[go: up one dir, main page]
More Web Proxy on the site http://driver.im/
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.

  • Article
  • Published:

A molecular mechanotransduction pathway regulates collective migration of epithelial cells

Nature Cell Biology volume 17, pages 276–287 (2015)Cite this article

Subjects

Abstract

Collective movement of epithelial cells drives essential multicellular organization during various fundamental physiological processes encompassing embryonic morphogenesis, cancer and wound healing. Yet the molecular mechanism that ensures the coordinated movement of many cells remains elusive. Here we show that a tumour suppressor protein, merlin, coordinates collective migration of tens of cells, by acting as a mechanochemical transducer. In a stationary epithelial monolayer and also in three-dimensional human skin, merlin localizes to cortical cell–cell junctions. During migration initiation, a fraction of cortical merlin relocalizes to the cytoplasm. This relocalization is triggered by the intercellular pulling force of the leading cell and depends on the actomyosin-based cell contractility. Then in migrating cells, taking its cue from the intercellular pulling forces, which show long-distance ordering, merlin coordinates polarized Rac1 activation and lamellipodium formation on the multicellular length scale. Together, these results provide a distinct molecular mechanism linking intercellular forces to collective cell movements in migrating epithelia.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Merlin supports collective migration of epithelial cells.
Figure 2: Importance of dynamic relocalization and phosphorylation of merlin.
Figure 3: Contractile pulling force across cell–cell boundary relocalizes cortical merlin to the cytoplasm.
Figure 4: Association of cortical merlin with the TJ complex is critical for collective migration.
Figure 5: Merlin supports collective migration through the angiomotin–Rich1–Rac1 axis independent of the Hippo pathway.
Figure 6: Merlin acts as a mechanotransducer to convert cell–cell pulling forces into polarized Rac1 activation.
Figure 7: Merlin regulates lamellipodium formation to ensure coherent cell movement.
Figure 8: The proposed model underlying the directional cell movement and merlin–Rac1 interaction.

Similar content being viewed by others

References

  1. Rorth, P. Fellow travellers: emergent properties of collective cell migration. EMBO Rep. 13, 984–991 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445–457 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Friedl, P., Locker, J., Sahai, E. & Segall, J. E. Classifying collective cancer cell invasion. Nat. Cell Biol. 14, 777–783 (2012).

    Article  PubMed  Google Scholar 

  4. Ramel, D., Wang, X. B., Laflamme, C., Montell, D. J. & Emery, G. Rab11 regulates cell–cell communication during collective cell movements. Nat. Cell Biol. 15, 317–324 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Theveneau, E. et al. Chase-and-run between adjacent cell populations promotes directional collective migration. Nat. Cell Biol. 15, 763 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Simpson, K. J. et al. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat. Cell Biol. 10, 1027–1038 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Poujade, M. et al. Collective migration of an epithelial monolayer in response to a model wound. Proc. Natl Acad. Sci. USA 104, 15988–15993 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Angelini, T. E., Hannezo, E., Trepat, X., Fredberg, J. J. & Weitz, D. A. Cell Migration Driven by Cooperative Substrate Deformation Patterns. Phys. Rev. Lett. 104, 168104 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Petitjean, L. et al. Velocity Fields in a Collectively Migrating Epithelium. Biophys. J. 98, 1790–1800 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Gaggioli, C. et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat. Cell Biol. 9, 1392–1400 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Xu, N., Keung, B. & Myat, M. M. Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase. Dev. Biol. 321, 88–100 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, X. B., He, L., Wu, Y. I., Hahn, K. M. & Montell, D. J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell. Biol. 12, 591–597 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hidalgo-Carcedo, C. et al. Collective cell migration requires suppression of actomyosin at cell–cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6. Nat. Cell Biol. 13, 49–58 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Ng, M. R., Besser, A., Danuser, G. & Brugge, J. S. Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility. J. Cell Biol. 199, 545–563 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Reffay, M. et al. Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells. Nat. Cell Biol. 16, 217–223 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Peglion, F., Llense, F. & Etienne-Manneville, S. Adherens junction treadmilling during collective migration. Nat. Cell Biol. 16, 639–651 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Tambe, D. T. et al. Collective cell guidance by cooperative intercellular forces. Nat. Mater. 10, 469–475 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Trepat, X. et al. Physical forces during collective cell migration. Nat. Phys. 5, 426–430 (2009).

