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. 1984 Jun 1;98(6):2091–2106. doi: 10.1083/jcb.98.6.2091

In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface

PMCID: PMC2113047  PMID: 6373789

Abstract

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.

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Selected References

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  1. Ainsworth S. K., Karnovsky M. J. An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections. J Histochem Cytochem. 1972 Mar;20(3):225–229. doi: 10.1177/20.3.225. [DOI] [PubMed] [Google Scholar]
  2. Ali I. U., Mautner V., Lanza R., Hynes R. O. Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein. Cell. 1977 May;11(1):115–126. doi: 10.1016/0092-8674(77)90322-1. [DOI] [PubMed] [Google Scholar]
  3. Bornstein P., Ash J. F. Cell surface-associated structural proteins in connective tissue cells. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2480–2484. doi: 10.1073/pnas.74.6.2480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen W. T., Singer S. J. Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol. 1982 Oct;95(1):205–222. doi: 10.1083/jcb.95.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clark R. A., DellaPelle P., Manseau E., Lanigan J. M., Dvorak H. F., Colvin R. B. Blood vessel fibronectin increases in conjunction with endothelial cell proliferation and capillary ingrowth during wound healing. J Invest Dermatol. 1982 Nov;79(5):269–276. doi: 10.1111/1523-1747.ep12500076. [DOI] [PubMed] [Google Scholar]
  6. Clark R. A., Dvorak H. F., Colvin R. B. Fibronectin in delayed-type hypersensitivity skin reactions: associations with vessel permeability and endothelial cell activation. J Immunol. 1981 Feb;126(2):787–793. [PubMed] [Google Scholar]
  7. Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Furcht L. T., Smith D., Wendelschafer-Crabb G., Mosher D. F., Foidart J. M. Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization. J Histochem Cytochem. 1980 Dec;28(12):1319–1333. doi: 10.1177/28.12.7014712. [DOI] [PubMed] [Google Scholar]
  9. Gabbiani G., Chaponnier C., Hüttner I. Cytoplasmic filaments and gap junctions in epithelial cells and myofibroblasts during wound healing. J Cell Biol. 1978 Mar;76(3):561–568. doi: 10.1083/jcb.76.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gabbiani G., Hirschel B. J., Ryan G. B., Statkov P. R., Majno G. Granulation tissue as a contractile organ. A study of structure and function. J Exp Med. 1972 Apr 1;135(4):719–734. doi: 10.1084/jem.135.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gabbiani G. Reparative processes in mammalian wound healing: the role of contractile phenomena. Int Rev Cytol. 1977;48:187–219. doi: 10.1016/s0074-7696(08)61745-3. [DOI] [PubMed] [Google Scholar]
  12. Gabbiani G. The role of contractile proteins in wound healing and fibrocontractive diseases. Methods Achiev Exp Pathol. 1979;9:187–206. [PubMed] [Google Scholar]
  13. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  14. Giloh H., Sedat J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. doi: 10.1126/science.7112126. [DOI] [PubMed] [Google Scholar]
  15. Gilula N. B., Reeves O. R., Steinbach A. Metabolic coupling, ionic coupling and cell contacts. Nature. 1972 Feb 4;235(5336):262–265. doi: 10.1038/235262a0. [DOI] [PubMed] [Google Scholar]
  16. Goldman R. D., Chojnacki B., Yerna M. J. Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid. J Cell Biol. 1979 Mar;80(3):759–766. doi: 10.1083/jcb.80.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grinnell F., Billingham R. E., Burgess L. Distribution of fibronectin during wound healing in vivo. J Invest Dermatol. 1981 Mar;76(3):181–189. doi: 10.1111/1523-1747.ep12525694. [DOI] [PubMed] [Google Scholar]
  18. Grinnell F., Feld M. K. Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin. Cell. 1979 May;17(1):117–129. doi: 10.1016/0092-8674(79)90300-3. [DOI] [PubMed] [Google Scholar]
  19. Hayashi M., Yamada K. M. Differences in domain structures between plasma and cellular fibronectins. J Biol Chem. 1981 Nov 10;256(21):11292–11300. [PubMed] [Google Scholar]
  20. Heggeness M. H., Ash J. F., Singer S. J. Transmembrane linkage of fibronectin to intracellular actin-containing filaments in cultured human fibroblasts. Ann N Y Acad Sci. 1978 Jun 20;312:414–417. doi: 10.1111/j.1749-6632.1978.tb16822.x. [DOI] [PubMed] [Google Scholar]
  21. Herman I. M., Crisona N. J., Pollard T. D. Relation between cell activity and the distribution of cytoplasmic actin and myosin. J Cell Biol. 1981 Jul;90(1):84–91. doi: 10.1083/jcb.90.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hirokawa N., Tilney L. G. Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear. J Cell Biol. 1982 Oct;95(1):249–261. doi: 10.1083/jcb.95.1.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hynes R. O., Destree A. T. Relationships between fibronectin (LETS protein) and actin. Cell. 1978 Nov;15(3):875–886. doi: 10.1016/0092-8674(78)90272-6. [DOI] [PubMed] [Google Scholar]
