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Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second

Nature Materials volume 16, pages 1262–1270 (2017)Cite this article

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A Corrigendum to this article was published on 19 December 2017

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Abstract

Integrin-mediated mechanosensing of the extracellular environment allows cells to control adhesion and signalling. Whether cells sense and respond to force immediately upon ligand-binding is unknown. Here, we report that during adhesion initiation, fibroblasts respond to mechanical load by strengthening integrin-mediated adhesion to fibronectin (FN) in a biphasic manner. In the first phase, which depends on talin and kindlin as well as on the actin nucleators Arp2/3 and mDia, FN-engaged α5β1 integrins activate focal adhesion kinase (FAK) and c-Src in less than 0.5 s to steeply strengthen α5β1- and αV-class integrin-mediated adhesion. When the mechanical load exceeds a certain threshold, fibroblasts decrease adhesion and initiate the second phase, which is characterized by less steep adhesion strengthening. This unique, biphasic cellular adhesion response is mediated by α5β1 integrins, which form catch bonds with FN and signal to FN-binding integrins to reinforce cell adhesion much before visible adhesion clusters are formed.

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Figure 1: Fibroblast adhesion to fibronectin (FN) increases biphasically with the retraction speed applied to separate cell and substrate.
Figure 2: Speed-dependent rupture forces required to separate single αV-class and α5β1 integrins bound to fibronectin.
Figure 3: How fibroblasts strengthen adhesion in response to mechanical load depends on integrin activity and contact time with the substrate.
Figure 4: Fibroblasts require talin, F-actin polymerization to strengthen adhesion in response to mechanical load.
Figure 5: Integrin activation in response to mechanical load depends on FAK and c-Src signalling.
Figure 6: Instant strengthening of fibroblast adhesion to FN in response to mechanical load.

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  • 21 November 2017

    In the version of this Article originally published, Fig. 6 was not cited; a citation to it has now been added at the end of the first paragraph of 'Discussion'. This has been corrected in all versions of the Article.

References

  1. Schwartz, M. A. & DeSimone, D. W. Cell adhesion receptors in mechanotransduction. Curr. Opin. Cell. Biol. 20, 551–556 (2008).

    Article  CAS  Google Scholar 

  2. Butcher, D. T., Alliston, T. & Weaver, V. M. A tense situation: forcing tumour progression. Nat. Rev. Cancer 9, 108–122 (2009).

    Article  CAS  Google Scholar 

  3. Ross, T. D. et al. Integrins in mechanotransduction. Curr. Opin. Cell. Biol. 25, 613–618 (2013).

    Article  CAS  Google Scholar 

  4. Humphrey, J. D., Dufresne, E. R. & Schwartz, M. A. Mechanotransduction and extracellular matrix homeostasis. Nat. Rev. Mol. Cell Biol. 15, 802–812 (2014).

    Article  CAS  Google Scholar 

  5. Iskratsch, T., Wolfenson, H. & Sheetz, M. P. Appreciating force and shape—the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol. 15, 825–833 (2014).

    Article  CAS  Google Scholar 

  6. Hynes, R. O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

    Article  CAS  Google Scholar 

  7. Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell Sci. 119, 3901–3903 (2006).

    Article  CAS  Google Scholar 

  8. Leiss, M., Beckmann, K., Girós, A., Costell, M. & Fässler, R. The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr. Opin. Cell. Biol. 20, 502–507 (2008).

    Article  CAS  Google Scholar 

  9. Benito-Jardón, M. et al. The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes. eLIFE 6, e22264 (2017).

    Article  Google Scholar 

  10. Taubenberger, A., Cisneros, D. A., Puech, P.-H., Müller, D. J. & Franz, C. M. Revealing early steps of α2β1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy. Mol. Biol. Cell 18, 1634–1644 (2007).

    Article  CAS  Google Scholar 

  11. Changede, R., Xu, X., Margadant, F. & Sheetz, M. P. Nascent integrin adhesions form on all matrix rigidities after integrin activation. Dev. Cell 35, 614–621 (2015).