    Article  CAS  Google Scholar 

  19. Angelini, T. E. et al. Glass-like dynamics of collective cell migration. Proc. Natl Acad. Sci. USA 108, 4714–4719 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Bernascone, I. & Martin-Belmonte, F. Crossroads of Wnt and Hippo in epithelial tissues. Trends Cell Biol. 23, 380–389 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Bosveld, F. et al. Mechanical control of morphogenesis by Fat/Dachsous/Four-Jointed planar cell polarity pathway. Science 336, 724–727 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Fehon, R. G., McClatchey, A. I. & Bretscher, A. Organizing the cell cortex: the role of ERM proteins. Nat. Rev. Mol. Cell Biol. 11, 276–287 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hamaratoglu, F. et al. The tumour-suppressor genes NF2/Merlin and expanded act through Hippo signalling to regulate cell proliferation and apoptosis. Nat. Cell Biol. 8, 27–36 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Li, W., Cooper, J., Karajannis, M. A. & Giancotti, F. G. Merlin: a tumour suppressor with functions at the cell cortex and in the nucleus. EMBO Rep. 13, 204–215 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cole, B. K., Curto, M., Chan, A. W. & McClatchey, A. I. Localization to the cortical cytoskeleton is necessary for Nf2/Merlin-dependent epidermal growth factor receptor silencing. Mol. Cell Biol. 28, 1274–1284 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Wozniak, M. A. & Chen, C. S. Mechanotransduction in development: a growing role for contractility. Nat. Rev. Mol. Cell Biol. 10, 34–43 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Tse, J. M. et al. Mechanical compression drives cancer cells toward invasive phenotype. Proc. Natl Acad. Sci. USA 109, 911–916 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Serra-Picamal, X. et al. Mechanical waves during tissue expansion. Nat. Phys. 8, 628–634 (2012).

    Article  CAS  Google Scholar 

  31. Yi, C. L. et al. A Tight junction-associated merlin-angiomotin complex Mediates Merlin’s regulation of mitogenic signaling and tumor suppressive functions. Cancer Cell 19, 527–540 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lallemand, D., Saint-Amaux, A. L. & Giovannini, M. Tumor-suppression functions of merlin are independent of its role as an organizer of the actin cytoskeleton in Schwann cells. J. Cell Sci. 122, 4141–4149 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Machacek, M. et al. Coordination of Rho GTPase activities during cell protrusion. Nature 461, 99–103 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Fenteany, G., Janmey, P. A. & Stossel, T. P. Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets. Curr. Biol. 10, 831–838 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Okada, T., Lopez-Lago, M. & Giancotti, F. G. Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane. J. Cell Biol. 171, 361–371 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Guo, L., Moon, C., Niehaus, K., Zheng, Y. & Ratner, N. Rac1 Controls Schwann Cell Myelination through cAMP and NF2/merlin. J. Neurosci. 32, 17251–17261 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gladden, A. B., Hebert, A. M., Schneeberger, E. E. & McClatchey, A. I. The NF2 tumor suppressor, Merlin, regulates epidermal development through the establishment of a junctional polarity complex. Dev. Cell 19, 727–739 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hebert, A. M., DuBoff, B., Casaletto, J. B., Gladden, A. B. & McClatchey, A. I. Merlin/ERM proteins establish cortical asymmetry and centrosome position. Genes Dev. 26, 2709–2723 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Flaiz, C., Kaempchen, K., Matthies, C. & Hanemann, C. O. Actin-rich protrusions and nonlocalized GTPase activation in Merlin-deficient schwannomas. J. Neuropathol. Exp. Neurol. 66, 608–616 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Shaw, R. J. et al. The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling. Dev. Cell 1, 63–72 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Farooqui, R. & Fenteany, G. Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J. Cell Sci. 118, 51–63 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Schubert, R. A., Dodd, I. B., Egan, J. B. & Shearwin, K. E. Cro’s role in the CI-Cro bistable switch is critical for lambda’s transition from lysogeny to lytic development. Genes Dev. 21, 2461–2472 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Huang, B. et al. The three-way switch operation of Rac1/RhoA GTPase-based circuit controlling amoeboid-hybrid-mesenchymal transition. Sci. Rep. 4, 6449 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Saito, K., Ozawa, Y., Hibino, K. & Ohta, Y. FilGAP, a Rho/Rho-associated protein kinase-regulated GTPase-activating protein for Rac, controls tumor cell migration. Mol. Biol. Cell 23, 4739–4750 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sanz-Moreno, V. et al. Rac activation and inactivation control plasticity of tumor cell movement. Cell 135, 510–523 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Alfthan, K., Heiska, L., Gronholm, M., Renkema, G. H. & Carpen, O. Cyclic AMP-dependent protein kinase phosphorylates merlin at serine 518 independently of p21-activated kinase and promotes merlin-ezrin heterodimerization. J. Biol. Chem. 279, 18559–18566 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Jin, H. C., Sperka, T., Herrlich, P. & Morrison, H. Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase. Nature 442, 576–579 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Safferling, K. et al. Wound healing revised: a novel reepithelialization mechanism revealed by in vitro and in silico models. J. Cell Biol. 203, 691–709 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Trepat, X. & Fredberg, J. J. Plithotaxis and emergent dynamics in collective cellular migration. Trends Cell Biol. 21, 638–646 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lukeman, R., Li, Y. X. & Edelstein-Keshet, L. Inferring individual rules from collective behavior. Proc. Natl Acad. Sci. USA 107, 12576–12580 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Sumino, Y. et al. Large-scale vortex lattice emerging from collectively moving microtubules. Nature 483, 448–452 (2012).

    Article  CAS  PubMed  Google Scholar 

  53. Yates, C. A. et al. Inherent noise can facilitate coherence in collective swarm motion. Proc. Natl Acad. Sci. USA 106, 5464–5469 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Fang, X. F. et al. The SOX2 response program in glioblastoma multiforme: an integrated ChIP-seq, expression microarray, and microRNA analysis. BMC Genomics 12, 11 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Dang, I. et al. Inhibitory signalling to the Arp2/3 complex steers cell migration. Nature 503, 281–284 (2013).

    Article  CAS  PubMed  Google Scholar 

  56. Jungbauer, S., Gao, H. J., Spatz, J. P. & Kemkemer, R. Two characteristic regimes in frequency-dependent dynamic reorientation of fibroblasts on cyclically stretched substrates. Biophys. J. 95, 3470–3478 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Harris, A. R. & Charras, G. T. Experimental validation of atomic force microscopy-based cell elasticity measurements. Nanotechnology 22, 345102 (2011).

    Article  PubMed  Google Scholar 

  58. Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Feder, T. J., BrustMascher, I., Slattery, J. P., Baird, B. & Webb, W. W. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70, 2767–2773 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Das, T., Maiti, T. K. & Chakraborty, S. Augmented stress-responsive characteristics of cell lines in narrow confinements. Integr. Biol. 3, 684–695 (2011).

    Article  CAS  Google Scholar 

  61. Rausch, S. et al. Polarizing cytoskeletal tension to induce leader cell formation during collective cell migration. Biointerphases 8, 32 (2013).

    Article  PubMed  Google Scholar 

  62. Tseng, Q. et al. Spatial organization of the extracellular matrix regulates cell–cell junction positioning. Proc. Natl Acad. Sci. USA 109, 1506–1511 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Tambe, D. T. et al. Monolayer stress microscopy: limitations, artifacts, and accuracy of recovered intercellular stresses. PLoS ONE 8, e55172 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to B. Geiger and F. Frischknecht for critical review of the manuscript and advice. This work was supported by the Max Planck Society. J.P.S. is the Weston Visiting Professor at the Weizmann Institute of Science and is a member of the Heidelberg Cluster of Excellence CellNetworks. This work was supported by CellNetworks EcTop2 and the Grassroot Project of the Max Planck Institute for Intelligent Systems. Part of this work was supported by the BMBF/MPG network MaxSynBio.