  24. Hynes R. O., Destree A. T., Wagner D. D. Relationships between microfilaments, cell-substratum adhesion, and fibronectin. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):659–670. doi: 10.1101/sqb.1982.046.01.062. [DOI] [PubMed] [Google Scholar]
  25. Hølund B., Clemmensen I., Junker P., Lyon H. Fibronectin in experimental granulation tissue. Acta Pathol Microbiol Immunol Scand A. 1982 May;90(3):159–165. doi: 10.1111/j.1699-0463.1982.tb00077_90a.x. [DOI] [PubMed] [Google Scholar]
  26. Kefalides N. A., Alper R., Clark C. C. Biochemistry and metabolism of basement membranes. Int Rev Cytol. 1979;61:167–228. doi: 10.1016/s0074-7696(08)61998-1. [DOI] [PubMed] [Google Scholar]
  27. Klebe R. J. Isolation of a collagen-dependent cell attachment factor. Nature. 1974 Jul 19;250(463):248–251. doi: 10.1038/250248a0. [DOI] [PubMed] [Google Scholar]
  28. Kurkinen M., Vaheri A., Roberts P. J., Stenman S. Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest. 1980 Jul;43(1):47–51. [PubMed] [Google Scholar]
  29. Little C. D., Chen W. T. Masking of extracellular collagen and the co-distribution of collagen and fibronectin during matrix formation by cultured embryonic fibroblasts. J Cell Sci. 1982 Jun;55:35–50. doi: 10.1242/jcs.55.1.35. [DOI] [PubMed] [Google Scholar]
  30. Matsudaira P. T., Burgess D. R. Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol. 1979 Dec;83(3):667–673. doi: 10.1083/jcb.83.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mukherjee T. M., Staehelin L. A. The fine-structural organization of the brush border of intestinal epithelial cells. J Cell Sci. 1971 May;8(3):573–599. doi: 10.1242/jcs.8.3.573. [DOI] [PubMed] [Google Scholar]
  33. Pfeffer L. M., Wang E., Tamm I. Interferon effects on microfilament organization, cellular fibronectin distribution, and cell motility in human fibroblasts. J Cell Biol. 1980 Apr;85(1):9–17. doi: 10.1083/jcb.85.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pitelka D. R., Hamamoto S. T., Taggart B. N. Basal lamina and tissue recognition in malignant mammary tumors. Cancer Res. 1980 May;40(5):1600–1611. [PubMed] [Google Scholar]
  35. Postlethwaite A. E., Keski-Oja J., Balian G., Kang A. H. Induction of fibroblast chemotaxis by fibronectin. Localization of the chemotactic region to a 140,000-molecular weight non-gelatin-binding fragment. J Exp Med. 1981 Feb 1;153(2):494–499. doi: 10.1084/jem.153.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Repesh L. A., Fitzgerald T. J., Furcht L. T. Fibronectin involvement in granulation tissue and wound healing in rabbits. J Histochem Cytochem. 1982 Apr;30(4):351–358. doi: 10.1177/30.4.6174568. [DOI] [PubMed] [Google Scholar]
  38. Ruoslahti E., Hayman E. G., Engvall E., Cothran W. C., Butler W. T. Alignment of biologically active domains in the fibronectin molecule. J Biol Chem. 1981 Jul 25;256(14):7277–7281. [PubMed] [Google Scholar]
  39. Sekiguchi K., Hakomori S. Functional domain structure of fibronectin. Proc Natl Acad Sci U S A. 1980 May;77(5):2661–2665. doi: 10.1073/pnas.77.5.2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Singer I. I. Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts. J Cell Biol. 1982 Feb;92(2):398–408. doi: 10.1083/jcb.92.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Singer I. I. Fibronexus formation is an early event during fibronectin-induced restoration of more normal morphology and substrate adhesion patterns in transformed hamster fibroblasts. J Cell Sci. 1982 Aug;56:1–20. doi: 10.1242/jcs.56.1.1. [DOI] [PubMed] [Google Scholar]
  42. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  43. Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
  44. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  45. Vaccaro C. A., Brody J. S. Structural features of alveolar wall basement membrane in the adult rat lung. J Cell Biol. 1981 Nov;91(2 Pt 1):427–437. doi: 10.1083/jcb.91.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vaheri A., Kurkinen M., Lehto V. P., Linder E., Timpl R. Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4944–4948. doi: 10.1073/pnas.75.10.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wagner D. D., Hynes R. O. Topological arrangement of the major structural features of fibronectin. J Biol Chem. 1980 May 10;255(9):4304–4312. [PubMed] [Google Scholar]
  48. Willingham M. C., Yamada K. M., Yamada S. S., Pouysségur J., Pastan I. Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts. Cell. 1977 Mar;10(3):375–380. doi: 10.1016/0092-8674(77)90024-1. [DOI] [PubMed] [Google Scholar]
  49. Wood G. S., Warnke R. Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem. 1981 Oct;29(10):1196–1204. doi: 10.1177/29.10.7028859. [DOI] [PubMed] [Google Scholar]
  50. Yamada K. M., Olden K. Fibronectins--adhesive glycoproteins of cell surface and blood. Nature. 1978 Sep 21;275(5677):179–184. doi: 10.1038/275179a0. [DOI] [PubMed] [Google Scholar]
  51. Yamada K. M., Yamada S. S., Pastan I. Cell surface protein partially restores morphology, adhesiveness, and contact inhibition of movement to transformed fibroblasts. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1217–1221. doi: 10.1073/pnas.73.4.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]

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