    Article  CAS  Google Scholar 

  12. Robertson, J. et al. Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling. Nat. Commun. 6, 6265 (2015).

    Article  CAS  Google Scholar 

  13. Schiller, H. B., Friedel, C. C., Boulegue, C. & Fässler, R. Quantitative proteomics of the integrin adhesome show a myosin II-dependent recruitment of LIM domain proteins. EMBO Rep. 12, 259–266 (2011).

    Article  CAS  Google Scholar 

  14. Schiller, H. B. et al. β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments. Nat. Cell Biol. 15, 625–636 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding. Science 323, 638–641 (2009).

    Article  CAS  Google Scholar 

  17. Sawada, Y. et al. Force sensing by extension of the Src family kinase substrate, p130Cas. Cell 127, 1015–1026 (2006).

    Article  CAS  Google Scholar 

  18. Wang, Y. et al. Visualizing the mechanical activation of Src. Nature 434, 1040–1045 (2005).

    Article  CAS  Google Scholar 

  19. Parsons, J. T., Horwitz, A. R. & Schwartz, M. A. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 11, 633–643 (2010).

    Article  CAS  Google Scholar 

  20. Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically activated integrin switch controls α5β1 function. Science 323, 642–644 (2009).

    Article  CAS  Google Scholar 

  21. Kong, F., García, A. J., Mould, A. P., Humphries, M. J. & Zhu, C. Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 1275–1284 (2009).

    Article  CAS  Google Scholar 

  22. Thomas, W. Catch bonds in adhesion. Annu. Rev. Biomed. Eng. 10, 39–57 (2008).

    Article  CAS  Google Scholar 

  23. Friedrichs, J. et al. A practical guide to quantify cell adhesion using single-cell force spectroscopy. Methods 60, 169–178 (2013).

    Article  CAS  Google Scholar 

  24. Helenius, J., Heisenberg, C-P., Gaub, H. E. & Müller, D. J. Single-cell force spectroscopy. J. Cell Sci. 121, 1785–1791 (2008).

    Article  CAS  Google Scholar 

  25. Balcioglu, H. E., van Hoorn, H., Donato, D. M., Schmidt, T. & Danen, E. H. J. The integrin expression profile modulates orientation and dynamics of force transmission at cell–matrix adhesions. J. Cell Sci. 128, 1316–1326 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Kong, F. et al. Cyclic mechanical reinforcement of integrin-ligand interactions. Mol. Cell 49, 1060–1068 (2013).

    Article  CAS  Google Scholar 

  28. Theodosiou, M. et al. Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin. eLIFE 5, e10130 (2016).

    Article  Google Scholar 

  29. Bharadwaj, M. et al. αV-class integrins exert dual roles on α5β1 integrins to strengthen adhesion to fibronectin. Nat. Commun. 8, 14348 (2017).

    Article  CAS  Google Scholar 

  30. Na, S. et al. Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc. Natl Acad. Sci. USA 105, 6626–6631 (2008).

    Article  CAS  Google Scholar 

  31. Tzima, E. et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437, 426–431 (2005).

    Article  CAS  Google Scholar 

  32. Shattil, S. J., Kim, C. & Ginsberg, M. H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010).

    Article  CAS  Google Scholar 

  33. Roca-Cusachs, P., Gauthier, N. C., del Rio, A. & Sheetz, M. P. Clustering of α5β1 integrins determines adhesion strength whereas αvβ3 and talin enable mechanotransduction. Proc. Natl Acad. Sci. USA 106, 16245–16250 (2009).

    Article  CAS  Google Scholar 

  34. Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 14, 1057–1067 (2012).

    Article  CAS  Google Scholar 

  35. Sun, Z. et al. Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation. Nat. Cell Biol. 18, 941–953 (2016).

    Article  CAS  Google Scholar 

  36. Das, M., Subbayya Ithychanda, S., Qin, J. & Plow, E. F. Mechanisms of talin-dependent integrin signaling and crosstalk. BBA-Biomembr. 1838, 579–588 (2014).

    Article  CAS  Google Scholar 

  37. Calderwood, D. A., Campbell, I. D. & Critchley, D. R. Talins and kindlins: partners in integrin-mediated adhesion. Nat. Rev. Mol. Cell Biol. 14, 503–517 (2013).

    Article  CAS  Google Scholar 

  38. Priddle, H. et al. Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells. J. Cell Biol. 142, 1121–1133 (1998).

    Article  CAS  Google Scholar 

  39. Zhang, X. et al. Talin depletion reveals independence of initial cell spreading from integrin activation and traction. Nat. Cell Biol. 10, 1062–1068 (2008).

    Article  CAS  Google Scholar 

  40. Moser, M., Legate, K. R., Zent, R. & Fässler, R. The tail of integrins, talin, and kindlins. Science 324, 895–899 (2009).

    Article  CAS  Google Scholar 

  41. Lawson, C. et al. FAK promotes recruitment of talin to nascent adhesions to control cell motility. J. Cell Biol. 196, 223–232 (2012).

    Article  CAS  Google Scholar 

  42. Nader, G., Ezratty, E. J. & Gundersen, G. G. FAK, talin and PIPKIγ regulate endocytosed integrin activation to polarize focal adhesion assembly. Nat. Cell Biol. 18, 491–503 (2016).

    Article  CAS  Google Scholar 

  43. Schubert, R. et al. Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy. FEBS Lett. 588, 3639–3648 (2014).

    Article  CAS  Google Scholar 

  44. Friedrichs, J., Helenius, J. & Müller, D. J. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nat. Protoc. 5, 1353–1361 (2010).

    Article  CAS  Google Scholar 

  45. Yu, M., Strohmeyer, N., Wang, J., Müller, D. J. & Helenius, J. Increasing throughput of AFM-based single cell adhesion measurements through multisubstrate surfaces. Beilstein J. Nanotech. 6, 157–166 (2015).

    Article  Google Scholar 

  46. Hutter, J. L. & Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).

    Article  CAS  Google Scholar 

  47. Müller, D. J., Helenius, J., Alsteens, D. & Dufrêne, Y. F. Force probing surfaces of living cells to molecular resolution. Nat. Chem. Biol. 5, 383–390 (2009).

    Article  Google Scholar 

  48. Krieg, M., Helenius, J., Heisenberg, C.-P. & Müller, D. J. A bond for a lifetime: employing membrane nanotubes from living cells to determine receptor-ligand kinetics. Angew. Chem. Int. Ed. 47, 9775–9777 (2008).

    Article  CAS  Google Scholar 

  49. Sheetz, M. P. Cell control by membrane–cytoskeleton adhesion. Nat. Rev. Mol. Cell Biol. 2, 392–396 (2001).

    Article  CAS  Google Scholar 

  50. R Core Team. R: A language and environment for statistical computing. (R Foundation for Statistical Computing, 2016); http://www.R-project.org

  51. Zar, J. H. Biostatistical Analysis (Pearson Education Limited, 2014).

    Google Scholar 

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Acknowledgements

We thank S. Weiser and J. Thoma for fibronectin fragment cloning, expression and purification, the Single Cell Facility (SCF) of the Department of Biosystems Science and Engineering of the ETH Zürich for help with flow cytometry, N. Beerenwinkel for help with statistical questions and J. Helenius and J. Polleux for critical discussions. This work was supported by the European Research Council (Grant Agreement no. 322652), Deutsche Forschungsgemeinschaft (SFB865-B3), and Swiss National Science Foundation (Grant 31003A_138063).

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zürich, 4058 Basel, Switzerland

    Nico Strohmeyer, Mitasha Bharadwaj & Daniel J. Müller

  2. Department of Biochemistry and Molecular Biology, Estructura de Reserca Interdisciplinar en Biotechnologia i Biomedicina, Universitat de València, 46100 Burjassot, Spain

    Mercedes Costell

  3. Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

    Reinhard Fässler

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Contributions

N.S., M.B., R.F. and D.J.M. designed the experiments and wrote the paper. N.S. performed and analysed most experiments. M.B. performed initial experiments. R.F. and M.C. provided reagents and/or analytical tools. All authors discussed the experiments, read and approved the manuscript.

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Correspondence to Daniel J. Müller.

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Strohmeyer, N., Bharadwaj, M., Costell, M. et al. Fibronectin-bound α5β1 integrins sense load and signal to reinforce adhesion in less than a second. Nature Mater 16, 1262–1270 (2017). https://doi.org/10.1038/nmat5023

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