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Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium

Abstract

Epithelial repair and regeneration are driven by collective cell migration and division. Both cellular functions involve tightly controlled mechanical events, but how physical forces regulate cell division in migrating epithelia is largely unknown. Here we show that cells dividing in the migrating zebrafish epicardium exert large cell–extracellular matrix (ECM) forces during cytokinesis. These forces point towards the division axis and are exerted through focal adhesions that connect the cytokinetic ring to the underlying ECM. When subjected to high loading rates, these cytokinetic focal adhesions prevent closure of the contractile ring, leading to multi-nucleation through cytokinetic failure. By combining a clutch model with experiments on substrates of different rigidity, ECM composition and ligand density, we show that failed cytokinesis is triggered by adhesion reinforcement downstream of increased myosin density. The mechanical interaction between the cytokinetic ring and the ECM thus provides a mechanism for the regulation of cell division and polyploidy that may have implications in regeneration and cancer.

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The data that support the findings of this study are available from the corresponding authors on reasonable request.

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References

  1. 1.

    Jopling, C. et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464, 606–609 (2010).

  2. 2.

    Raya, Á. et al. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc. Natl Acad. Sci. USA 100, 11889–11895 (2003).

  3. 3.

    Wang, J. et al. The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion. Development 138, 3421–3430 (2011).

  4. 4.

    Lien, C.-L., Harrison, M. R., Tuan, T.-L. & Starnes, V. A. Heart repair and regeneration: recent insights from zebrafish studies. Wound Repair Regen. 20, 638–646 (2012).

  5. 5.

    Lepilina, A. et al. A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127, 607–619 (2006).

  6. 6.

    Wei, K. et al. Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature 525, 479–485 (2015).

  7. 7.

    Huang, G. N. et al. C/EBP transcription factors mediate epicardial activation during heart development and injury. Science 338, 1599–1603 (2012).

  8. 8.

    Peralta, M., González-Rosa, J. M., Marques, I. J. & Mercader, N. The epicardium in the embryonic and adult zebrafish. J. Dev. Biol. 2, 101–116 (2014).

  9. 9.

    Wang, J., Cao, J., Dickson, A. L. & Poss, K. D. Epicardial regeneration is guided by cardiac outflow tract and hedgehog signalling. Nature 522, 226–230 (2015).

  10. 10.

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

  11. 11.

    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).

  12. 12.

    Das, T. et al. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat. Cell Biol. 17, 276–287 (2015).

  13. 13.

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

  14. 14.

    du Roure, O. et al. Force mapping in epithelial cell migration. Proc. Natl Acad. Sci. USA 102, 2390–2395 (2005).

  15. 15.

    Kim, J., Rubin, N., Huang, Y., Tuan, T.-L. & Lien, C.-L. In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix. Nat. Protoc. 7, 247–255 (2012).

  16. 16.

    Garcia-Puig, A. et al. Proteomics analysis of extracellular matrix remodeling during zebrafish heart regeneration. Preprint at https://www.biorxiv.org/content/10.1101/588251v1 (2019).

  17. 17.

    Cao, J. et al. Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue. Dev. Cell 42, 600–615 (2017).

  18. 18.

    Bazellières, E. et al. Control of cell–cell forces and collective cell dynamics by the intercellular adhesome. Nat. Cell Biol. 17, 409–420 (2015).

  19. 19.

    Brugués, A. et al. Forces driving epithelial wound healing. Nat. Phys. 10, 683–690 (2014).

  20. 20.

    Kim, J. H. et al. Propulsion and navigation within the advancing monolayer sheet. Nat. Mater. 12, 856–863 (2013).

  21. 21.

    Cadart, C., Zlotek-Zlotkiewicz, E., Le Berre, M., Piel, M. & Matthews, H. K. Exploring the function of cell shape and size during mitosis. Dev. Cell 29, 159–169 (2014).

  22. 22.

    Lancaster, O. M. et al. Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation. Dev. Cell 25, 270–283 (2013).

  23. 23.

    Cramer, L. P. & Mitchison, T. J. Investigation of the mechanism of retraction of the cell margin and rearward flow of nodules during mitotic cell rounding. Mol. Biol. Cell 8, 109–119 (1997).

  24. 24.

    Maddox, A. S. & Burridge, K. RhoA is required for cortical retraction and rigidity during mitotic cell rounding. J. Cell Biol. 160, 255–265 (2003).

  25. 25.

    Dao, V. T., Dupuy, A. G., Gavet, O., Caron, E. & de Gunzburg, J. Dynamic changes in Rap1 activity are required for cell retraction and spreading during mitosis. J. Cell Sci. 122, 2996–3004 (2009).

  26. 26.

    Goody, M. F., Kelly, M. W., Lessard, K. N., Khalil, A. & Henry, C. A. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis. Dev. Biol. 344, 809–826 (2010).

  27. 27.

    Sidhaye, J. & Norden, C. Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis. eLife 6, e22689 (2017).

  28. 28.

    Davoli, T. & de Lange, T. The causes and consequences of polyploidy in normal development and cancer. Annu. Rev. Cell Dev. Biol. 27, 585–610 (2011).

  29. 29.

    Losick, VickiP., Fox, DonaldT. & Spradling, AllanC. Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium. Curr. Biol. 23, 2224–2232 (2013).

  30. 30.

    Campinho, P. et al. Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly. Nat. Cell Biol. 15, 1405 (2013).

  31. 31.

    Lens, S. M. A. & Medema, R. H. Cytokinesis defects and cancer. Nat. Rev. Cancer 19, 32–45 (2019).

  32. 32.

    Fujiwara, T. et al. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437, 1043–1047 (2005).

  33. 33.

    Caldwell, C. M., Green, R. A. & Kaplan, K. B. APC mutations lead to cytokinetic failures in vitro and tetraploid genotypes in Min mice. J. Cell Biol. 178, 1109–1120 (2007).

  34. 34.

    Riveline, D. et al. Focal contacts as mechanosensors. J. Cell Biol. 153, 1175–1186 (2001).

  35. 35.

    Elosegui-Artola, A. et al. Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity. Nat. Cell Biol. 18, 540–548 (2016).

  36. 36.

    Oria, R. et al. Force loading explains spatial sensing of ligands by cells. Nature 552, 219–224 (2017).

  37. 37.

    Elosegui-Artola, A. et al. Rigidity sensing and adaptation through regulation of integrin types. Nat. Mater. 13, 631–637 (2014).

  38. 38.

    Chan, C. E. & Odde, D. J. Traction dynamics of filopodia on compliant substrates. Science 322, 1687–1691 (2008).

  39. 39.

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

  40. 40.

    Uroz, M. et al. Regulation of cell cycle progression by cell–cell and cell–matrix forces. Nat. Cell Biol. 20, 646–654 (2018).

  41. 41.

    Dix, C. L. et al. The role of mitotic cell-substrate adhesion re-modeling in animal cell division. Dev. Cell 45, 132–145 (2018).

  42. 42.

    Kamranvar, S. A., Gupta, D. K., Huang, Y., Gupta, R. K. & Johansson, S. Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody. Oncotarget 7, 30820–30830 (2016).

  43. 43.

    Jones, M. C., Askari, J. A., Humphries, J. D. & Humphries, M. J. Cell adhesion is regulated by CDK1 during the cell cycle. J. Cell Biol. 217, 3203–3218 (2018).

  44. 44.

    Lock, J. G. et al. Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis. Nat. Cell Biol. 20, 1290–1302 (2018).

  45. 45.

    Pellinen, T. et al. Integrin trafficking regulated by Rab21 is necessary for cytokinesis. Dev. Cell 15, 371–385 (2008).

  46. 46.

    Taneja, N. et al. Focal adhesions control cleavage furrow shape and spindle tilt during mitosis. Sci. Rep. 6, 29846 (2016).

  47. 47.

    Bastos, R. N., Penate, X., Bates, M., Hammond, D. & Barr, F. A. CYK4 inhibits Rac1-dependent PAK1 and ARHGEF7 effector pathways during cytokinesis. J. Cell Biol. 198, 865–880 (2012).

  48. 48.

    He, X., Chen, X., Li, B., Ji, J. & Chen, S. FAK inhibitors induce cell multinucleation and dramatically increase pro‐tumoral cytokine expression in RAW 264.7 macrophages. FEBS Lett. 591, 3861–3871 (2017).

  49. 49.

    Burton, K. & Taylor, D. L. Traction forces of cytokinesis measured with optically modified elastic substrata. Nature 385, 450–454 (1997).

  50. 50.

    Tanimoto, H. & Sano, M. Dynamics of traction stress field during cell division. Phys. Rev. Lett. 109, 248110 (2012).

  51. 51.

    Ermis, A. et al. Tetraploidization is a physiological enhancer of wound healing. Eur. Surg. Res. 30, 385–392 (1998).

  52. 52.

    Schoenfelder, K. P. & Fox, D. T. The expanding implications of polyploidy. J. Cell Biol. 209, 485–491 (2015).

  53. 53.

    Normand, G. & King, R. W. Understanding cytokinesis failure. Adv. Exp. Med. Biol. 676, 27–55 (2010).

  54. 54.

    Zhang, W. & Robinson, D. N. Balance of actively generated contractile and resistive forces controls cytokinesis dynamics. Proc. Natl Acad. Sci. USA 102, 7186–7191 (2005).

  55. 55.

    Descovich, C. P. et al. Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis. Mol. Biol. Cell 29, 622–631 (2018).

  56. 56.

    Polte, T. R., Eichler, G. S., Wang, N. & Ingber, D. E. Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress. Am. J. Physiol. Cell Physiol. 286, C518–C528 (2004).

