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

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.

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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.

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.

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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.

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Authors

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.

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Correspondence to Marek Mlodzik.

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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

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