Author information

Authors and Affiliations

  1. Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Stuttgart 70569, Germany

    Tamal Das, Sebastian Rausch, Heike Boehm & Joachim P. Spatz

  2. Department of Biophysical Chemistry, University of Heidelberg, Heidelberg 69120, Germany

    Tamal Das, Sebastian Rausch, Heike Boehm & Joachim P. Spatz

  3. Hamamatsu Tissue Imaging and Analysis (TIGA) Center, BIOQUANT, University of Heidelberg, Heidelberg 69120, Germany

    Kai Safferling & Niels Grabe

  4. Department of Medical Oncology, NCT National Center for Tumor Diseases, University of Heidelberg, Heidelberg 69120, Germany

    Kai Safferling & Niels Grabe

Authors
  1. Tamal Das
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kai Safferling
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sebastian Rausch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Niels Grabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heike Boehm
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joachim P. Spatz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.D., H.B. and J.P.S. conceived the project, analysed the data and wrote the manuscript. T.D. carried out all experiments, except those experiments that involve wound healing in the 3D skin model, which were carried out by K.S. and N.G. S.R. provided technical help. All authors went through the manuscript and provided feedback.

Corresponding authors

Correspondence to Tamal Das or Joachim P. Spatz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Effect of Merlin depletion on velocity correlation function and correlations lengths.

(a) Representative images showing the sheet migration of MDCK monolayer at the indicated times after the removal of confinement. Scale bars, 100 μm. (b–c) Time-lapse phase-contrast images (b) of migrating epithelial monolayer were analysed by PIV to determine the velocity vectors (c). Scale bars, 150 μm. Magnified panel (c, Right, Scale bar, 50 μm) shows a highly correlated velocity domain. (d) Lateral and axial velocity correlation functions were calculated as indicated by the expressions. (e) Representative lateral (Left) and axial (Right) velocity correlation function. Correlation length is the distance where correlation function becomes zero. (f) (Left) While lateral correlation function in DECMA-1 treated cells (cells with reduced intercellular connection; green squares) showed a drastic decay in comparison to control cells (red circles), (Right) no such trend was observed in axial correlation function. (g) Lateral and axial velocity correlation lengths of DECMA-1 treated and control cells. (n = 15 samples from 3 independent experiments in all cases; Lateral control versus DECMA-1, P = 7.79 × 10−26; Axial control versus DECMA-1, P = 0.51; Lateral DECMA-1 versus axial DECMA-1, P = 1.49 × 10−12; Wilcoxon rank-sum test) ∗∗∗P < 0.001, NS: not significant (Wilcoxon rank-sum test). In box plots central mark is the median, and the edges of the box are the 1st and 3rd quartiles. Whiskers extend to the most extreme non-outlier data points. (h) Synopsis of the results from the screening experiments in HaCaT cells. Values written in the color-coded table are the mean correlation length. n = 6 samples from 3 independent experiments performed with each siRNA (Supplementary Table 1). Lack of response due to failure in gene knockdown cannot be ruled out. (i) Comparison of the representative lateral velocity correlation functions in Merlin-depleted versus scramble siRNA-transfected (control) MDCK cells showed a drastic decrease of correlation in Merlin-depleted cells. (j) Color map of velocity vectors revealed highly correlated velocity domains (indicated with arrow) in scramble siRNA-transfected MDCK cells. (k) In contrast, no such domain could be observed in Merlin-depleted cells. In (j, k), scale bars, 100 μm. Panels (a–c, e,f, i-k) show the representative image of 3 independent experiments.

Supplementary Figure 5 Effect of Merlin-depletion on cell migration and integrity of cell–cell junctions.

