Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics

Nature volume 466, pages 263–266 (2010)Cite this article

Subjects

Abstract

Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ∼2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Vinculin tension sensor (VinTS) constructs.
Figure 2: Responses to mechanical force.
Figure 3: Forces across vinculin during cell migration.
Figure 4: Tension on vinculin in dynamic FAs.

Similar content being viewed by others

References

  1. Orr, A. W., Helmke, B. P., Blackman, B. R. & Schwartz, M. A. Mechanisms of mechanotransduction. Dev. Cell 10, 11–20 (2006)

    Article  CAS  Google Scholar 

  2. Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318, 279–283 (2007)

    Article  ADS  CAS  Google Scholar 

  3. Bershadsky, A. D., Balaban, N. Q. & Geiger, B. Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell Dev. Biol. 19, 677–695 (2003)

    Article  CAS  Google Scholar 

  4. 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 

  5. Bakolitsa, C. et al. Structural basis for vinculin activation at sites of cell adhesion. Nature 430, 583–586 (2004)

    Article  ADS  CAS  Google Scholar 

  6. Ziegler, W. H., Gingras, A. R., Critchley, D. R. & Emsley, J. Integrin connections to the cytoskeleton through talin and vinculin. Biochem. Soc. Trans. 36, 235–239 (2008)

    Article  CAS  Google Scholar 

  7. Galbraith, C. G., Yamada, K. M. & Sheetz, M. P. The relationship between force and focal complex development. J. Cell Biol. 159, 695–705 (2002)

    Article  CAS  Google Scholar 

  8. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an Mdia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001)

    Article  CAS  Google Scholar 

  9. Xu, W., Baribault, H. & Adamson, E. D. Vinculin knockout results in heart and brain defects during embryonic development. Development 125, 327–337 (1998)

    CAS  PubMed  Google Scholar 

  10. Alenghat, F. J. et al. Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer. Biochem. Biophys. Res. Commun. 277, 93–99 (2000)

    Article  CAS  Google Scholar 

  11. Mierke, C. T. et al. Mechano-coupling and regulation of contractility by the vinculin tail domain. Biophys. J. 94, 661–670 (2008)

    Article  ADS  CAS  Google Scholar 

  12. Hu, K. et al. Differential transmission of actin motion within focal adhesions. Science 315, 111–115 (2007)

    Article  ADS  CAS  Google Scholar 

  13. Ji, L., Lim, J. & Danuser, G. Fluctuations of intracellular forces during cell protrusion. Nature Cell Biol. 10, 1393–1400 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Shemesh, T., Geiger, B., Bershadsky, A. D. & Kozlov, M. M. Focal adhesions as mechanosensors: a physical mechanism. Proc. Natl Acad. Sci. USA 102, 12383–12388 (2005)

    Article  ADS  CAS  Google Scholar 

  16. 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  ADS  CAS  Google Scholar 

  17. Meng, F., Suchyna, T. M. & Sachs, F. A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ. FEBS J. 275, 3072–3087 (2008)

    Article  CAS  Google Scholar 

  18. Day, R. N., Booker, C. F. & Periasamy, A. Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J. Biomed. Opt. 13, 031203 (2008)

    Article  ADS  Google Scholar 

  19. Becker, N. et al. Molecular nanosprings in spider capture-silk threads. Nature Mater. 2, 278–283 (2003)

    Article  ADS  CAS  Google Scholar 

  20. Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043–1057 (2007)

    Article  CAS  Google Scholar 

  21. Chen, H. et al. Spatial distribution and functional significance of activated vinculin in living cells. J. Cell Biol. 169, 459–470 (2005)

    Article  CAS  Google Scholar 

  22. Cohen, D. M. et al. A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions. J. Biol. Chem. 281, 16006–16015 (2006)

    Article  CAS  Google Scholar 

  23. Parsons, M. et al. Quantification of integrin receptor agonism by fluorescence lifetime imaging. J. Cell Sci. 121, 265–271 (2008)

    Article  CAS  Google Scholar 

  24. Cai, Y. et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys. J. 91, 3907–3920 (2006)

    Article  ADS  CAS  Google Scholar 

  25. Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006)

    Article  ADS  CAS  Google Scholar 

  26. Kolega, J. Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction. Mol. Biol. Cell 14, 4745–4757 (2003)

    Article  CAS  Google Scholar 

  27. Periasamy, A., Wallrabe, H., Chen, Y. & Barroso, M. Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy. Methods Cell Biol. 89, 569–598 (2008)

    Article  CAS  Google Scholar 

  28. Zamir, E. et al. Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112, 1655–1669 (1999)

    CAS  PubMed  Google Scholar 

  29. Zaidel-Bar, R., Milo, R., Kam, Z. & Geiger, B. A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions. J. Cell Sci. 120, 137–148 (2007)

