Abstract
Cell contacts provide spatial cues that polarize early embryos and epithelial cells. The homophilic adhesion protein E-cadherin is required for contact-induced polarity in many cells. However, it is debated whether E-cadherin functions instructively as a spatial cue, or permissively by ensuring adequate adhesion so that cells can sense other contact signals. In Caenorhabditis elegans, contacts polarize early embryonic cells by recruiting the RhoGAP PAC-1 to the adjacent cortex, inducing PAR protein asymmetry. Here we show that the E-cadherin HMR-1, which is dispensable for adhesion, functions together with the α-catenin HMP-1, the p120 catenin JAC-1, and the previously uncharacterized linker PICC-1 (human CCDC85A-C) to bind PAC-1 and recruit it to contacts. Mislocalizing the HMR-1 intracellular domain to contact-free surfaces draws PAC-1 to these sites and depolarizes cells, demonstrating an instructive role for HMR-1 in polarization. Our findings identify an E-cadherin-mediated pathway that translates cell contacts into cortical polarity by directly recruiting a symmetry-breaking factor to the adjacent cortex.
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Acknowledgements
We thank A. Fisher (University of Pittsburgh, USA), K. Gieseler (Université Claude Bernard Lyon 1, France), G. Hermann (Lewis and Clark College, USA), O. Hobert (Columbia University Medical Center, USA), J. Hubbard (New York University School of Medicine, USA), T. Hyman (Max Planck Institute for Molecular Cell Biology and Genetics, Germany) and E. Jorgensen (University of Utah, USA) for their generous gifts of strains, plasmids or antibodies. Special thanks to M. Burel for help in examining GFP–PAC-1ΔPH localization. Thanks to L. Christiaen, T. Hurd, and members of the Nance laboratory for comments on the manuscript. This study was financially supported by a National Science Foundation Graduate Research Fellowship under grant No. 12-A0-00-000165-01 (D.K.), an American Heart Association postdoctoral fellowship (Y.Z.), and NIH grants R01GM098492 (J.N.), R01GM078341 (J.N.) and T32HD007520 (D.C.A.).
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D.K., D.C.A. and J.N. designed, executed and analysed the experiments. J.Y.Y., Y.Z. and J.N. designed, executed and analysed the co-immunoprecipitation experiments. D.K. and J.N. wrote the manuscript with input from all authors.
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Supplementary Figure 3 GFP-PAC-1 transgene expression levels and controls for GFP-PAC-1N localization in the absence of endogenous PAC-1.
(a) Expression levels of GFP-PAC-1 transgenes and derivatives, measured by fluorescence intensity (after background subtraction) at the four-cell stage (see Methods), expressed in arbitrary fluorescence units. PAC-1 amino acids present in each GFP fusion protein are indicated (ΔPH lacks the PH domain only). The box represents first and third quartiles, bars represent maximum and minimum values, and the line within the box is the mean fluorescence intensity (full length n = 11 embryos, ΔPH n = 13, 392-838 n = 11, 575-1604 n = 13, 2-610 n = 12, 1-574 n = 14). While expression levels vary between transgenes, contact localization is evident even from those with the lowest expression levels (ex. full-length GFP-PAC-1 and GFP-PAC-12–610). Therefore, lack of apparent contact localization for GFP-PAC-1392–838 and GFP-PAC-1575–1604 is not due to low expression level. (b) Full-length GFP-PAC-1 in control (empty vector) RNAi embryos localizes to cell contacts (arrow, 20/20 embryos). (c) In pac-1(3′ RNAi) embryos GFP-PAC-1 is not detected (19/19 embryos), indicating that RNAi effectively depletes PAC-1. Scale bars, 10 μm.
Supplementary Figure 4 Rescue of pac-1 polarity defects by GFP-PAC-1ΔPH.
(a) Wild-type, (b) pac-1(xn6), and (c) pac-1(xn6); GFP-PAC-1ΔPH 6–8 cell embryos stained for PAR-6. (d) PAR-6 asymmetry is lost in pac-1 mutants but is rescued by expression of GFP-PAC-1ΔPH. PAR-6 asymmetry was quantified by determining the polarity index, defined as the ratio of PAR-6 levels at contact-free surfaces versus half of the PAR-6 level at cell contacts (see Methods). Circles represent individual data points and the gray line indicates the average (wild type n = 8 embryos, pac-1 n = 10, pac-1; GFP-PAC-1ΔPHn = 8). Measured by Mann–Whitney U-test, pac-1(xn6) embryos have a polarity index significantly lower than wild type (∗∗∗p < 0.001) and pac-1(xn6); GFP-PAC-1ΔPH (∗∗∗p < 0.001). The polarity index of pac-1(xn6); GFP-PAC-1ΔPH is marginally significantly different from wild type (∗p < 0.05). Samples were pooled from two independent experiments. Scale bars, 10 μm.
Supplementary Figure 5 Comparison of GFP-PAC-1N in hmp-2(RNAi) and hmp-1(RNAi) embryos.
