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β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments

Abstract

How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of β1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with β1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1–RhoA pathway coupled to the formin mDia1 but not myosin II, and α5β1 integrins to a RhoA–Rock–myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5β1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.

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Figure 1: Different morphologies and adhesive functions of pKO-αv, pKO- β1 and pKO- αv1cells.
Figure 2: αv-class integrins cooperate with α5β1 for myosin II reinforcement on stiff fibronectin-coated substrates.
Figure 3: αv-class integrins accumulate in adhesion areas exposed to high traction force and cooperate with α5β1 for rigidity sensing on fibronectin.
Figure 4: Composition and stoichiometry of the adhesome is determined by the individual integrin and myosin II activity.
Figure 5: αv- and β1-mediated activation of myosin II requires ILK and GEF-H1.
Figure 6: Integrin-specific phosphorylation landscapes on adhesion to fibronectin.
Figure 7: Activation of Rock is α5β1-dependent.
Figure 8: Model of α5β1 and αv-class integrin cooperation during rigidity sensing.

References

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

    Article  CAS  Google Scholar 

  2. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).

    Article  CAS  Google Scholar 

  3. Avraamides, C. J., Garmy-Susini, B. & Varner, J. A. Integrins in angiogenesis and lymphangiogenesis. Nat. Rev. Cancer 8, 604–617 (2008).

    Article  CAS  Google Scholar 

  4. Liu, H. et al. MYC suppresses cancer metastasis by direct transcriptional silencing of α(v) and β(3) integrin subunits. Nat. Cell Biol. 14, 567–574 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Leiss, M., Beckmann, K., Giros, 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 

  7. Yang, J. T., Rayburn, H. & Hynes, R. O. Embryonic mesodermal defects in α5 integrin-deficient mice. Development 119, 1093–1105 (1993).

    CAS  PubMed  Google Scholar 

  8. Bader, B. L., Rayburn, H., Crowley, D. & Hynes, R. O. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all αv integrins. Cell 95, 507–519 (1998).

    Article  CAS  Google Scholar 

  9. Yang, J. T. et al. Overlapping and independent functions of fibronectin receptor integrins in early mesodermal development. Dev. Biol. 215, 264–277 (1999).

    Article  CAS  Google Scholar 

  10. Zamir, E. et al. Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2, 191–196 (2000).

    Article  CAS  Google Scholar 

  11. Ballestrem, C., Hinz, B., Imhof, B. A. & Wehrle-Haller, B. Marching at the front and dragging behind: differential αvβ3-integrin turnover regulates focal adhesion behavior. J. Cell Biol. 155, 1319–1332 (2001).

    Article  CAS  Google Scholar 

  12. Danen, E. H., Sonneveld, P., Brakebusch, C., Fässler, R. & Sonnenberg, A. The fibronectin-binding integrins α5β1 and αvβ3 differentially modulate RhoA–GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis. J. Cell Biol. 159, 1071–1086 (2002).

    Article  CAS  Google Scholar 

  13. White, D. P., Caswell, P. T. & Norman, J. C. αvβ3 and α5β1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration. J. Cell Biol. 177, 515–525 (2007).

    Article  CAS  Google Scholar 

  14. Morgan, M. R., Byron, A., Humphries, M. J. & Bass, M. D. Giving off mixed signals-distinct functions of α(5)β(1) and α(v)β(3) integrins in regulating cell behaviour. IUBMB Life 61, 731–738 (2009).

    Article  CAS  Google Scholar 

  15. Van der Flier, A. et al. Endothelial α5 and αv integrins cooperate in remodeling of the vasculature during development. Development 137, 2439–2449 (2010).

    Article  CAS  Google Scholar 

  16. Choi, C. K. et al. Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 10, 1039–1050 (2008).

    Article  CAS  Google Scholar 

  17. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    Article  CAS  Google Scholar 

  18. Bershadsky, A., Kozlov, M. & Geiger, B. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr. Opin. Cell Biol. 18, 472–481 (2006).

    Article  CAS  Google Scholar 

  19. 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 

  20. Kuo, J. C., Han, X., Hsiao, C. T., Yates Iii, J. R. & Waterman, C. M. Analysisof the myosin II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation. Nat. Cell Biol. 13, 383–393 (2011).

    Article  CAS  Google Scholar 

  21. Lammermann, T. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008).

    Article  Google Scholar 

  22. Bhadriraju, K. et al. Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension. Exp. Cell Res. 313, 3616–3623 (2007).

    Article  CAS  Google Scholar 

  23. Danen, E. H. et al. Integrins control motile strategy through a Rho-cofilin pathway. J. Cell Biol. 169, 515–526 (2005).

    Article  CAS  Google Scholar 

  24. Worth, D. C. et al. αvβ3 integrin spatially regulates VASP and RIAM to control adhesion dynamics and migration. J. Cell Biol. 189, 369–383 (2010).

    Article  CAS  Google Scholar 

  25. Huttenlocher, A. & Horwitz, A. R. Integrins in cell migration. Cold Spr. Harbor Perspec. Biol. 3, a005074 (2011).

    Google Scholar 

  26. Thery, M. et al. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc. Natl Acad. Sci. USA 103, 19771–19776 (2006).

    Article  CAS  Google Scholar 

  27. Thery, M. Micropatterning as a tool to decipher cell morphogenesis and functions. J. Cell Sci. 123, 4201–4213 (2010).

    Article  CAS  Google Scholar 

  28. Tseng, Q. et al. A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels. Lab on a Chip 11, 2231–2240 (2011).

    Article  CAS  Google Scholar 

  29. 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 

  30. Zaidel-Bar, R., Itzkovitz, S., Ma’ayan, A., Iyengar, R. & Geiger, B. Functional atlas of the integrin adhesome. Nat. Cell Biol. 9, 858–867 (2007).

    Article  CAS  Google Scholar 

  31. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotech. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  32. Zaidel-Bar, R. & Geiger, B. The switchable integrin adhesome. J. Cell Sci. 123, 1385–1388 (2010).

    Article  CAS  Google Scholar 

  33. Meves, A. et al. {β}1 integrin cytoplasmic tyrosines promote skin tumorigenesis independent of their phosphorylation. Proc. Natl Acad. Sci. USA 108, 15213–15218 (2011).

    Article  CAS  Google Scholar 

  34. Sakai, T. et al. Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation. Genes Dev. 17, 926–940 (2003).

    Article  CAS  Google Scholar 

  35. Guilluy, C. et al. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat. Cell Biol. 13, 722–727 (2011).

    Article  Google Scholar 

  36. Montanez, E., Wickstrom, S. A., Altstatter, J., Chu, H. & Fässler, R. α-parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling. EMBO J. 28, 3132–3144 (2009).

    Article  CAS  Google Scholar 

  37. Montanez, E. et al. Kindlin-2 controls bidirectional signaling of integrins. Genes Dev. 22, 1325–1330 (2008).

    Article  CAS  Google Scholar 

  38. Holzapfel, G., Wehland, J. & Weber, K. Calcium control of actin-myosin based contraction in triton models of mouse 3T3 fibroblasts is mediated by the myosin light chain kinase (MLCK)-calmodulin complex. Exp. Cell Res. 148, 117–126 (1983).

    Article  CAS  Google Scholar 

  39. Fincham, V. J., James, M., Frame, M. C. & Winder, S. J. Active ERK/MAP kinase is targeted to newly forming cell–matrix adhesions by integrin engagement and v-Src. EMBO J. 19, 2911–2923 (2000).

    Article  CAS  Google Scholar 

  40. 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 

  41. Oakes, P. W., Beckham, Y., Stricker, J. & Gardel, M. L. Tension is required but not sufficient for focal adhesion maturation without a stress fibre template. J. Cell Biol. 196, 363–374 (2012).

    Article  CAS  Google Scholar 

  42. Stricker, J., Aratyn-Schaus, Y., Oakes, P. W. & Gardel, M. L. Spatiotemporal constraints on the force-dependent growth of focal adhesions. Biophys. J. 100, 2883–2893 (2011).

    Article  CAS  Google Scholar 

  43. Roca-Cusachs, P., Gauthier, N. C., Del Rio, A. & Sheetz, M. P. Clustering of α(5)β(1) integrins determines adhesion strength whereas α(v)β(3) and talin enable mechanotransduction. Proc. Natl Acad. Sci. USA 106, 16245–16250 (2009).

    Article  CAS  Google Scholar 

  44. Kong, F., Garcia, 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 

  45. Schiller, H. B., Szekeres, A., Binder, B. R., Stockinger, H. & Leksa, V. Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling αvβ3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor. Mol. Biol. Cell 20, 745–756 (2009).

