Abstract
How do cells degrade their surrounding matrix? Constitutive and induced cellular secretion of several classes of proteinases have been implicated in extracellular degradation (1) and many of these proteinases have the ability to degrade extracellular matrix proteins in vitro. However, clear evidence of focal matrix degradation adjacent to cells in which enzyme synthesis occurs is sparse. In this chapter we describe a method we have developed that allows unambiguous detection of focal extracellular matrix (ECM) degradation below cells, although at the same time localizing cellular expression of enzyme: we illustrate the use of this method to demonstrate focal proteolysis by cells overexpressing the membrane-type matrix metalloproteinase-1 (MT1-MMP) on the cell surface.
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References
Murphy G. and Reynolds J. J. (1993) Extracellular matrix degradation, in Connective Tissue and its Heritable Disorders (Royce P. M. and Steinmann B., eds.), Wiley-Liss, Inc, New York, pp. 287–316.
Sato H., Takino T., Okada Y., Cao J., Shinagawa A., Yamamoto E., and Seiki M. (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370, 61–65.
Strongin A. Y., Collier I., Bannikov G., Marmer B. L., Grant G. A., and Goldberg G. I. (1995) Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J. Biol. Chem. 270, 5331–5338.
Atkinson S. J., Crabbe T., Cowell S., Ward R. V., Butler M. J., Sato H., Seiki M., Reynolds J. J., and Murphy G. (1995) Intermolecular autolytic cleavage can contribute to the activation of progelatinase A by cell membranes. J. Biol. Chem. 270, 30,479–30,485.
Butler G. S., Butler M. J., Atkinson S. J., Will H., Tamura T., Van Westrum S. S., Crabbe T., Clements J., D’Ortho M. P., and Murphy G. (1998) The TIMP2 membrane type 1 metalloproteinase “receptor” regulates the concentration and efficient activation of progelatinase A. J. Biol. Chem. 273, 871–880.
Imai K., Ohuchi E., Aoki T., Nomura H., Fujii Y., Sato H., Seiki M., and Okada Y. (1996) Membrane-type matrix metalloproteinase 1 is a gelatinolytic enzyme and is secreted in a complex with tissue inhibitor of metalloproteinases 2. Cancer Res. 56, 2707–2710.
Pei D. Q. and Weiss S. J. (1996) Transmembrane-deletion mutants of the membrane-type matrix metalloproteinase-1 process progelatinase A and express intrinsic matrix-degrading activity. J. Biol. Chem. 271, 9135–9140.
Ohuchi E., Imai K., Fujii Y., Sato H., Seiki M., and Okada Y. (1997) Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J. Biol. Chem. 272, 2446–2451.
D’Ortho M.-P., Will H., Atkinson S., Butler G. S., Messant A., Gavrilovic J., Smith B., Timpl R., Zardi L., and Murphy G. (1997) Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur. J. Biochem. 250, 751–757.
D’Ortho M.-P., Stanton H., Butler M., Atkinson S. J., Murphy G., and Hembry R. M. (1998) MT1-MMP on the cell surface causes focal degradation of gelatin films. FEBSLett. 421, 159–164.
Nakahara H., Howard L., Thompson E. W., Sato H., Seiki M., Yeh Y. Y., and Chen W. T. (1997) Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cell invasion. Proc. Natl. Acad. Sci. USA 94, 7959–7964.
Pilcher B. K., Dumin J. A., Sudbeck B. D., Krane S. M., Welgus H. G., and Parks W. C. (1997) The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J. Cell Biol. 137, 1445–1457.
Palecek S. P., Schmidt C. E., Lauffenburger D. A., and Horwitz A. F. (1996) Integrin dynamics on the tail region of migrating fibroblasts. J. Cell Sci. 109, 941–952.
Owen C. A. and Campbell E. J. (1995) Neutrophil proteinases and matrix degradation. The cell biology of pericellular proteolysis. Seminars in Cell Biology 6, 367–376.
Albrecht-Buehler G. (1977) The phagokinetic tracks of 3T3 cells. Cell 11, 395–404.
Chen D. and Okayama H. (1987) High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752.
Hembry R. M., Murphy G., and Reynolds J. J. (1985) Immunolocalization of tissue inhibitor of metalloproteinases (TIMP) in human cells. Characterization and use of a specific antiserum. J. Cell Sci. 73, 105–119.
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© 2001 Humana Press Inc.
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Hembry, R.M. (2001). Detection of Focal Proteolysis Using Texas-Red-Gelatin. In: Clark, I.M. (eds) Matrix Metalloproteinase Protocols. Methods in Molecular Biology™, vol 151. Humana Press. https://doi.org/10.1385/1-59259-046-2:417
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DOI: https://doi.org/10.1385/1-59259-046-2:417
Publisher Name: Humana Press
Print ISBN: 978-0-89603-733-5
Online ISBN: 978-1-59259-046-9
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