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.

  • Protocol
  • Published:

Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy

Abstract

Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here we present a protocol to study integrin-mediated adhesion of HeLa cells to collagen type I using SCFS. We describe procedures for (i) functionalization of AFM cantilevers with the lectin concanavalin A and supports with collagen, (ii) cell handling and attachment to the AFM cantilever, (iii) measurement of adhesion forces and (iv) data analysis and interpretation. Although designed to measure HeLa cell adhesion to collagen, the protocol can be modified for other cell lines and ECM proteins. Compared with other SCFS assays (for example, optical tweezer, biomembrane force probe), AFM-based SCFS has a more versatile force detection range, and it can therefore be used to address a broader range of biological questions. The protocol can be completed in 2–3 d.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Experimental SCFS setup.
Figure 2: Preparation of collagen-coated mica supports.
Figure 3: Attachment of a cell to the AFM cantilever.
Figure 4: Data analysis steps.
Figure 5: Anticipated results.

Similar content being viewed by others

References

  1. Adams, J.C. Regulation of protrusive and contractile cell–matrix contacts. J. Cell Sci. 115, 257–265 (2002).

    CAS  PubMed  Google Scholar 

  2. Lauffenburger, D.A. & Wells, A. Getting a grip: new insights for cell adhesion and traction. Nat. Cell Biol. 3, E110–E112 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Fassler, R. & Meyer, M. Consequences of lack of beta 1 integrin gene expression in mice. Genes Dev. 9, 1896–1908 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Lohler, J., Timpl, R. & Jaenisch, R. Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death. Cell 38, 597–607 (1984).

    Article  CAS  PubMed  Google Scholar 

  5. Monkley, S.J. et al. Disruption of the talin gene arrests mouse development at the gastrulation stage. Dev. Dyn. 219, 560–574 (2000).

    Article  CAS  PubMed  Google Scholar 

  6. Klebe, R.J. Isolation of a collagen-dependent cell attachment factor. Nature 250, 248–251 (1974).

    Article  CAS  PubMed  Google Scholar 

  7. Amano, M. et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275, 1308–1311 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Ridley, A.J. & Hall, A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399 (1992).

    Article  CAS  PubMed  Google Scholar 

  9. Sieg, D.J. et al. FAK integrates growth-factor and integrin signals to promote cell migration. Nat. Cell Biol. 2, 249–256 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Kaplanski, G. et al. Granulocyte–endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow. Biophys. J. 64, 1922–1933 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Garcia, A.J., Ducheyne, P. & Boettiger, D. Quantification of cell adhesion using a spinning disc device and application to surface-reactive materials. Biomaterials 18, 1091–1098 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Reyes, C.D. & Garcia, A.J. A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces. J Biomed. Mater. Res. 67, 328–333 (2003).

    Article  Google Scholar 

  13. Florin, E.L., Moy, V.T. & Gaub, H.E. Adhesion forces between individual ligand–receptor pairs. Science 264, 415–417 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Moy, V.T., Florin, E.L. & Gaub, H.E. Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Lee, G.U., Kidwell, D.A. & Colton, R.J. Sensing discrete streptavidin–biotin interactions with atomic force microscopy. Langmuir 10, 354–357 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Rose, D.M., Alon, R. & Ginsberg, M.H. Integrin modulation and signaling in leukocyte adhesion and migration. Immunol. Rev. 218, 126–134 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Benoit, M., Gabriel, D., Gerisch, G. & Gaub, H.E. Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat. Cell Biol. 2, 313–317 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Benoit, M. & Gaub, H.E. Measuring cell adhesion forces with the atomic force microscope at the molecular level. Cells Tissues Organs 172, 174–189 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Horton, M., Charras, G. & Lehenkari, P. Analysis of ligand–receptor interactions in cells by atomic force microscopy. J. Recept. Signal Transduct. Res. 22, 169–190 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Lehenkari, P.P. & Horton, M.A. Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. Biochem. Biophys. Res. Commun. 259, 645–650 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Grandbois, M., Dettmann, W., Benoit, M. & Gaub, H.E. Affinity imaging of red blood cells using an atomic force microscope. J. Histochem. Cytochem. 48, 719–724 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Sung, K.L., Sung, L.A., Crimmins, M., Burakoff, S.J. & Chien, S. Determination of junction avidity of cytolytic T cell and target cell. Science 234, 1405–1408 (1986).

