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

Nature Cell Biology volume 15, pages 625–636 (2013)Cite this article

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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- αv/β1cells.
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.

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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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  1. Herbert B. Schiller and Michaela-Rosemarie Hermann: These authors contributed equally to this work

Authors and Affiliations

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

    Herbert B. Schiller, Michaela-Rosemarie Hermann, Julien Polleux, Zhiqi Sun, Aurelia Raducanu & Reinhard Fässler

  2. Laboratoire de Physiologie Cellulaire et Végétale, Institut de Recherche en Technologies et Sciences pour le Vivant, CNRS/UJF/INRA/CEA, 17 Rue des Martyrs, 38054 Grenoble, France

    Timothée Vignaud & Manuel Théry

  3. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

    Sara Zanivan & Matthias Mann

  4. Institute for Informatics, Ludwig-Maximilians-Universität München, 80333 Munich, Germany

    Caroline C. Friedel

  5. Institute of Experimental Physics, University of Ulm, 89069 Ulm, Germany

    Kay-E. Gottschalk

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Contributions

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-αv/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 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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