(a) Representative migration tracks of cells, transfected either with scramble siRNA (Left) or with Merlin siRNA1 (Right). (b–d) Directional persistence of cell migration (b, computed as the ratio of the distance between two points by the actual trajectory), individual cell speed (c), and collective speed (d) in control and Merlin-depleted MDCK monolayers. In (b) and (c), n = number of cells from 3 independent experiments (mean ± s.e.m; b, P = 3.55 × 10−57; c, P = 4.66 × 10−21; Wilcoxon rank-sum test). In (d), n = number of samples from 3 independent experiments (mean ± s.e.m; P = 3.84 × 10−16; Wilcoxon rank-sum test). (e) Mean individual cell speed in confluent control and Merlin-depleted MDCK cell monolayer. (n = 5 samples from 5 independent experiments in all cases, P = 0.07; Wilcoxon rank-sum test) For each sample, 20 or more cells were counted. In box plots central mark is the median, and the edges of the box are the 1st and 3rd quartiles. Whiskers extend to the most extreme non-outlier data points. (f) Phase-contrast image (Left) and traction force landscape (Right) of a MDCK monolayer moving towards right side. Scale bars, 100 μm. (g) Distribution of the axial (w.r.t. the direction of collective migration) traction forces showed broad-tailed distribution in both control (green circles) and Merlin-depleted (red squares) cells. (h) Adherens and tight junctions as marked by E-cadherin and ZO-1, respectively, in control and Merlin-depleted MDCK cells. Scale bars, 20 μm. (i) Z-section of confocal images showing the distribution of E-cadherin and ZO-1. Scale bars, 10 μm. (j) FRAP curve showing E-cadherin recovery after photobleaching in E-cadherin-GFP transfected control MDCK cells. t1/2 is the time required to attain 50% of the final recovered intensity (mobile fraction). n = 10 independent experiments; mean ± s.e.m. (k, l) Immobile fraction (k) and t1/2 (l) in control and Merlin siRNA1 transfected cells (by either forward or reverse transfection method). In (k,l), n = 50 cells from 3 independent experiments in all cases (mean ± s.e.m; k, Control versus Forward, P = 0.53; Control versus Reverse, P = 0.08; l, Control versus Forward, P = 0.65; Control versus Reverse, P = 0.28; Wilcoxon rank-sum test). ∗∗∗P < 0.001, NS: not significant (Wilcoxon rank-sum test). Panels (a, f, i) show the representative image of 3 independent experiments.

Supplementary Figure 6 Localization of Merlin and representative polarity complex and cell–cell junction associated proteins during collective migration.

(a) Expression pattern of Merlin within the moonlayer (Left panels), or at the leading edge (Right panels) at different time points after the confinement removal. Images show a gradual propagation of Merlin relocalization inside the monolayer with time. (b) Cytoplasmic localization of Merlin (Left—Fix1: sequential fixing and permeabilization) could not be observed in detergent-extracted samples (Right—Fix2: simultaneous fixing and permeabilization). (c) Merlin relocalization correlates with the velocity profile (representative) across the monolayer, from the leading edge towards the centre. (d) Expression pattern of Merlin upon cycloheximide treatment at 0 and 2 h after the confinement removal. Results supported Merlin relocalization over de novo synthesis of cytoplasmic Merlin during migration. (e) Localization of Par3 in stationary (Top) and migrating (Middle) wild-type MDCK cells and in Merlin-depleted cells (Bottom) in stationary condition. (f) Western blot showing Par3 depletion in MDCK cells with Par3 specific siRNAs. (g) Merlin and ZO-1 localization in control and Par3-depleted cells. Par3 siRNA2 showed similar effect. (h) Expression of E-cadherin and α-catenin in stationary and migrating MDCK monolayers. In (a–h) scale bars, 100 μm. (i) Localization of tight junction complex proteins Angiomotin and Patj in stationary and migrating MDCK cells. Scale bars, 25 μm. Panels (a-i) show the representative image of 3 independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 The excisional 3D in vitro wound model using precast epiderm full thickness cultures and dermal equivalents.

