32, 5373–5380 (1993).

    Article  CAS  Google Scholar 

  20. Kooijman, E. E. et al. An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins. J. Biol. Chem. 282, 11356–11364 (2007).

    Article  CAS  Google Scholar 

  21. Lemmon, M. A. Membrane recognition by phospholipid-binding domains. Nature Rev. Mol. Cell Biol. 9, 99–111 (2008).

    Article  CAS  Google Scholar 

  22. Penton, A., Wodarz, A. & Nusse, R. A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin. Genetics 161, 747–762 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Zeng, X. et al. Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions. Development 135, 367–375 (2008).

    Article  CAS  Google Scholar 

  24. Jenny, A., Darken, R. S., Wilson, P. A. & Mlodzik, M. Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. EMBO J. 22, 4409–4420 (2003).

    Article  CAS  Google Scholar 

  25. Bizzarri, R. et al. Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies. Biophys. J. 90, 3300–3314 (2006).

    Article  CAS  Google Scholar 

  26. Schwarz-Romond, T., Merrifield, C., Nichols, B. J. & Bienz, M. The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles. J. Cell Sci. 118, 5269–5277 (2005).

    Article  CAS  Google Scholar 

  27. Lang, K., Wagner, C., Haddad, G., Burnekova, O. & Geibel, J. Intracellular pH activates membrane-bound Na(+)/H(+) exchanger and vacuolar H(+)-ATPase in human embryonic kidney (HEK) cells. Cell. Physiol. Biochem. 13, 257–262 (2003).

    Article  CAS  Google Scholar 

  28. Sandvig, K., Olsnes, S., Petersen, O. W. & van Deurs, B. Acidification of the cytosol inhibits endocytosis from coated pits. J. Cell Biol. 105, 679–689 (1987).

    Article  CAS  Google Scholar 

  29. Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A. & Chen, J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 294, 1942–1945 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Bloomington Stock Center for fly strains, S. Grinstein, J. Orlowski, T. Kirchhausen and R. Bizzarri for cDNA constructs. We are grateful to A. Jenny and Y. Wang for the cloning of cDNA constructs, and U. Weber for analysis of the embryonic/early larval lethal phenotype. We thank C. Iomini, R. Krauss, S. Sokol and D. del Alamo for reading the manuscript, members of the Mlodzik laboratory for discussions and S. Okello, G. Garcia and M. Stricker for technical support. The work has been supported by NIH grant to M.M. RO1 GM62917. M.S. was a recipient of EMBO and DFG long-term fellowships.

Author information

Authors and Affiliations

  1. Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, 10029, NY, USA

    Matias Simons, William J. Gault, Thomas J. Klein & Marek Mlodzik

  2. Department of Medicine, Division of Gastroenterology, University Hospital Heidelberg, Heidelberg, D-69120, Germany

    Daniel Gotthardt

  3. Department of Pediatrics, Mount Sinai School of Medicine, One Gustave L. Levy Place, NY, 10029, NY, USA

    Rajeev Rohatgi & Lisa M. Satlin

  4. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, 38105, TN, USA

    Youming Shao, Ho-Jin Lee & Jie Zheng

  5. Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, 61801, IL, USA

    Ai-Luen Wu, Yimin Fang & Jie Chen

  6. Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK

    Julian T. Dow

  7. Division of Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, Heidelberg, D-69120, Germany

    Michael Boutros

Authors
  1. Matias Simons
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. William J. Gault
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Gotthardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rajeev Rohatgi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas J. Klein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Youming Shao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ho-Jin Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ai-Luen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yimin Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lisa M. Satlin
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Julian T. Dow
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jie Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Michael Boutros
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Marek Mlodzik
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S. coordinated the project, designed and conducted the RNAi screen, performed the experimental work and data analysis and wrote the manuscript. W.J.G. performed the pupal dissections. W.J.G. and D.G. assisted with experiments. T.J.K. helped with the RNAi screen assay. R.R. and L.S.M. performed pH measurements. Y.S., H.-J.L. and J.Z. purified the DEP domain and performed electrostatic potential calculations. A.-L.W., Y.F. and J.C. performed SUV lipid binding assays. J.T.D. provided Nhe2 tools. M.B. designed and conducted the RNAi screen. M.M. coordinated the project, assisted with planning the experiments and data analysis and wrote the manuscript.

Corresponding author

Correspondence to Marek Mlodzik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1209 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Simons, M., Gault, W., Gotthardt, D. et al. Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization. Nat Cell Biol 11, 286–294 (2009). https://doi.org/10.1038/ncb1836

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1836

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing