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Quantifying cell-generated mechanical forces within living embryonic tissues

A Corrigendum to this article was published on 27 February 2014

This article has been updated

Abstract

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN μm−2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

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Figure 1: Oil microdroplets as force transducers.
Figure 2: Measure of cell-generated mechanical stresses in epithelial and mesenchymal cell aggregates.
Figure 3: Ensemble statistics of droplet deformations in cell aggregates.
Figure 4: Measurement of cell-generated mechanical stresses in living mandibles.
Figure 5: Statistics of droplet deformations in living mandibles.

Change history

  • 05 February 2014

    In the version of this article initially published, the current affiliation of author Ralph Sperling was not included. His current affiliation is the Fraunhofer ICT-IMM, Mainz, Germany. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Thompson, D.W. On Growth and Form 2nd ed. (Dover, 1942).

  2. Mammoto, T. & Ingber, D.E. Mechanical control of tissue and organ development. Development 137, 1407–1420 (2010).

    Article  CAS  Google Scholar 

  3. 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  Google Scholar 

  4. Blanchard, G.B. et al. Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation. Nat. Methods 6, 458–464 (2009).

    Article  CAS  Google Scholar 

  5. Butler, L.C. et al. Cell shape changes indicate a role for extrinsic tensile forces in Drosophila germ-band extension. Nat. Cell Biol. 11, 859–864 (2009).

    Article  CAS  Google Scholar 

  6. Beloussov, L.V. Mechanically based generative laws of morphogenesis. Phys. Biol. 5, 015009 (2008).

    Article  Google Scholar 

  7. Lecuit, T. & Lenne, P.-F. Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis. Nat. Rev. Mol. Cell Biol. 8, 633–644 (2007).

    Article  CAS  Google Scholar 

  8. Ingber, D.E. Mechanical control of tissue growth: function follows form. Proc. Natl. Acad. Sci. USA 102, 11571–11572 (2005).

    Article  CAS  Google Scholar 

  9. Shraiman, B.I. Mechanical feedback as a possible regulator of tissue growth. Proc. Natl. Acad. Sci. USA 102, 3318–3323 (2005).

    Article  CAS  Google Scholar 

  10. Ingber, D.E. & Jamieson, J.D. in Gene Expression During Normal and Malignant Differentiation (eds. Andersson, L.C., Gahmberg, C.G. & Ekblom, P.) (Academic Press, Orlando, Florida, USA, 1985).

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Parker, K.K. et al. Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces. FASEB J. 16, 1195 (2002).

    Article  CAS  Google Scholar 

  14. Maniotis, A.J., Chen, C.S. & Ingber, D.E. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc. Natl. Acad. Sci. USA 94, 849–854 (1997).

    Article  CAS  Google Scholar 

  15. Théry, M., Jiménez-Dalmaroni, A., Racine, V., Bornens, M. & Jülicher, F. Experimental and theoretical study of mitotic spindle orientation. Nature 447, 493–496 (2007).

    Article  Google Scholar 

  16. Puliafito, A. et al. Collective and single cell behavior in epithelial contact inhibition. Proc. Natl. Acad. Sci. USA 109, 739–744 (2012).

    Article  CAS  Google Scholar 

  17. Montel, F. et al. Stress clamp experiments on multicellular tumor spheroids. Phys. Rev. Lett. 107, 188102 (2011).

    Article  Google Scholar 

  18. Chen, C.S., Mrksich, M., Huang, S., Whitesides, G.M. & Ingber, D.E. Geometric control of cell life and death. Science 276, 1425–1428 (1997).

    Article  CAS  Google Scholar 

  19. Singhvi, R. et al. Engineering cell shape and function. Science 264, 696–698 (1994).

    Article  CAS  Google Scholar 

  20. Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  21. Farge, E. Mechanical induction of Twist in the Drosophila foregut/stomodeal primordium. Curr. Biol. 13, 1365–1377 (2003).

    Article  CAS  Google Scholar 

  22. Desprat, N., Supatto, W., Pouille, P.-A., Beaurepaire, E. & Farge, E. Tissue deformation modulates twist expression to determine anterior midgut differentiation in Drosophila embryos. Dev. Cell 15, 470–477 (2008).

    Article  CAS  Google Scholar 

  23. Puech, P.-H. et al. Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy. J. Cell Sci. 118, 4199 (2005).

    Article  CAS  Google Scholar 

  24. Krieg, M. et al. Tensile forces govern germ-layer organization in zebrafish. Nat. Cell Biol. 10, 429–436 (2008).

    Article  CAS  Google Scholar 

  25. Maître, J.-L. & Heisenberg, C.-P. The role of adhesion energy in controlling cell-cell contacts. Curr. Opin. Cell Biol. 23, 508–514 (2011).

    Article  Google Scholar 

  26. Guevorkian, K., Gonzalez-Rodriguez, D., Carlier, C., Dufour, S. & Brochard-Wyart, F. Mechanosensitive shivering of model tissues under controlled aspiration. Proc. Natl. Acad. Sci. USA 108, 13387–13392 (2011).

    Article  CAS  Google Scholar 

  27. Wang, N., Butler, J.P. & Ingber, D.E. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 (1993).

    Article  CAS  Google Scholar 

  28. Stabley, D.R., Jurchenko, C., Marshall, S.S. & Salaita, K.S. Visualizing mechanical tension across membrane receptors with a fluorescent sensor. Nat. Methods 9, 64–67 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Harris, A.K., Wild, P. & Stopak, D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177–179 (1980).

    Article  CAS  Google Scholar 

  31. Dembo, M. & Wang, Y.-L. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 76, 2307–2316 (1999).

    Article  CAS  Google Scholar 

  32. Tan, J.L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl. Acad. Sci. USA 100, 1484–1489 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Legant, W.R. et al. Measurement of mechanical tractions exerted by cells in three-dimensional matrices. Nat. Methods 7, 969–971 (2010).

    Article  CAS  Google Scholar 

  35. Gjorevski, N. & Nelson, C.M. Mapping of mechanical strains and stresses around quiescent engineered three-dimensional epithelial tissues. Biophys. J. 103, 152–162 (2012).

    Article  CAS  Google Scholar 

  36. Rauzi, M. & Lenne, P.-F. Cortical forces in cell shape changes and tissue morphogenesis. Curr. Top. Dev. Biol. 95, 93–144 (2011).

    Article  Google Scholar 

  37. Rauzi, M., Verant, P., Lecuit, T. & Lenne, P.-F. Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis. Nat. Cell Biol. 10, 1401–1410 (2008).

    Article  CAS  Google Scholar 

  38. Behrndt, M. et al. Forces driving epithelial spreading in zebrafish gastrulation. Science 338, 257–260 (2012).

    Article  CAS  Google Scholar 

  39. Hutson, M.S. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300, 145–149 (2003).

    Article  CAS  Google Scholar 

  40. Boukellal, H., Campàs, O., Joanny, J.-F., Prost, J. & Sykes, C. Soft Listeria: actin-based propulsion of liquid drops. Phys. Rev. E 69, 061906 (2004).

    Article  Google Scholar 

  41. Trichet, L., Campàs, O., Sykes, C. & Plastino, J. VASP governs actin dynamics by modulating filament anchoring. Biophys. J. 92, 1081–1089 (2007).

    Article  CAS  Google Scholar 

  42. Keese, C.R. & Giaever, I. Cell growth on liquid microcarriers. Science 219, 1448–1449 (1983).

    Article  CAS  Google Scholar 

  43. Riess, J.G. & Krafft, M.P. Fluorinated materials for in vivo oxygen transport (blood substitutes), diagnosis and drug delivery. Biomaterials 19, 1529–1539 (1998).

    Article  CAS  Google Scholar 

  44. Krafft, M.P. & Riess, J.G. Chemistry, physical chemistry, and uses of molecular fluorocarbon–hydrocarbon diblocks, triblocks, and related compounds–unique apolar components for self-assembled colloid and interface engineering. Chem. Rev. 109, 1714–1792 (2009).

