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Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions

Nature Cell Biology volume 17, pages 1282–1293 (2015)Cite this article

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Abstract

Actomyosin at the epithelial zonula adherens (ZA) generates junctional tension for tissue integrity and morphogenesis. This requires the RhoA GTPase, which establishes a strikingly stable active zone at the ZA. Mechanisms must then exist to confer robustness on junctional RhoA signalling at the population level. We now identify a feedback network that generates a stable mesoscopic RhoA zone out of dynamic elements. The key is scaffolding of ROCK1 to the ZA by myosin II. ROCK1 protects junctional RhoA by phosphorylating Rnd3 to prevent the cortical recruitment of the Rho suppressor, p190B RhoGAP. Combining predictive modelling and experimentation, we show that this network constitutes a bistable dynamical system that is realized at the population level of the ZA. Thus, stability of the RhoA zone is an emergent consequence of the network of interactions that allow myosin II to feedback to RhoA.

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Figure 1: Myosin II supports a stable Rho zone at the ZA.
Figure 2: p190B RhoGAP disrupts the junctional Rho zone when NMII is inactivated.
Figure 3: Myosin antagonizes the junctional localization of Rnd3.
Figure 4: Rnd3 recruits p190B to the ZA when NMII is inhibited.
Figure 5: ROCK1 phosphorylates Rnd3 to support junctional RhoA signalling.
Figure 6: Myosin II scaffolds ROCK1 at the epithelial ZA.
Figure 7: Bistable properties of junctional RhoA.

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Acknowledgements

We thank our laboratory colleagues for their unstinting support and advice during the course of this project, and our colleagues elsewhere for their kind gifts of reagents. We also thank M. Zerial and L. Tucker-Kellogg for thought-provoking discussions, M. Naghibosadat for her help with cloning and E. Moussa for his help in nano-ablation analysis. This work was supported by the National Health and Medical Research Council of Australia (1044041, 1037320, 1067405), The Kids Cancer Project of the Oncology Research Foundation, the EMPathy National Collaborative Research Program (CG-10-04) of the National Breast Cancer Foundation (Australia), a University of Queensland Early Career Research Grant to G.A.G. (2012003354) and an ANZ Trustees PhD Scholarship in Medical Research to R.P.

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  1. Guillermo A. Gomez and Alpha S. Yap: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Rashmi Priya, Guillermo A. Gomez, Srikanth Budnar, Suzie Verma, Hayley L. Cox, Nicholas A. Hamilton & Alpha S. Yap

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Contributions

R.P., G.A.G. and A.S.Y. conceived the project and designed the experiments. R.P. performed most of the experiments in collaboration with G.A.G. for nano-ablation, FRAP and Rho biosensor experiments, with assistance from S.V. for western blotting and immunoprecipitation experiments. G.A.G. and N.A.H. performed the mathematical modelling. H.L.C. performed experiments. S.B. performed the cloning and characterization of AHPH mutants. R.P. and G.A.G. analysed the data. R.P., G.A.G. and A.S.Y. wrote the paper.

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Correspondence to Guillermo A. Gomez or Alpha S. Yap.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Myosin II supports a stable Rho zone at the ZA.

(a) RhoA, E-cadherin and ZO-1 localization in TCA-fixed MCF-7 cells. (b) Domain organization of human full-length Anillin (FL-Anillin) and the respective constructs used in this study. The residues are numbered according to the Uniprot Id Q9NQW6. FBD (Formin binding domain); MBD (Myosin binding domain); ABD (Actin binding domain); AHD (Anillin homology domain), which is composed of RBD (Rho binding domain) and C2 domain. The AHPH (location biosensor) is composed of AHD and PH (Pleckstrin homology domain). (c) Representative images and fluorescence line-scan analysis of GFP-AHPH and endogenous RhoA at the ZA of MCF-7 cells. White line (merged image) represents the position where fluorescence intensity was measured. (d) Localization of RFP-UtrCH and GFP-tagged AHPH, AHPH + C3-T (C3 Transferase, 1 μg ml−1; 3 h), Δ AH, AHPHC2mut, AHPHA740D, AHPHE758K and AHPHA740D E758K in MCF-7 cells. (e) Lysates from control or ECT2 knockdown MCF-7 cells immunoblotted for ECT2 and GAPDH (loading control). (f) GTP-RhoA distribution in control or ECT2 knockdown MCF-7 cells. (g) Control (Ctrl) or E-cadherin RNAi (E-cad KD) MCF-7 cells fixed and immunostained for GFP (GFP AHPH), E-cadherin (E-cad) and ZO-1. (h) Lysates from control (Ctrl) or E-cadherin RNAi (E-cad KD) MCF-7 cells immunoblotted for E-cadherin and β-tubulin (loading control). (i) GTP-RhoA pull-down from control (Ctrl) or E-cadherin RNAi (E-cad KD) or NMIIA RNAi (NMIIA KD) MCF-7 cells using RBD domain of Rhotekin and immunoblotted for RhoA and β-tubulin. (j) NMIIA and E-cadherin localization in MCF-7 cells treated with vehicle (DMSO) or blebbistatin (BLB, 100 μM, 2 h). (k) RhoA localization in Caco2 and MDCK treated with DMSO or blebbistatin (100 μM, 2 h). (l,m) Lysates from Control (Ctrl), NMIIA knockdown or NMIIB knockdown MCF-7 cells immunoblotted for NMIIA, NMIIB and β-tubulin (loading control). Scale bars, 10 μm. DIC images corresponding to Supplementary Fig. 1d are shown in Supplementary Fig. 7.

