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Adhesion Protein Protocols pp 67–81Cite as

Dynamic Assessment of Cell-Matrix Mechanical Interactions in Three-Dimensional Culture

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 370))

Abstract

Cell-matrix mechanical interactions play a defining role in a range of biological processes such as developmental morphogenesis and wound healing. Despite current agreement that fibroblasts exert mechanical forces on the extracellular matrix (ECM) to promote structural organization of the collagen architecture, the underlying mechanisms of force generation and transduction to the ECM are not completely understood. Investigation of these processes has been limited, in part, by the technical challenges associated with simultaneous imaging of cell activity and fibrillar collagen organization. To overcome these limitations, we have developed an experimental model in which cells expressing proteins tagged with enhanced green fluorescent protein are plated inside fibrillar collagen matrices, and high magnification time-lapse differential interference contrast and fluorescent imaging is then performed. Using this system, focal adhesion movement and reorganization in isolated cells can be directly correlated with collagen matrix deformation and changes in the mechanical behavior of fibroblasts can be assessed over time.

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References

  1. Harris, A. K., Wild, P., and Stopak, D. (1980) Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208, 177–189.

    Article  CAS  PubMed  Google Scholar 

  2. Harris, A. K., Stopak, D., and Wild, P. (1981) Fibroblast traction as a mechanism for collagen morphogenesis. Nature 290, 249–251.

    Article  CAS  PubMed  Google Scholar 

  3. Harris, A. K. (1986) Cell traction in relationship to morphogenesis and malignancy. Dev. Biol. 3, 339–357.

    CAS  Google Scholar 

  4. Stopak, D. and Harris, A. K. (1982) Connective tissue morphogenesis by fibroblast traction. Dev. Biol. 90, 383–398.

    Article  CAS  PubMed  Google Scholar 

  5. Lee, J., Leonard, M., Oliver, T., Ishihara, A., and Jacobson, K. (1994) Traction forces generated by locomoting keratocytes. J. Cell Biol. 127, 1957–1964.

    Article  CAS  PubMed  Google Scholar 

  6. Oliver, T., Dembo, M., and Jacobson, K. (1995) Traction forces in locomoting cells. Cell Motil. Cytoskeleton 31, 225–240.

    Article  CAS  PubMed  Google Scholar 

  7. Balaban, N. Q., Schwarz, U. S., Riveline, D., et al. (2001) Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3, 466–472.

    Article  CAS  PubMed  Google Scholar 

  8. Pelham, R. J. Jr. and Wang, Y. (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13,661–13,665.

    Article  CAS  PubMed  Google Scholar 

  9. Pelham, R. J. and Wang, Y. (1999) High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate. Mol. Biol. Cell 10, 935–945.

    CAS  PubMed  Google Scholar 

  10. Wang, Y. L. and Pelham, R. J. Jr. (1998) Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489–496.

    Article  CAS  PubMed  Google Scholar 

  11. Munevar, S., Wang, Y., and Dembo, M. (2001) Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts. Biophys. J. 80, 1744–1757.

    Article  CAS  PubMed  Google Scholar 

  12. Pelham, R. J. Jr. and Wang, Y. L. (1998) Cell locomotion and focal adhesions are regulated by the mechanical properties of the substrate. Biol. Bull. 194, 348–350.

    Article  CAS  PubMed  Google Scholar 

  13. Beningo, K. A., Dembo, M., Kaverina, I., Small, J. V., and Wang, Y. L. (2001) Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J. Cell Biol. 153, 881–888.

    Article  CAS  PubMed  Google Scholar 

  14. Beningo, K. A., Dembo, M., and Wang, Y. L. (2004) Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors. Proc. Natl. Acad. Sci. 101, 18,024–18,029.

    Article  CAS  PubMed  Google Scholar 

  15. Bard, J. B. L. and Hay, E. D. (1975) The behavior of fibroblasts from the developing avian cornea: morphology and movement in situ and in vitro. J. Cell Biol. 67, 400–418.

    Article  CAS  PubMed  Google Scholar 

  16. Cukierman, E., Pankov, R., and Yamada, K. M. (2002) Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 14, 633–639.

    Article  CAS  PubMed  Google Scholar 

  17. Cukierman, E., Pankov, R., Stevens, D. R., and Yamada, K. M. (2001) Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712.

    Article  CAS  PubMed  Google Scholar 

  18. Doane, K. J. and Birk, D. E. (1991) Fibroblasts retain their tissue phenotype when grown in three-dimensional collagen gels. Exp. Cell Res. 195, 432–442.

    Article  CAS  PubMed  Google Scholar 

  19. Friedl, P. and Brocker, E.-B. (2000) The biology of cell locomotion within three-dimensional extracellular matrix. Cell. Mol. Life Sci. 57, 41–64.

    Article  CAS  PubMed  Google Scholar 

  20. Tomasek, J. J., Hay, E. D., and Fujiwara, K. (1982) Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: distribution of actin, α-actinin and myosin. Dev. Biol. 92, 107–122.