    Article  CAS  Google Scholar 

  46. Azioune, A., Carpi, N., Tseng, Q., Thery, M. & Piel, M. Protein micropatterns: a direct printing protocol using deep UVs. Meth. Cell Biol. 97, 133–146 (2010).

    Article  CAS  Google Scholar 

  47. Rape, A. D., Guo, W. H. & Wang, Y. L. The regulation of traction force in relation to cell shape and focal adhesions. Biomaterials 32, 2043–2051 (2011).

    Article  CAS  Google Scholar 

  48. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  CAS  Google Scholar 

  49. Wisniewski, J. R., Zougman, A. & Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 8, 5674–5678 (2009).

    Article  CAS  Google Scholar 

  50. Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).

    Article  CAS  Google Scholar 

  51. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  Google Scholar 

  52. Luber, C. A. et al. Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. Immunity 32, 279–289 (2010).

    Article  CAS  Google Scholar 

  53. Olsen, J. V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell Proteomics 4, 2010–2021 (2005).

    Article  CAS  Google Scholar 

  54. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protocols 4, 698–705 (2009).

    Article  CAS  Google Scholar 

  55. Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protocols 4, 44–57 (2009).

    Article  Google Scholar 

  56. Xenarios, I. et al. DIP, the database of interacting proteins: a research tool for studying cellular networks of protein interactions. Nucleic Acids Res. 30, 303–305 (2002).

    Article  CAS  Google Scholar 

  57. Aranda, B. et al. The IntAct molecular interaction database in 2010. Nucleic Acids Res. 38, D525–D531 (2010).

    Article  CAS  Google Scholar 

  58. Ceol, A. et al. MINT, the molecular interaction database: 2009 update. Nucleic Acids Res. 38, D532–D539 (2010).

    Article  CAS  Google Scholar 

  59. Breitkreutz, B. J. et al. The BioGRID Interaction Database: 2008 update. Nucleic Acids Res. 36, D637–D640 (2008).

    Article  CAS  Google Scholar 

  60. Prasad, T. S., Kandasamy, K. & Pandey, A. Human protein reference database and human proteinpedia as discovery tools for systems biology. Methods Mol. Biol. 577, 67–79 (2009).

    Article  CAS  Google Scholar 

  61. Kanehisa, M., Goto, S., Sato, Y., Furumichi, M. & Tanabe, M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 40, D109–D114 (2012).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Cox for software tool development, U. Kuhn and C. Boulegue (MPIB) and A. F. Christ (CNRS/UJF/INRA/CEA) for excellent technical support, and A. Meves, T. Geiger and D. Boettiger for discussions. H.B.S. was a fellow of the European Molecular Biology Organisation (EMBO) and M-R.H. a fellow of the Boehringer Ingelheim fonds. The work was financially supported by the ERC, DFG and the Max Planck Society.

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R.F. initiated the project; R.F. and H.B.S. designed the experiments and wrote the paper; H.B.S., M-R.H., T.V., S.Z., J.P., Z.S. and A.R. performed experiments; H.B.S., M-R.H., T.V., S.Z., K-E.G., C.C.F. and R.F. analysed data; J.P., M.T., K-E.G. and M.M. provided important reagents and/or analytical tools; all authors read and approved the manuscript.

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Correspondence to Reinhard Fässler.

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Time-lapse movie of pKO-αv cells plated on FN.

Cells were plated on FN-coated (5 μg ml−1; blocked with 1% BSA) tissue culture dishes in the presence of 10% serum and video tracked over 20 h with a frame rate of 1 picture every 4 min. Pictures were acquired with a phase contrast microscope at ×20 magnification. (MOV 2361 kb)

Time-lapse movie of pKO-αv1 cells plated on FN.

Cells were plated on FN-coated (5 μg ml−1; blocked with 1% BSA) tissue culture dishes in the presence of 10% serum and video tracked over 20 h with a frame rate of 1 picture every 4 min. Pictures were acquired with a phase contrast microscope at ×20 magnification. (MOV 2014 kb)

Time-lapse movie of pKO-β1 cells plated on FN.

Cells were plated on FN-coated (5 μg ml−1; blocked with 1% BSA) tissue culture dishes in the presence of 10% serum and video tracked over 20 h with a frame rate of 1 picture every 4 min. Pictures were acquired with a phase contrast microscope at ×20 magnification. (MOV 2709 kb)

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Schiller, H., Hermann, MR., Polleux, J. 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). https://doi.org/10.1038/ncb2747

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