(a) Precast epiderm full thickness (EFT) skin cultures were wounded twice with a 2 mm biopsy punch. The cultures were connected on top to a prepared dermal equivalent (DE) using 6 μl unpolymerized rat tail collagen I as ‘glue’. (b) After wounding cultures were placed into a 12-well ThinCert insert. (c) Histology of the wound cultures. H&E stained sections showed punched wounds with clear defined wound margins (arrowheads). The underlying DE was seamlessly attached providing the wound matrix. (dotted line = basement membrane). (d–e) Merlin expression in histological sections of human skin equivalent in 48 h (d) and 72 h (e) post wounding tissue. Merlin shows predominant cytoplasmic localization in migrating cells. (f) Merlin expression in histological sections of human skin shows localization to cell–cell contact. (g) MDA-MB-231 cells were used as negative control for Merlin expression. (h–j) E-cadherin localization in an unwounded (h) skin equivalent and back (i) and tongue region (j) of a wounded human skin equivalent (48 h post wounding). Scale bars, 1 cm (b), 1 mm (c), 100 μm (d-g) and 50 μm (h-j). Panels (b–j) show the representative image of 3 independent experiments.

Supplementary Figure 8 Inhibiting actomyosin based cell contractility stabilizes cell-cell junctions.

(a) FRAP curve showing E-cadherin recovery after photobleaching in E-cadherin-GFP transfected control MDCK cells (mean ± s.e.m; n = 50 cells from 3 independent experiments). (b) Comparing the FRAP curve of Blebbistatin-treated cells with that of control cells showed lower mobile fraction in former (mean ± s.e.m; n = 50 cells from 3 independent experiments in all cases). (c) Immobile fraction of E-cadherin-GFP in cells treated with DMSO (control), Blebbistatin (Bleb., 50 μM), or Y27632 (Y, 30 μM). mean ± s.e.m; n = 50 cells from 3 independent experiments in all cases (Control versus Bleb., P = 2.91 × 10−12; Control versus Y, P = 3.35 × 10−10; Wilcoxon rank-sum test). (d) Adherens and tight junction markers E-cadherin and ZO-1, respectively, in control, Blebbistatin-, or Y27632-treated cells. Scale bars, 20 μm. (e) Z-section images showing adherens and tight junctions in control or treated cells. Scale bars, 10 μm. (f) Localization of endogenous Merlin in Blebbistatin- or Y27632-treated cells. Scale bars, 100 μm.∗∗∗P < 0.001 (Wilcoxon rank-sum test). Panels (d–f) show the representative image of 3 independent experiments.

Supplementary Figure 9 Interaction with cortical actin cytoskeleton and membrane-localization stability of Merlin mutants.

(a) Schematic diagram of the FRET constructs used in Merlin-actin interaction experiments. (b) Representative image showing Merlin-cortical actin interaction in a TGFP:hMerlin-FL and TRFP:LifeAct transfected MDCK cell. Scale bars, 8 μm. (c) Mean FRET index for indicated constructs. Only E1-M was found incapable of interacting with cortical actin. (n = 7 independent experiments in all cases; hMerlin-FL versus E1-M, P = 6.67 × 10−9; Wilcoxon rank-sum test) (d) A representative FRAP curve showing the prebleach and recovery phases. Intensity was normalized to ensure average prebleach intensity = 1 and intensity immediately after bleaching = 0. Immobile fraction is the non-recovered fraction of intensity. Squares and line represent the experimental points and theoretical fitting respectively. (e) Comparison of immobile fraction for different Merlin mutants. M-EzABD showed a very high immobile fraction, while M325 was highly mobile. (n = 8 independent experiments in all cases; For hMerlin-FL versus M325, M437, hEzrin-FL, E1-M, E123-M, M-EzCC, and M-EzABD, P = 5.95 × 10−53, 8.73 × 10−38, 4.69 × 10−17, 6.53 × 10−25, 2.11 × 10−39, 4.07 × 10−36, and 1.6 × 10−63 respectively; Wilcoxon rank-sum test) Red plus sign represents the outliers that is data falling outside 2.698 times of standard deviation. ∗∗∗ P < 0.001 (Wilcoxon rank-sum test). In box plots (c,e) central mark is the median, and the edges of the box are the 1st and 3rd quartiles. Whiskers extend to the most extreme non-outlier data points. Panels b and d show the representative image of 7 and 8 independent experiments respectively.