(a–c) GFP-PAC-1N in live embryos of the indicated genotype; images were captured at the same exposure. (d) Quantification of GFP-PAC-1N contact enrichment. Circles represent data points from individual embryos, and the average is a gray line. ∗∗p < 0.01; n.s. = not significantly different, Mann–Whitney U test. (Control n = 13 embryos, hmp-2(RNAi) n = 9, hmp-1(RNAi) n = 9). Scale bars, 10 μm.
Supplementary Figure 6 Genetic requirements for catenin localization.
Control embryos are wild-type embryos fed on bacteria containing empty RNAi vector. (a,b) HMP-1 immunostaining at cell contacts in control embryos (40/40 embryos) or in the cytoplasm of hmp-2(RNAi) embryos (35/35 embryos). (c,d) α-JAC-1 antiserum immunostains cell contacts in wild-type embryos (30/30 embryos) but not in jac-1(xn15) embryos (42/42 embryos). α-JAC-1 antiserum also stains nuclei non-specifically, as evidenced by the loss of contact staining, but not nuclear staining, in jac-1 mutant embryos (d). (e,f) GFP-JAC-1 at cell contacts in a live control embryo (41/41 embryos) and in the cytoplasm in a live hmr-1 embryo (25/25 embryos). (g) HMP-1 immunostaining in a hmp-1(RNAi) embryo. (h) HMP-1 immunostaining in a hmr-1 embryo, where HMP-1 localizes to the cytoplasm (45/45 embryos). (i) Schematic of the jac-1 gene, with exons shown as rectangles and introns as chevrons. Region encoding the Armadillo (Arm) repeats, which contains the peptide used to generate α-JAC-1 antiserum, is indicated. The dashed region indicates the extent of jac-1 deleted in the xn15 allele. Scale bars, 10 μm.
Supplementary Figure 7 Catenin and HMR-1 localization following catenin depletion.
Control embryos are wild-type embryos fed on bacteria containing RNAi empty vector. (a,b) HMP-1-GFP at cell contacts in wild-type and jac-1 embryos. (c) Quantification of HMP-1-GFP contact enrichment in wild-type and jac-1 embryos (wild type n = 12 embryos, jac-1 n = 12). Circles represent individual values and gray lines the mean. HMP-1-GFP levels at contacts in the two genotypes are not significantly different (p = 0.06, Mann–Whitney U test). (d,e) GFP-JAC-1 at cell contacts in a control and hmp-1(RNAi) embryo. (f) Quantification of GFP-JAC-1 contact enrichment in control and hmp-1(RNAi) embryos. (control n = 13 embryos, hmp-1(RNAi) n = 12). GFP-JAC-1 levels at contacts in the two genotypes are not significantly different (p = 0.3, Mann–Whitney U test). (g–j) HMR-1-GFP expression in live four-cell embryos of the indicated genotype. (k) Quantification of HMR-1-GFP levels at cell contacts in embryos of the indicated genotype (control n = 18 embryos, hmp-1(RNAi) n = 33, jac-1 n = 17, jac-1 + hmp-1(RNAi)n = 17). None of the mean values are significantly different from control (p > 0.05, Mann–Whitney U test). Control samples and hmp-1(RNAi) embryos were pooled from two independent experiments. (l,m) Endogenous HMR-1 immunostaining at cell contacts in control (91/91 embryos) and jac-1(xn15) + hmp-1(RNAi) (133/133 embryos). Scale bars, 10 μm.
Supplementary Figure 8 picc-1 gene and regulation of PICC-1 localization.
(a) Schematic of the picc-1 gene, with exons shown as rectangles and introns as chevrons. The dashed region indicates portion of the picc-1 gene deleted in the xn14 allele. (b) Region of conservation between PICC-1 and homologues in human (CCDC85A-C; CCDC85B is also known as DIPA) and Drosophila (CG17265). Black shading indicates three or more identical residues; gray shading indicates similar residues. (c,d) GFP-PICC-1 localization in a hmr-1 mutant embryo (n = 50 embryos) and a hmp-1(RNAi) embryo (n = 31 embryos). Compare to wild-type localization in Figure 4a. Scale bars, 10 μm.
Supplementary Figure 9 Two-hybrid controls and reciprocal immunoprecipitations.
(a) Control two-hybrid experiments, performed as described in Figure 5 legend. (b) Immunoprecipitation of PICC-1-GFP from embryonic lysates showing co-immunoprecipitation of JAC-1 species. The experiment was performed seven times, and a representative example is shown. (c) Immunoprecipitation of mCherry-HA-PAC-1 from embryonic lysates showing co-immunoprecipitation of PICC-1-GFP. The experiment was performed four times, and a representative example is shown. Tubulin levels in the input (total embryonic lysate) are shown as a loading control. Uncropped blots are shown in Supplementary Figure 8.
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Klompstra, D., Anderson, D., Yeh, J. et al. An instructive role for C. elegans E-cadherin in translating cell contact cues into cortical polarity. Nat Cell Biol 17, 726–735 (2015). https://doi.org/10.1038/ncb3168
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DOI: https://doi.org/10.1038/ncb3168
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