  57. 57.

    van der Zwaag, D. et al. Super resolution imaging of nanoparticles cellular uptake and trafficking. ACS Appl. Mater. Interfaces 8, 6391–6399 (2016).

  58. 58.

    Christensen, A. et al. Friction-limited cell motility in confluent monolayer tissue. Phys. Biol. 15, 066004 (2018).

  59. 59.

    Balaban, N. Q. et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466–472 (2001).

  60. 60.

    Srinivasan, M. & Walcott, S. Binding site models of friction due to the formation and rupture of bonds: state–function formalism, force–velocity relations, response to slip velocity transients and slip stability. Phys. Rev. E 80, 046124 (2009).

  61. 61.

    Sabass, B. & Schwarz, U. S. Modeling cytoskeletal flow over adhesion sites: competition between stochastic bond dynamics and intracellular relaxation. J. Phys. Condens. Matter 22, 194112 (2010).

  62. 62.

    Harland, B., Walcott, S. & Sun, S. X. Adhesion dynamics and durotaxis in migrating cells. Phys. Biol. 8, 015011 (2011).

  63. 63.

    Sens, P. Rigidity sensing by stochastic sliding friction. Europhys. Lett. 104, 38003 (2013).

  64. 64.

    Ghibaudo, M. et al. Traction forces and rigidity sensing regulate cell functions. Soft Matter 4, 1836–1843 (2008).

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Acknowledgements

The authors thank N. Castro and C. Garcia-Pastor for technical assistance, C. Norden, E. Martí and K. Poss for sharing mutant zebrafish and J. Muñoz’s custom FEM-platform EMBRYO, which has been utilized to implement the numerical model. M.U., A.G.-P. and I.T. were partially supported by pre-doctoral fellowships from the Spanish Ministry of Economy and Competitiveness ((MINECO)/FEDER BES-2013-062633, BES-2013-064698 and FPU-AP2010-5071, respectively). This work was supported by MINECO/FEDER (BFU2016-79916-P and BFU2014-52586-REDT to P.R.-C., BFU2015-65074-P to X.T., SAF2015-69706-R to A.R., BFU2016-75101-P and RYC-2014-15559 to V.C.), the Generalitat de Catalunya (2014-SGR-927 to X.T., 2014-SGR-1460 and PERIS SLT002/16/00234 to A.R. and the CERCA Programme), Instituto de Salud Carlos III-ISCIII/FEDER (Red de Terapia Celular—TerCel RD16/0011/0024 to A.R.), the European Research Council (CoG-616480 to X.T.), the European Commission (grant agreeement SEP-210342844 to P.R.-C. and X.T.), Obra Social ‘La Caixa’, and the prize ICREA Academia for excellence in research (P.R.-C.). IBEC is recipient of a Severo Ochoa Award of Excellence from MINECO.

Author information

M.U., A.R. and X.T. conceived the study and designed experiments. M.U., A.G.-P., I.T., J.F.A. and A.M.-L. performed experiments. M.U. and A.E.-A. analysed data. A.E.-A., V.C. and P.R.-C. carried out theoretical modelling. M.U., S.P. and L.A. carried out STORM imaging. All authors discussed the results. M.U. and X.T. wrote the manuscript with feedback from all authors. X.T. oversaw the project.

Ethics

All experiments were performed in accordance with relevant guidelines and regulations, and conducted following procedures approved by the Ethics Committee on Experimental Animals of the PRBB (CEEA-PRBB).

Competing interests

The authors declare no competing interests.

Correspondence to Xavier Trepat.

Supplementary information

  1. Supplementary Information

    Supplementary Figs. 1–4, Supplementary Video Captions 1–10, Supplementary Table 1

  2. Reporting Summary

  3. Supplementary Video 1

    Collective cell migration of a monolayer of epicardial cells explanting from the heart

  4. Supplementary Video 2

    Myosin dynamics during cell migration (cells expressing Myosin-GFP)

  5. Supplementary Video 3

    Myosin dynamics with tractions overlaid after a follower cell spontaneously dies

  6. Supplementary Video 4

    Phase contrast images of a monolayer of epicardial cells with overlaid tractions

  7. Supplementary Video 5

    Tractions during a successful division overlaid on phase contrast images

  8. Supplementary Video 6

    Tractions during a successful division overlaid on myosin-GFP images

  9. Supplementary Video 7

    Paxillin-GFP during a successful cell division

  10. Supplementary Video 8

    Tractions during a failed division overlaid on myosin-GFP images

  11. Supplementary Video 9

    Paxillin images during a failed cell division

  12. Supplementary Video 10

    Calculation of average traction and myosin intensity

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Fig. 1: Zebrafish epicardium uses an unconventional mechanism of collective migration.
Fig. 2: Mechanics of epicardial migration.
Fig. 3: Epicardial cells exert traction forces during cytokinesis.
Fig. 4: A large number of cells are multinucleated due to cytokinetic failure.
Fig. 5: Reinforcement of CFAs leads to cytokinetic failure.