Supplementary Figure 10 Essentiality of Rac1 activation in collective migration.

(a) Phase contrast images of wound closing by control (Left panels), Rac1 inhibitor (NSC 23766)-treated (Middle panels), Rac1-depleted (Right panels) MDCK cells at different time points after confinement removal. Both NSC 23766-treated and Rac1-depleted cells showed negligible migration activity within the investigated time frame. Scale bars, 200 μm. (b) Western blot confirming Rac1-depletion in MDCK cells with Rac1-specific siRNA. (c) Percentage of cells with cryptic lamellipodium, 2 h after removal of confinement, with first 20 layers of cells behind the leading edge. (n = 5 independent experiments; P = 4.92 × 10−8; Wilcoxon rank-sum test). In box plots central mark is the median, and the edges of the box are the 1st and 3rd quartiles. Whiskers extend to the most extreme non-outlier data points. (d) The mutant, M-EzABD, inhibits Rac1 activation even in migration promoting condition. MDCK cells, transfected as indicated, were grown to confluency for 18 h and then were allowed to migrate for 3 h. Western blots showing GTP bound Rac1 (active Rac1) and total Rac1. (e) Bars represent relative Rac1 activation results from densitometric analysis. (mean ± s.e.m; n = 3 independent experiments; P = 2.61 × 10−10; Wilcoxon rank-sum test). Statistics source data are given in Supplementary Table 3. (f) Localization of endogenous Merlin in Jasplakinolide-treated stationary and migrating cells. Even though enhanced actin binding with the M-EzABD construct prevents relocalization, stabilized cortical actin cytoskeleton does not prevent Merlin relocalization. Scale bar, 100 μm. ∗∗∗P < 0.001 (Wilcoxon rank-sum test). Panels (a, b, d, f) show the representative image of 3 independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.

Supplementary Figure 11 Polarization of lamellipodium formation and Rac1 activation in migrating epithelium.

(a–b) Representative images showing the orientations of cryptic lamellipodia in control and Merlin-depleted MDCK cells. (a) In stationary condition, Merlin-depleted MDCK cells showed higher tendency for lamellipodial protrusion formation as compared to control cells. White arrowheads indicate the lamellipodia. (b) During migration, Merlin-depleted MDCK cells showed a significantly decreased alignment in their lamellipodia as compared to control MDCK cells. White arrows approximately indicate the direction of lamellipodia with respect to the cell centre. Scale bars, 25 μm. (c) Orientations of lamellipodia with respect to the global migration direction in MDCK cells, transfected as indicated. hMerlin-FL, E1-M, and E123-M were found capable of polarizing lamellipodium formation in migrating monolayer of MDCK cells. n = 154 (Scramble control), 178 (siRNA3), 112 (hMerlin-FL), 119 (M325), 116 (M487), 108 (E1-M), 105 (E123-M), and 114 (M-EzCC) cells from 3 independent experiments. (d) Orientations of lamellipodia in control and blebbistatin-treated MDCK cells. n = 121 (control) and 117 (blebbistatin) cells from 3 independent experiments. (e) Distribution of θ (angle between Rac1 activation direction and group migration direction) in control and blebbistatin-treated MDCK cells. n = 205 (control) and 198 (blebbistatin) cells from 3 independent experiments. Panels (a, b) show the representative image of 3 independent experiments.

Supplementary Figure 12 Uncropped Western Blot images.

The corresponding main or supplementary figure numbers are shown. Blue boxes highlight the cropped segment presented in main or supplementary figures. Loading controls are provided.

Supplementary Table 1 List of siRNAs used in the study.
Full size table
Supplementary Table 2 List of primary antibodies.
Full size table
Supplementary Table 3 Statistics source data.
Full size table

Supplementary information

Supplementary Information

Supplementary Information (PDF 2262 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Das, T., Safferling, K., Rausch, S. et al. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat Cell Biol 17, 276–287 (2015). https://doi.org/10.1038/ncb3115

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3115

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