Supplementary Figure 3 p190B RhoGAP degrades the junctional Rho zone when NMII is inactivated.

(a–e) ECT2 (a), MgcRacGAP (RacGAP) (b), p190A Rho GAP (p190A), Myosin IIA and Myosin IIB (c,e) localization in control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells. (f,g) Lysates from control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells immunoblotted for p190B Rho GAP and β-tubulin (loading control). (h) Rac 1 localization in control (Ctrl), NMIIA RNAi (IIA KD), NMIIB RNAi (IIB KD), DMSO or blebbistatin (BLB, 100 μM, 2 h) treated MCF-7 cells. (i) MCF-7 cells transfected with non-targeting control siRNA or Rac1 siRNA (Rac1KD) were treated with DMSO or with blebbistatin (BLB; 100 μM, 2 h), then fixed and stained for p190B Rho GAP. (j) Lysates from MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or transfected with siRNA against Rac1 (Rac1KD) were immunoblotted for Rac1 and β-tubulin (loading control). (k) MCF-7 cells were treated with DMSO or blebbistatin (BLB; 100 μM, 2 h) alone or in combination with Rac inhibitors EHT1864 (10 μM, 12 h) or NSC23766 (50 μM, 12 h), fixed with methanol and stained for p190B GAP. (l) MCF-7 cells transduced with lentivirus encoding non-targeting control shRNA (Ctrl), NMIIA (IIAKD) or NMIIB (IIB KD) shRNA were transfected with the Raichu-Rac FRET biosensor and FRET measurements were performed as described in the Methods section. Average emission ratios were calculated at the apical junctions. Data represent mean ± s.e.m. of 30 contacts (n = 30) and the result is representative of two independent experiments. Statistical information, P values and source data are in Supplementary Table 2. (m) MCF-7 cells were treated with DMSO or treated with blebbistatin (BLB) (100 μM, 2 h) alone or in combination with Src inhibitors SU6656 (10 μM) or PP2 (10 μM)) for 4 h. Cells were fixed with methanol and stained for p190B GAP. Scale bars, 10 μm.

Supplementary Figure 4 Rnd3 recruits p190B to the ZA when NMII is inhibited.

(a) MCF-7 cells transfected with non-targeting control siRNA or siRNA against Rnd3 (Rnd3 KD) or Rnd1 (Rnd1 KD) and treated with vehicle control DMSO or blebbistatin (BLB; 100 μM, 2 h) were fixed with methanol and stained for p190B Rho GAP and E-cadherin. Representative confocal images were acquired at the apical junctions. (b) Lysates from MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or siRNA targeted against Rnd3 (Rnd3 KD) were immunoblotted for Rnd3 and β-tubulin (loading control). (c) Lysates from MCF-7 cells transfected with Rnd3siRNA along with GFP-tagged Rnd3 constructs (WT, AIIA, S240A, T55V) were immunoblotted for GFP and β-tubulin (loading control). (d) MCF-7 cells transfected with non-targeting siRNA (Ctrl siRNA) or Rnd3 siRNA (Rnd3 KD) and reconstituted with low levels of GFP, or GFP-tagged WT Rnd3 (WT), AIIA Rnd3 (AIIA), S240A Rnd3 (S240A), or T55V Rnd3 (T55V) were treated with either DMSO or blebbistatin (BLB) (100 μM, 2 h), fixed with TCA and stained for RhoA. Asterisks (∗) indicate GFP-tagged Rnd3 expressing cells. (e) MCF-7 cells transfected with GFP-AHPH and non-targeting siRNA (Ctrl siRNA) or Rnd3 siRNA (Rnd3 KD) and reconstituted with low levels of mCherry, or mCherry-tagged WT Rnd3 (WT), AIIA Rnd3 (AIIA), S240A Rnd3 (S240A), T55V Rnd3 (T55V) were treated with either DMSO or blebbistatin (BLB, 100 μM, 2 h) fixed with TCA and stained for GFP. Asterisks (∗) indicate mCherry-tagged Rnd3 expressing cells. Scale bars, 10 μm.