    Article  CAS  PubMed  Google Scholar 

  21. Abbott, A. (2003) Biology’s new dimension. Nature 424, 870–872.

    Article  CAS  PubMed  Google Scholar 

  22. Bell, E., Ivarsson, B., and Merril, C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vivo. Proc. Natl. Acad. Sci. USA 76, 1274–1278.

    Article  CAS  PubMed  Google Scholar 

  23. Elsdale, T. and Bard, J. (1972) Collagen substrata for studies on cell behavior. J. Cell Biol. 54, 626–637.

    Article  CAS  PubMed  Google Scholar 

  24. Grinnell, F. and Lamke, C. R. (1984) Reorganization of hydrated collagen lattices by human skin fibroblasts. J. Cell Sci. 66, 51–63.

    CAS  PubMed  Google Scholar 

  25. Grinnell, F. (2000) Fibroblast-collagen matrix contraction: growth-factor signalling and mechanical loading. Trends Cell Biol. 10, 362–365.

    Article  CAS  PubMed  Google Scholar 

  26. Cheema, U., Yang, S.-Y., Mudera, V., Goldspink, G. G., and Brown, R. A. (2003) 3-D in vitro model of early skeletal muscle development. Cell Motil. Cytoskeleton 54, 226–236.

    Article  CAS  PubMed  Google Scholar 

  27. Eastwood, M., McGrouther, D. A., and Brown, R. A. (1994) A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell matrix mechanical signalling. Biochim. Biophys. Acta 1201, 186–192.

    CAS  PubMed  Google Scholar 

  28. Kolodney, M. S. and Elson, E. L. (1993) Correlation of myosin light chain phosphorylation with isometric contraction of fibroblasts. J. Biol. Chem. 268, 23,850–23,855.

    CAS  PubMed  Google Scholar 

  29. Wakatsuki, T. and Elson, E. L. (2003) Reciprocal interactions between cells and extracellular matrix during remodeling of tissue constructs. Biophys. Chem. 100, 593–605.

    Article  CAS  PubMed  Google Scholar 

  30. Arora, P. D., Narani, N., and McCulloch, C. A. G. (1999) The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts. Am. J. Pathol. 154, 871–882.

    Article  CAS  PubMed  Google Scholar 

  31. Brown, R. A., Prajapati, R., McGrouther, D. A., Yannas, I. V., and Eastwood, M. (1998) Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J. Cell. Physiol. 175, 323–332.

    Article  CAS  PubMed  Google Scholar 

  32. Freyman, T. M., Yannas, I. V., Yokoo, R., and Gibson, L. J. (2002) Fibroblast contractile force is independent of the stiffness which resists the contraction. Exp. Cell Res. 272, 153–162.

    Article  CAS  PubMed  Google Scholar 

  33. Parizi, M., Howard, E. W., and Tomasek, J. J. (2000) Regulation of LPA-promoted myofibroblast contraction: role of rho, myosin light chain kinase, and myosin light chain phosphotase. Exp. Cell Res. 254, 210–220.

    Article  CAS  PubMed  Google Scholar 

  34. Rosenfeldt, H., Lee, D. J., and Grinnell, F. (1998) Increased c-fos mRNA expression by human fibroblasts contracting stressed collagen matrices. Mol. Cell. Biol. 18, 2659–2667.

    CAS  PubMed  Google Scholar 

  35. Shreiber, D. I., Enever, P. A. J., and Tranquillo, R. T. (2001) Effects of PDGF-BB on rat dermal fibroblast behavior in mechnically stressed and unstressed collagen and fibrin gels. Exp. Cell Res. 266, 155–166.

    Article  CAS  PubMed  Google Scholar 

  36. Skuta, G., Ho, C.-H., and Grinnell, F. (1999) Increased myosin light chain phosphorylation is not required for growth factor stimulation of collagen matrix contraction. J. Biol. Chem. 274, 30,163–30,168.

    Article  CAS  PubMed  Google Scholar 

  37. Vaughan, M. B., Howard, E. W., and Tomasek, J. J. (2000) Transforming growth factor-?1 promotes the morphological and functional differentiation of the myofibroblast. Exp. Cell Res. 257, 180–189.

    Article  CAS  PubMed  Google Scholar 

  38. Grinnell, F., Ho, C.-H., Tamariz, E., Lee, D. J., and Skuta, G. (2003) Dendritic fibroblasts in three-dimensional collagen matrices. Mol. Cell. Biol. 14, 384–395.

    Article  CAS  Google Scholar 

  39. Abe, M., Ho, C.-H., Kamm, K. E., and Grinnell, F. (2003) Different molecular motors mediate platelet-derived growth factor and lysophosphatidic acid-mediated floating collagen matrix contraction. J. Biol. Chem. 278, 47,707–47,712.

    Article  CAS  PubMed  Google Scholar 

  40. Petroll, W. M. and Ma, L. (2003) Direct, dynamic assessment of cell-matrix interactions inside fibrillar collagen lattices. Cell Motil. Cytoskeleton 55, 254–264.