    Article  CAS  Google Scholar 

  45. de Gennes, P.-G., Brochard-Wyart, F. & Quéré, D. Capillarity and Wetting Phenomena: Drops, Bubbles, Pearls, Waves (Springer, 2003).

  46. Do Carmo, M.P. Differential Geometry of Curves and Surfaces (Prentice Hall, 1976).

  47. Basan, M., Risler, T., Joanny, J.-F., Sastre-Garau, X. & Prost, J. Homeostatic competition drives tumor growth and metastasis nucleation. HFSP J. 3, 265–272 (2009).

    Article  Google Scholar 

  48. 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).

    Article  CAS  Google Scholar 

  49. Lew, R.R., Levina, N.N., Walker, S.K. & Garrill, A. Turgor regulation in hyphal organisms. Fungal Genet. Biol. 41, 1007–1015 (2004).

    Article  CAS  Google Scholar 

  50. Ashok, B., Arleth, L., Hjelm, R.P., Rubinstein, I. & Önyüksel, H. In vitro characterization of PEGylated phospholipid micelles for improved drug solubilization: effects of PEG chain length and PC incorporation. J. Pharm. Sci. 93, 2476–2487 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the SysCODE consortium for postdoctoral financial support for O.C. and for interesting discussions with several of its members. We thank C. Jorcyk (Boise State University) for providing premalignant mammary epithelial M28 cells and B. Ristenpart for the Matlab code used to analyze data obtained with the pendant drop method. O.C. thanks all members of the Ingber lab for their help and support, J. Gros for help with imaging, and F. Aguet for help with SteerableJ plugins. R.A.S. gratefully acknowledges funding from the German Research Foundation (Sp 1282/1-1). This work was supported by US National Institutes of Health grant RL1 DE019023-01 (to D.E.I.), the Wyss Institute for Biologically Inspired Engineering at Harvard University, the MacArthur Foundation and the Harvard NSF-MRSEC (L.M.).

Author information

Authors and Affiliations

Authors

Contributions

D.E.I., O.C. and L.M. defined the project; O.C. conceived of the droplets as force transducers; O.C. and D.E.I. designed the technique; T.M. and D.O. provided dissected mouse mandibles; O.C. and S.H. microinjected droplets into mouse mandibles; O.C., R.A.S. and D.A.W. designed and synthesized new fluorocarbon-hydrocarbon block copolymers; O.C. and A.G.B. did the initial tests of the technique using cell aggregates; O.C. performed force measurements in cell-drop aggregates and living mouse mandibles; O.C. performed confocal measurements; O.C. analyzed the data; D.O. and R.M. provided transgenic mice; and O.C., L.M. and D.E.I. wrote the paper.

Corresponding authors

Correspondence to Otger Campàs or Donald E Ingber.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 and Supplementary Notes 1–3 (PDF 4059 kb)

Effect of myosin II inhibition on droplet deformations

Time-lapse showing the effect of myosin II inhibition on droplet deformations. Myosin II was inhibited using blebbistatin (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Droplets rounded up as a consequence on myosin II inhibition, indicating a substantial decrease in the ability of cells to generate forces. (MP4 1457 kb)

Effect of actin polymerization inhibition on droplet deformations

Time-lapse showing the effect of actin polymerization inhibition on droplet deformations. Actin polymerization was inhibited using cytochalasin D (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Droplets rounded up as a consequence on actin polymerization inhibition, indicating a substantial decrease in the ability of cells to generate forces. (MP4 1464 kb)

Effect of cell disruption on droplet deformations

Time-lapse showing the effect of cell disruption on droplet deformations. Cells were disrupted with the detergent sodium dodecyl sulfate (see Online Methods: 'Perturbation of cellular forces with drugs'). The drug was added at t = 0. Cell aggregates disassembled completely in the presence of the drug and droplets rounded up immediately as a consequence, indicating that cell-generated forces were causing the droplet deformations. (MP4 1137 kb)

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Campàs, O., Mammoto, T., Hasso, S. et al. Quantifying cell-generated mechanical forces within living embryonic tissues. Nat Methods 11, 183–189 (2014). https://doi.org/10.1038/nmeth.2761

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