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An apical MRCK-driven morphogenetic pathway controls epithelial polarity

Nature Cell Biology volume 19, pages 1049–1060 (2017)Cite this article

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Abstract

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC–Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC–Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.

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Figure 1: MRCK activates apical actomyosin contractility that controls apical morphogenesis.
Figure 2: MRCK/gek regulates apical myosin activation and morphogenesis in differentiating Drosophila photoreceptors.
Figure 3: MRCK functions as an effector of Dbl3–Cdc42 signalling.
Figure 4: Cdc42 activates MRCK apically, stimulating apical myosin-II activation and differentiation.
Figure 5: Dbl3 and Cdc42 drive apical actomyosin activation and aPKC recruitment.
Figure 6: Apical activation of Cdc42 by Dbl3 antagonizes junctional RhoA-stimulated myosin-II activation.
Figure 7: MRCK-stimulated myosin-II activation drives PAR separation at tight junctions.
Figure 8: MRCK-activated actomyosin contractility drives apical PAR segregation and polarization of cytosolic factors.

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Acknowledgements

This work was supported by the BBSRC (BB/L007584/1 and BB/N014855/1) and the Wellcome Trust (099173/Z/12/Z). Work in the F.P. laboratory, including support to E.V., was funded by an MRC grant (MC_UU_12018/3). The N2 A71 monoclonal antibodies, developed by E. Wieschaus, were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, Iowa 52242. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.

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Author notes

  1. Evi Vlassaks

    Present address: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

  2. Stephen Terry

    Present address: Randall Division of Cell and Molecular Biophysics, King’s College London, SE1 1UL, UK

Authors and Affiliations

  1. Institute of Ophthalmology, University College London, Bath Street, London EC1V 9EL, UK

    Ceniz Zihni, Stephen Terry, Maria Susana Balda & Karl Matter

  2. MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK

    Evi Vlassaks & Franck Pichaud

  3. Division of Cancer Studies, Section of Cell Biology and Imaging, King’s College London, London SE1 1UL, UK

    Jeremy Carlton

  4. Institute of Molecular and Cell Biology, A-STAR, 61 Biopolis Drive, Singapore 138673, Singapore

    Thomas King Chor Leung

  5. The Department of Anatomy, National University of Singapore, Singapore 119260, Singapore

    Thomas King Chor Leung

  6. Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK

    Michael Olson

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Contributions

C.Z. performed most of the vertebrate and E.V. the Drosophila experiments. All other authors performed particular subsets of experiments. C.Z., M.S.B. and K.M. designed the project and drafted the manuscript. All authors read and contributed to the final version of the manuscript.

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Correspondence to Maria Susana Balda or Karl Matter.

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Integrated supplementary information

Supplementary Figure 1 Quantification of microvilli induction and role of MRCK in apical morphogenesis of Caco-2 cells.

(a) Broad view of apical surface of MDCK cells by scanning electron microscopy after MRCK knockdown and after complementation with MRCKβ-flag. (b) Measurement of apical membrane brush border cluster induction using threshold function of Nikon imaging software. (c,d) Levels of microvilli induction in human Caco-2 intestinal epithelial cells following MRCK knockdown without or with complementation with MRCKβ-flag. Panel d is based on n = 3 independent experiments and shows the data points, means ± 1 s.d. (in black), the total number of cells analysed for each type of sample across all experiments, and P-values derived from t-tests.

Supplementary Figure 2 Inhibition of ROCK does not affect Dbl3-induced apical actomyosin activation.

Confocal analysis of apical pMLC activity and F-actin in MDCK cells upon induction of Dbl3 expression by tetracycline in a conditional cell line in the absence or presence of the ROCK inhibitor GSK269962. Arrowheads point to apical cortex. Panel b is based on n = 3 independent experiments and shows the data points, means ± 1 s.d. (in black), the total number of cells analysed for each type of sample across all experiments, and P-values derived from t-tests. Scale bars: 10 μm.

Supplementary Figure 3 MRCKβ expression does not rescue loss of apical Myosin-II activation and differentiation in Dbl3-depleted MDCK cells.