    Article  PubMed  Google Scholar 

  41. Petroll, W. M., Ma, L., and Jester, J. V. (2003) Direct correlation of collagen matrix deformation with focal adhesion dynamics in living corneal fibroblasts. J. Cell Sci. 116, 1481–1491.

    Article  CAS  PubMed  Google Scholar 

  42. Vishwanath, M., Ma, L., Jester, J. V., Otey, C. A., and Petroll, W. M. (2003) Modulation of corneal fibroblast contractility within fibrillar collagen matrices. Invest. Ophthalmol. Vis. Sci. 44, 4724–4735.

    Article  PubMed  Google Scholar 

  43. Petroll, W. M., Vishwanath, M., and Ma, L. (2004) Corneal fibroblasts respond rapidly to changes in local mechanical stress. Invest. Ophthalmol. Vis. Sci. 45, 3466–3474.

    Article  PubMed  Google Scholar 

  44. Kaverina, I., Krylyshkina, O., and Small, J. V. (1999) Microtubule targeting of substrate contacts promotes their relaxation and dissociation. J. Cell Biol. 146, 1033–1044.

    Article  CAS  PubMed  Google Scholar 

  45. Kaverina, I., Krylyshkina, O., Gimona, M., Beningo, K., Wang, Y. L., and Small, J. V. (2000) Enforced polarisation and locomotion of fibroblasts lacking microtubules. Curr. Biol. 10, 739–742.

    Article  CAS  PubMed  Google Scholar 

  46. Rottner, K., Krause, M., Gimona, M., Small, J. V., and Wehland, J. (2001) Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions. Mol. Biol. Cell 12, 3103–3113.

    CAS  PubMed  Google Scholar 

  47. Moller-Pedersen, T., Cavanagh, H. D., Petroll, W. M., and Jester, J. V. (1998) Corneal haze development after PRK is regulated by volume of stromal tissue removal. Cornea 17, 627–639.

    Article  CAS  PubMed  Google Scholar 

  48. Moller-Pedersen, T., Cavanagh, H. D., Petroll, W. M., and Jester, J. V. (1998) Neutralizing antibody to TGF? modulates stromal fibrosis but not regression of photoablative effect following PRK. Curr. Eye Res. 17, 736–747.

    Article  CAS  PubMed  Google Scholar 

  49. Petroll, W. M., Cavanagh, H. D., Barry-Lane, P., Andrews, P., and Jester, J. V. (1993) Quantitative analysis of stress fiber orientation during corneal wound contraction. J. Cell Sci. 104, 353–363.

    PubMed  Google Scholar 

  50. Edlund, E., Lotano, M. A., and Otey, C. A. (2001) Dynamics of α-actinin in focal adhesions and stress fibers visualized with α-actinin green fluorescent protein. Cell Motil. Cytoskeleton 48, 190–200.

    Article  CAS  PubMed  Google Scholar 

  51. Friedl, P., Noble, P. B., and Zanker, K. S. (1995) T lymphocyte locomotion in a three-dimensional collagen matrix. J. Immunol. 154, 4973–4985.

    CAS  PubMed  Google Scholar 

  52. Friedl, P., Maaser, K., Klein, C. E., Niggemann, B., Krohne, G., and Zanker, K. S. (1997) Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. Cancer Res. 57, 2061–2070.

    CAS  PubMed  Google Scholar 

  53. Friedl, P., Zanker, K., and Brocker, E.-B. (1998) Cell migration strategies in 3-D extracellular matrix: differences in morphology, cell matrix interactions, and integrin function. Microsc. Res. Tech. 43, 369–378.

    Article  CAS  PubMed  Google Scholar 

  54. Hegerfeldt, Y., Tusch, M., Brocker, E.-B., and Friedl, P. (2002) Collective cell movement in primary melanoma explants: plasticity of cell-cell interaction, β1-integrin function, and migration strategies. Cancer Res. 62, 2125–2130.

    CAS  PubMed  Google Scholar 

  55. Wolf, K., Mazo, I., Leung, H., et al. (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J. Cell Biol. 160, 267–277.

    Article  CAS  PubMed  Google Scholar 

  56. Petroll, W. M., Cavanagh, H. D., and Jester, J. V. (2004) Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts. Scanning 26, 1–10.

    Article  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX

    W. Matthew Petroll

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  1. W. Matthew Petroll
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Editor information

Editors and Affiliations

  1. Medical Sciences Division, University of Oxford, Oxford, UK

    Amanda S. Coutts

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© 2007 Humana Press Inc., Totowa, NJ

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Petroll, W.M. (2007). Dynamic Assessment of Cell-Matrix Mechanical Interactions in Three-Dimensional Culture. In: Coutts, A.S. (eds) Adhesion Protein Protocols. Methods in Molecular Biology™, vol 370. Humana Press. https://doi.org/10.1007/978-1-59745-353-0_6

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  • DOI: https://doi.org/10.1007/978-1-59745-353-0_6

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-533-0

  • Online ISBN: 978-1-59745-353-0

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