Supplementary Figure 5 ROCK-1 phosphorylates Rnd3 to support junctional Rho signalling.

(a) Confluent MCF-7 cells were fixed with methanol and stained for ROCK-2 and E-cadherin. Representative images were acquired at the apical junctions of the cells by confocal microscopy. (b,c) Confluent MCF-7 cells were treated with vehicle (0.5% glycerol, Ctrl) or with Rho-inhibitor C3 Transferase (C3T, 1 μg ml−1) for 3 h, fixed with methanol and stained for ROCK-1 and ZO-1. Representative images were acquired at the apical junctions of the cells by confocal microscopy (b) and the fluorescence intensity at cell junctions was quantitated by linescan analysis (c). Data represent mean ± s.e.m. of three individual experiments (n = 3), ∗P < 0.05 Student’s t-test. Statistical information, P values and source data are in Supplementary Table 2. (d) HEK293T cells expressing GFP-Rnd3 were treated with either PBS or Y-27632 (30 μM, 1 h). Rnd3 was immunoprecipitated by GFP-trap and lysates were immunoblotted for GFP and phospho-serine. (e) MCF-7 cells transfected with Rnd3 siRNA and reconstituted with GFP tagged WT Rnd3 (WT), AIIA Rnd3 (AIIA), S240A Rnd3 (S240A) were treated with either PBS or Y-27632 (30 μM, 1 h). Representative images were acquired at the apical junctions of the cells by confocal microscopy. (f) Lysates from MCF-7 cells treated either with PBS or Y-27632 (30 μM, 1 h), or blebbistatin (BLB, 100 μM, 2 h) and blotted for Rnd3 and β-tubulin (loading control). (g) MCF-7 cells were transfected with non-targeting Ctrl siRNA (Ctrl) or ROCK-1 siRNA (ROCK-1KD) and 48 h later treated with either blebbistatin (100 μM, 2 h) alone (BLB) or in combination with ROCK-1 KD (ROCK-1 KD + BLB). Cells were fixed with methanol and stained for p190B Rho GAP or fixed with TCA and stained for RhoA. Representative images were acquired at the apical junctions of the cells by confocal microscopy. (h,i) Lysates from MCF-7 cells transfected with non-targeting control siRNA (ctrl) or ROCK-1 siRNA (ROCK-1 KD) and immunoblotted for ROCK-1, ROCK-2 and β-tubulin (loading control). Scale bars, 10 μm. Corresponding DIC images for 4e are shown in Supplementary Fig. 7.

Supplementary Figure 6 Myosin II scaffolds ROCK-1 at the epithelial Zonula Adherens.

(a,b) MCF-7 cells transduced with lentivirus encoding non-targeting control shRNA (Ctrl) or shRNA against NMIIA (IIAKD) or shRNA against NMIIB (IIB KD) were fixed with methanol and stained for ROCK-1, NMIIA and NMIIB. Representative images were acquired at the apical junctions of the cells by confocal microscopy. (c) HEK293T cells were transfected with GFP-ROCK-1 and treated with blebbistatin (BLB, 100 μM, 2 h). ROCK-1 was immunoprecipitated using GFP-TRAP and lysates were immunoblotted for NMIIA, NMIIB and GFP. (d) HEK293T cells were transfected with GFP-FL NMIIA (full-length) or GFP-NMIIA-ROD and treated with Y-27632 (30 μM, 1 h). Immunoprecipitation was performed using GFP-TRAP and lysates were immunoblotted for ROCK-1 and GFP. (e,g) Lysates from MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or NMIIA siRNA (NMIIAKD, e), Cdc42siRNA (Cdc42 KD, f), or N-WASP siRNA (N-WASP KD, g) and immunoblotted for NMIIA, Cdc42 or N-WASP, respectively, as well as β-tubulin or GAPDH (loading control). (h) MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or siRNA targeted against Cdc42 (Cdc42 KD) or N-WASP(N-WASP KD) were fixed with methanol and stained for ROCK-1 and ZO-1. Representative images were acquired at the apical junctions of the cells by confocal microscopy. (i) MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or siRNA targeted against Cdc42 (Cdc42 KD) or N-WASP (N-WASP KD were fixed with TCA and stained for RhoA. Representative images were acquired at the apical junctions of the cells by confocal microscopy. (j) MCF-7 cells transfected with non-targeting control siRNA (Ctrl) or siRNA targeted against Cdc42 (Cdc42 KD) or N-WASP (N-WASP KD) were fixed with methanol and stained for p190B Rho GAP, E-cadherin or ZO-1. Representative images were acquired at the apical junctions of the cells by confocal microscopy. Scale bars, 10 μm.

Supplementary Figure 7 Bistable properties of RhoA localization at cell–cell junctions.

(a) Using the model and constants as described in Supplementary Table 1, time course plots for Rnd3 against RhoA are shown for three values of KNMIIAROCK1 = 1.4: (left); KNMIIAROCK1 = 2.3 (centre); KNMIIAROCK1 = 1.4 (right) KNMIIAROCK1 = 0.5. In each, initial concentrations of RhoA and Rnd3 were set in a range between 0 and 3.5 and all other concentrations equal to unity. Simulations were run for t = 0 to t = 5,000, with each line corresponding to a time course. Arrows denote the direction of increased time and red circles denote stable points. (b,c) Convergence behaviour of Rnd3 and RhoA upon (b) varying initial values of NMIIA for the network in Fig. 7a and; (c) varying the strength of negative feedback from Rnd3 to ROCK-1 for the network present in Scheme 4 (Supplementary Note). Using the model and constants as in Supplementary Table 1, the convergence behaviour for initial values of Rnd3 and RhoA in the range 0 to 3.5 were examined, with all other initial concentrations set to 1 apart from NMIIA which was allowed to take values 0, 1, 2, 3, 4 for the simulations in (b); and for the Hill constant KRnd3ROCK1 which was allowed to take values 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30, and with initial concentration of NMIIA = 1, and b7 = 0.15 for simulations in (c). The red circles show the stable point concentrations for Rnd3 and RhoA that all initialisations converge towards. Each point in the plane then corresponds to an initialisation for Rnd3 and RhoA that will converge to one of the stable circles. Each line then denotes the boundary between those points in the plane that will converge to either the RhoA low/Rnd3 high state of junctional content and the RhoA high/Rnd3 low state). (d) A model for the maintenance of a robust Rho zone at the epithelial Zonula Adherens.

Supplementary Figure 8 Bright field/DIC images corresponding to Figs 1a, f, 2h, 3a, 3b, 5h and Supplementary Figs 1c, e, 4e.

Supplementary Figure 9 Uncropped western blots.

Supplementary Table 1 Sequences of siRNA and primers used for the study.
Full size table

Supplementary information

Supplementary Information

Supplementary Information (PDF 2428 kb)

Supplementary Note

Supplementary Information (PDF 19518 kb)

Supplementary Table 2

Statistical source data (XLSX 3890 kb)

GFP-AHPH localizes at the zonula adherens of epithelial cells.

Z-stacks of GFP-AHPH and RFP-UtrCH acquired by spinning disc confocal microscopy. (AVI 967 kb)

GFP-AHPH exhibits stability on the time scale of minutes.

Time-lapse imaging of GFP-APHPH (transfected in MCF-7 cells) acquired over a span of 30 min. (AVI 3294 kb)

ROCK-1 inhibition causes accumulation of p190B Rho GAP at the cell–cell junctions.

MCF-7 cells were transfected with GFP-p190B RhoGAP and time-lapsed imaging was performed briefly before and after addition of Y-27632 (30 μM) (20-min post-treatment). (AVI 3327 kb)

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Priya, R., Gomez, G., Budnar, S. et al. Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions. Nat Cell Biol 17, 1282–1293 (2015). https://doi.org/10.1038/ncb3239

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