(a–c) Levels of Myosin phosphorylation at the apical membrane domain (A) and basal membrane (B) and F-actin during polarization and differentiation of MDCK cells following Dbl3 siRNA transfection and with constitutive expression of MRCKβ-flag. Quantifications are based on n = 3 independent experiments and show the data points, means ± 1 s.d. (in black), the total number of cells analysed for each type of sample across all experiments, and P-values derived from t-tests show means ± 1 s.d., n + 3. (d) SEM images of the effect of MRCK knock down on microvilli induction and conditional expression of Dbl3-myc. Note, MRCKβ-flag does not rescue the Dbl3 depletion-induced phenotype. (e) Time course of tetracycline-induced Dbl3-myc expression in MDCK cells. Asterisks highlight time points with clear induction of Dbl3-myc expression that were used for the functional assays. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (f) Confocal z-sections showing representative images of either endogenous or constitutively expressed MRCKβ in MDCK cells following conditional tetracycline-inducible expression of Dbl3-myc. Constitutive expression of MRCKβ-flag accelerates activation of Myosin-II at the apical membrane, highlighted by white arrows. Asterisks highlight basolateral MRCK localization at earlier stages of polarization. (g) Confocal Z-sections showing representative images of pEzrinT567 localization in MDCK cells following conditional tetracycline-inducible expression of Dbl3-myc without or with constitutive expression of MRCKβ-flag. Constitutive expression of MRCKβ-flag accelerates enrichment of active Ezrin at the apical membrane domain, indicated by white arrowheads. Scale bars, 10 μ m.

Supplementary Figure 4 Cdc42 stimulates aPKC recruitment and antagonizes Rho-signalling.

(a) Conditional tetracycline-induced expression of Dbl3 in MDCK cells stimulates increased recruitment of aPKCζ. Shown are confocal xy. (b) Levels of protein expression during tetracycline-induced Dbl3-myc expression. Unprocessed original scans of blots are shown in Supplementary Fig. 8. (c) Quantification of levels of pEzrinT567D following tetracycline-induced Dbl3-myc expression. (d) Conditional tetracycline-induced expression of Dbl3 in MDCK cells stimulates loss of junctional p114RhoGEF and LULU-2. Shown are confocal z-sections. Junctions are indicated with white arrowheads. (e,f) Spontaneously polarizing MDCK cells were treated with control or Dbl3 siRNAs, or a myristoylated aPKCζ inhibitor. LULU-2 localization at tight junctions was then analysed by confocal microscopy using ZO-1 as a marker for tight junctions (indicated by arrowheads). Quantifications are based on n = 3 independent experiments and show the data points, means ± 1 s.d. (in black), the total number of cells analysed for each type of sample across all experiments, and P-values derived from t-tests. Scale bars, 10 μm.

Supplementary Figure 5 MRCK-activated Myosin-II motor activity drives apical polarization of Par6-aPKC complex.

(a) MDCK cells conditionally expressing tetracycline-inducible Dbl3-myc were analysed by confocal microscopy using co-localization software based on the Pearson’s correlation coefficient. Co-localization is calculated in grid 3 of the scatter charts. Bright green represents co-localization of aPKCζ and Par6β, which increases and is confined to the apical cortical membrane following polarization stimulated by conditional tetracycline inducible Dbl3-myc expression in MDCK cells and is dependent on Myosin-II motor activity. Coloured outlines of apical membrane domains represent calculation areas of co-localization coefficients. Scale bars: 10 μm. (b) Co-immunoprecipitation of Par6 and aPKC indicating formation of stable complexes that are independent of Myosin-II motor activity and Dbl3 expression. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 6 Dbl3-activated Myosin-II motor activity drives apical polarization and co-localization of brush border regulators.

(a–d) MDCK cells conditionally expressing tetracycline-inducible Dbl3-myc were analysed by confocal microscopy using co-localization software based on the Pearson’s correlation coefficient. Co-localization is calculated in grid 3 of the scatter charts. Bright green represents co-localization of Ezrin and SLK or NHERF-1 and SLK, which increases and is confined to the apical membrane domain following polarization stimulated by conditional tetracycline inducible Dbl3-myc expression in MDCK cells and is dependent on Myosin-II motor activity. Coloured outlines of apical membrane domains represent calculation areas of co-localization coefficients. Scale bars: 10 μm. (e) Expression levels of differentiation markers upon Dbl3 and Myosin manipulation. Unprocessed original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 Myosin-II activation mediates gek function in pupal photoreceptors.

(a–c) Confocal sections of a pupal retinas showing wild type cells (blue nuclei) and gek mutant cells (labelled with asterisks) stained for the apical markers aPKC and Moesin, or the junctional marker Arm. (d) Quantification of the effect of mutant gek on junctional levels of Arm (measurements from wild type and neighbouring mutant cells, paired within sections; based on n = 7 animals; P value was calculated with a t-test).

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Induction of polarized myosin activation by Cdc42.

Apical Cdc42 activation by conditional expression of Dbl3-myc in MDCK cells constitutively expressing EGFP-MLC was induced by adding tetracycline followed by recording image stacks every 5 min. Shown are projections of 4 images derived from the apical domain. The experiment was performed five times. (AVI 3413 kb)

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Zihni, C., Vlassaks, E., Terry, S. et al. An apical MRCK-driven morphogenetic pathway controls epithelial polarity. Nat Cell Biol 19, 1049–1060 (2017). https://doi.org/10.1038/ncb3592

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