    Article  CAS  PubMed  Google Scholar 

  24. Evans, E., Ritchie, K. & Merkel, R. Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys. J. 68, 2580–2587 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Evans, E.A., Waugh, R. & Melnik, L. Elastic area compressibility modulus of red cell membrane. Biophys. J. 16, 585–595 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Leckband, D. & Israelachvili, J. Intermolecular forces in biology. Q. Rev. Biophys. 34, 105–267 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Taubenberger, A. et al. Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy. Mol. Biol. Cell 18, 1634–1644 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Simon, A. & Durrieu, M.C. Strategies and results of atomic force microscopy in the study of cellular adhesion. Micron 37, 1–13 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Evans, E.A. & Calderwood, D.A. Forces and bond dynamics in cell adhesion. Science 316, 1148–1153 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Zhang, X., Wojcikiewicz, E. & Moy, V.T. Force spectroscopy of the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. Biophys. J. 83, 2270–2279 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Puech, P.H. et al. Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy. J. Cell Sci. 118, 4199–4206 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Tulla, M. et al. TPA primes alpha2beta1 integrins for cell adhesion. FEBS Lett. 582, 3520–3524 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Friedrichs, J. et al. Contributions of galectin-3 and -9 to epithelial cell adhesion analyzed by single cell force spectroscopy. J. Biol. Chem. 282, 29375–29383 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Friedrichs, J., Manninen, A., Muller, D.J. & Helenius, J. Galectin-3 regulates integrin alpha2beta1-mediated adhesion to collagen-I and -IV. J. Biol. Chem. 283, 32264–32272 (2008).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. Burnham, N.A. et al. Comparison of calibration methods for atomic-force microscopy cantilevers. Nanotechnology 14, 1–6 (2003).

    Article  CAS  Google Scholar 

  37. Janovjak, H., Struckmeier, J. & Muller, D.J. Hydrodynamic effects in fast AFM single-molecule force measurements. Eur. Biophys. J. 34, 91–96 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Kerssemakers, J.W. et al. Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Benoit, M. Cell adhesion measured by force spectroscopy on living cells. Methods Cell Biol. 68, 91–114 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Nugaeva, N. et al. Micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection. Biosens. Bioelectron. 21, 849–856 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Saif, M.T., Sager, C.R. & Coyer, S. Functionalized biomicroelectromechanical systems sensors for force response study at local adhesion sites of single living cells on substrates. Ann. Biomed. Eng. 31, 950–961 (2003).

    Article  PubMed  Google Scholar 

  42. Waite, J.H. & Tanzer, M.L. Polyphenolic substance of Mytilus edulis: novel adhesive containing L-dopa and hydroxyproline. Science 212, 1038–1040 (1981).

    Article  CAS  PubMed  Google Scholar 

  43. Waite, J.H. Evidence for a repeating 3,4-dihydroxyphenylalanine- and hydroxyproline-containing decapeptide in the adhesive protein of the mussel, Mytilus edulis L. J. Biol. Chem. 258, 2911–2915 (1983).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Taubenberger, M. Krieg, C. Franz and P.-H. Puech for support in the development of the technique. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF).

Author information

Authors and Affiliations

Authors

Contributions

J.F. and D.J.M. designed the study; J.F. designed and performed experiments and J.F. and J.H. analyzed the data. All the authors wrote the article.

Corresponding author

Correspondence to Daniel J Muller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Friedrichs, J., Helenius, J. & Muller, D. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nat Protoc 5, 1353–1361 (2010). https://doi.org/10.1038/nprot.2010.89

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.89

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

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