Confocal-FRAP Analysis of ECM Molecular Interactions

  • Timothy Hardingham
  • Philip Gribbon
Part of the Methods in Molecular Biology™ book series (MIMB, volume 139)

Abstract

Extracellular matrices (ECM) contain a mixture of fibrillar and nonfibrillar macromolecular components, which together form a composite structure (1, 2, 3). It is the ECM that defines the architecture, the form, and the biomechanical properties of different tissues (4,5). Among the nonfibrillar macromolecules, the highly charged proteoglycans and hyaluronan are major components that occur at high concentration and greatly influence the movement of solutes and water between the tissue and the circulation, and control the access to cells of nutrients, metabolites, growth factors, and chemokines (6, 7). This local environmental regulation may have important consequences on cellular functions, especially in tissues with large dense ECMs, such as articular cartilage.

Keywords

Fluorescence Recovery After Photobleaching Gentle Rotation Label Buffer Bleached Area Bean Trypsin Inhibitor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Winlove, C. P. and Parker, K. H. P. (1995) The physiological functions of extra-cellular matrix macromolecules, in Interstitium, Connective Tissue and Lymphat-ics (Reed, R. K., McHale, N. G., Bert, J. L., Winlove, C. P., and Laine, G. A., eds.), Portland, London, UK, pp. 137–165.Google Scholar
  2. 2.
    Comper, W. D. and Laurent, T. C. (1978) Physiological functions of connective tissue polysaccharides. Physiol. Rev. 58, 255–316.PubMedGoogle Scholar
  3. 3.
    Comper, W. D., ed. (1996) Extracellular Matrix. Harwood, Amsterdam, The Netherlands.Google Scholar
  4. 4.
    Maroudas, A. (1976) Balance between swelling pressure and collagen tension in normal and degenerate cartilage. Nature 260, 808–809.PubMedCrossRefGoogle Scholar
  5. 5.
    Grodzinsky, A. J. (1983) Electromechanical and physicochemical properties of connective tissue. CRC Critical Rev. Bioeng. 14, 133–199.Google Scholar
  6. 6.
    Hardingham, T. E. and Fosang, A. (1992) Proteoglycans: many forms and many functions. FASEB. J. 6, 861–870.PubMedGoogle Scholar
  7. 7.
    Urban, J. P. G., Holm, S., and Maroudas, A. (1982) Nutrition of the intervertebral disk: Effect of fluid flow on solute transport. Clin. Orthop. 170, 293–302.Google Scholar
  8. 8.
    Hardingham, T. E., Muir, H., Kwan, M. K., Lai, W. M., and Mow, V. C. (1987) Viscoelastic properties of proteoglycan solutions with varying proportions present as aggregates. J. Orthop. Res. 5, 36–46.PubMedCrossRefGoogle Scholar
  9. 9.
    Li, X. and Reed. W. F. (1991) Polyelectrolyte properties of proteoglycan mono-mers. J. Chem. Phys. 94, 4658–4580.Google Scholar
  10. 10.
    Sheehan, J. K. Arundel, C., and Phelps, C. F. (1983) Effects of the cations sodium, potassium and calcium on the interaction of hyaluronate chains: a light scattering and viscometric study. Int. J. Biol. Macromol. 5, 222–228.CrossRefGoogle Scholar
  11. 11.
    Harper, G. S., Comper, W. D. Preston, B. N., and Daivies, P. (1985) Concentra-tion dependence of proteoglycan diffusion. Biopolymers 24, 2165–2173.PubMedCrossRefGoogle Scholar
  12. 12.
    Axelrod, D., Koppel, D. E., Schlessinger, J. Elson, E., and Webb, W. W. (1976) Mobility measurements by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069.PubMedCrossRefGoogle Scholar
  13. 13.
    Kubitscheck, H., Wedekind, P., and Peters R. (1994) Lateral diffusion measure-ments at high spatial resolution by scanning microphotolysis in a confocal micro-scope. Biophys. J. 67. 946–965.CrossRefGoogle Scholar
  14. 14.
    Bayley, P. M. and Clough, B. (1995) Application of optical microscopy to cellu-lar-dynamics: studies of fluorescence photobleaching (FRAP) of erythrocyte-membrane proteins using the confocal microscope. J. Trace. Microprobe T. 13, 209–216.Google Scholar
  15. 15.
    Blonk, J. C. G., Don, A., Van Aalst, H., and Birmingham, J. J. (1993) Fluores-cence photobleaching in the confocal scanning light microscope. J. Microsc. 169, 363–374.Google Scholar
  16. 16.
    Gribbon, P. and Hardingham, T. E. (1998) Macromolecular diffusion of biologi-cal polymers measured by confocal fluorescence recovery after photobleaching. Biophys. J. 75. 1032–1039.PubMedCrossRefGoogle Scholar
  17. 17.
    Imhof, A., Van Blaadren, A., Maret, G., Mellema, J., and Dhont. J. K. G. (1994) A comparison between the long time self diffusion of and low shear viscosity of concentrated dispersions of charged colloidal silica spheres. J. Chem Phys. 100, 2170–2181.CrossRefGoogle Scholar
  18. 18.
    Peters, R., Brunger, A. and Schulten, K. (1981) Continuous fluorescence microphotolysis: a sensitive method for the study of diffusion processes in single cells. Proc. Natl. Acad. Sci. USA 78, 962–966.PubMedCrossRefGoogle Scholar
  19. 19.
    Hardingham, T. E., Ewins, R. J. F., and Muir, H. (1976) Cartilage proteoglycans: structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate. Biochem. J. 157. 127–143.PubMedGoogle Scholar
  20. 20.
    Gribbon, P., Heng, B. C., and Hardingham, T. E. (1999) The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence after photobleaching. Biophy. J. 77. 2210–2216.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Timothy Hardingham
    • 1
  • Philip Gribbon
    • 1
  1. 1.School of Biological SciencesUniversity of ManchesterManchesterUK

Personalised recommendations