    Article  CAS  Google Scholar 

  30. Crocker, J. C. & Hoffman, B. D. Multiple-particle tracking and two-point microrheology in cells. Methods Cell Biol. 83, 141–178 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Horwitz, M. Vicente-Manzanares, D. Schafer, L. K. Tamm and K. A. DeMali for reagents and M. Gardel for critical reading of the manuscript. This work was supported by USPHS grant U54 GM64346 to M.A.S., C.G. was supported by a Research Fellowship from the Deutsche Forschungsgemeinschaft (DFG, GR3399/1-1). B.D.H. was supported by USPHS training grant 5T32-HL007284 and an AHA Postdoctoral Fellowship. M.D.B., R.Z. and T.H. were supported by the US National Science Foundation Physics Frontier Center grant 0822613 and by USPHS grant R21 RR025341. T.H. is an investigator with the Howard Hughes Medical Institute. M.P. was supported by a Royal Society University Research Fellowship (UK). M.T.Y. was supported by an IGERT fellowship from the National Science Foundation (DGE-0221664).

Author information

Author notes

  1. Carsten Grashoff and Brenton D. Hoffman: These authors contributed equally to this work.

Authors and Affiliations

  1. Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, 22908, Virginia, USA

    Carsten Grashoff, Brenton D. Hoffman & Martin A. Schwartz

  2. Department of Microbiology, University of Virginia, Charlottesville, 22908, Virginia, USA

    Carsten Grashoff, Brenton D. Hoffman & Martin A. Schwartz

  3. Center for the Physics of Living Cells and Department of Physics, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Michael D. Brenner, Ruobo Zhou & Taekjip Ha

  4. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Michael D. Brenner & Taekjip Ha

  5. Randall Division of Cell and Molecular Biophysics, King’s College London, London, SE1 1UL, UK

    Maddy Parsons

  6. Department of Bioengineering, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Michael T. Yang & Christopher S. Chen

  7. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Mark A. McLean & Stephen G. Sligar

  8. Howard Hughes Medical Institute, Urbana, 61801, Illinois, USA

    Taekjip Ha

  9. Department of Biomedical Engineering, University of Virginia, Charlottesville, 22908, Virginia, USA

    Martin A. Schwartz

Authors
  1. Carsten Grashoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brenton D. Hoffman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael D. Brenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruobo Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maddy Parsons
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael T. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mark A. McLean
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stephen G. Sligar
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Christopher S. Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Taekjip Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Martin A. Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.A.S. conceived the general idea. C.G. conceived the use of the flagelliform sequence, designed and generated vinculin expression constructs, performed spectrofluorometry and all cell and imaging experiments. B.D.H. generated analysis tools and analysed the in vivo data. T.H., M.A.M. and S.G.S. conceived the sensor calibration scheme. M.D.B. designed and generated the tension sensor construct for the calibration. M.D.B. and R.Z. performed and analysed the single molecule experiments. T.H., R.Z., M.D.B and B.D.H. developed an algorithm to map in vitro and in vivo tension sensor data. M.P. performed and analysed FLIM experiments. M.T.Y and C.S.C. generated micropost arrays and analysed traction force data. C.G., B.D.H. and M.A.S. designed the in vivo experiments, discussed the results and wrote the paper with input from all authors.

Corresponding authors

Correspondence to Taekjip Ha or Martin A. Schwartz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with legends, Supplementary Notes 1-3 and References. (PDF 3336 kb)

Supplementary Movie 1

This movie shows a bovine aortic endothelial cell expressing VinTS. The colour bar indicates FRET index. Notice lower FRET index in protruding regions but high FRET index in retracting areas of the cell. The time-lapse covers a period of 48 min. Frame rate: 90 s. (MOV 1447 kb)

Supplementary Movie 2

This movie shows a bovine aortic endothelial cell expressing VinTL. The colour bar indicates FRET index. Notice that the FRET index is uniformly high. The time-lapse covers a period of 45 min. Frame rate: 90 s. (MOV 1226 kb)

Supplementary Movie 3

This movie shows a vinculin-/- cell expressing paxillin-EGFP. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 408 kb)

Supplementary Movie 4

This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 573 kb)

Supplementary Movie 5

This movie shows a vinculin-/- cell expressing paxillin-EGFP and myosin IIa. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 316 kb)

Supplementary Movie 6

This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP and myosin IIa. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 593 kb)

Supplementary Movie 7

This movie shows a vinculin-/- cell expressing paxillin-EGFP and RhoA-V14. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 381 kb)

Supplementary Movie 8

This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP and RhoA-V14. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 470 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Grashoff, C., Hoffman, B., Brenner, M. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010). https://doi.org/10.1038/nature09198

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09198

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing