• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Oct 1, 1982; 95(1): 205–222.
PMCID: PMC2112341

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts

Abstract

Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double immunoelectron- microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and alpha- actinin with respect to these sites. (a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections. (b) FA sites were immunolabeled intracellularly for vinculin and alpha- actinin, with vinculin labeling situated closer to the membrane than alpha-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites. (c) CC sites were labeled intracellularly for alpha-actinin but not vinculin and were labeled extracellularly for fibronectin. (d) ECM sites were characterized by large separations (often greater than 100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both alpha-actinin and vinculin, the other for alpha-actinin alone. (e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and alpha-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.

Full Text

The Full Text of this article is available as a PDF (9.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abercrombie M, Dunn GA. Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp Cell Res. 1975 Apr;92(1):57–62. [PubMed]
  • Abercrombie M, Heaysman JE, Pegrum SM. The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Exp Cell Res. 1971 Aug;67(2):359–367. [PubMed]
  • Ali IU, Mautner V, Lanza R, Hynes RO. 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. [PubMed]
  • Avnur Z, Geiger B. The removal of extracellular fibronectin from areas of cell-substrate contact. Cell. 1981 Jul;25(1):121–132. [PubMed]
  • Begg DA, Rodewald R, Rebhun LI. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. [PMC free article] [PubMed]
  • Bereiter-Hahn J, Fox CH, Thorell B. Quantitative reflection contrast microscopy of living cells. J Cell Biol. 1979 Sep;82(3):767–779. [PMC free article] [PubMed]
  • Bergmann JE, Tokuyasu KT, Singer SJ. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1746–1750. [PMC free article] [PubMed]
  • Birchmeier C, Kreis TE, Eppenberger HM, Winterhalter KH, Birchmeier W. Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4108–4112. [PMC free article] [PubMed]
  • Bornstein P, Ash JF. Cell surface-associated structural proteins in connective tissue cells. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2480–2484. [PMC free article] [PubMed]
  • Burridge K, Feramisco JR. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. [PubMed]
  • Chen LB, Murray A, Segal RA, Bushnell A, Walsh ML. Studies on intercellular LETS glycoprotein matrices. Cell. 1978 Jun;14(2):377–391. [PubMed]
  • Chen WT. Mechanism of retraction of the trailing edge during fibroblast movement. J Cell Biol. 1981 Jul;90(1):187–200. [PMC free article] [PubMed]
  • Chen WT, Singer SJ. Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7318–7322. [PMC free article] [PubMed]
  • Couchman JR, Rees DA. The behaviour of fibroblasts migrating from chick heart explants: changes in adhesion, locomotion and growth, and in the distribution of actomyosin and fibronectin. J Cell Sci. 1979 Oct;39:149–165. [PubMed]
  • CURTIS AS. THE MECHANISM OF ADHESION OF CELLS TO GLASS. A STUDY BY INTERFERENCE REFLECTION MICROSCOPY. J Cell Biol. 1964 Feb;20:199–215. [PMC free article] [PubMed]
  • Dutton AH, Tokuyasu KT, Singer SJ. Iron-dextran antibody conjugates: General method for simultaneous staining of two components in high-resolution immunoelectron microscopy. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3392–3396. [PMC free article] [PubMed]
  • Fox CH, Cottler-Fox MH, Yamada KM. The distribution of fibronectin in attachment sites of chick fibroblasts. Exp Cell Res. 1980 Dec;130(2):477–481. [PubMed]
  • Furcht LT, Mosher DF, Wendelschafer-Crabb G. Immunocytochemical localization of fibronectin (LETS proteins) on the surface of L6 myoblasts: light and electron microscopic studies. Cell. 1978 Feb;13(2):263–271. [PubMed]
  • Furcht LT, Wendelschafer-Crabb G, Mosher DF, Foidart JM. An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts. J Supramol Struct. 1980;13(1):15–33. [PubMed]
  • 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. [PubMed]
  • Geiger B, Singer SJ. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. [PubMed]
  • Geiger B, Tokuyasu KT, Singer SJ. Immunocytochemical localization of alpha-actinin in intestinal epithelial cells. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2833–2837. [PMC free article] [PubMed]
  • Geiger B, Tokuyasu KT, Dutton AH, Singer SJ. Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4127–4131. [PMC free article] [PubMed]
  • Geiger B, Dutton AH, Tokuyasu KT, Singer SJ. Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells. J Cell Biol. 1981 Dec;91(3 Pt 1):614–628. [PMC free article] [PubMed]
  • Greve JM, Gottlieb DI. Monoclonal antibodies which alter the morphology of cultured chick myogenic cells. J Cell Biochem. 1982;18(2):221–229. [PubMed]
  • Grinnell F, Bennett MH. Fibroblast adhesion on collagen substrata in the presence and absence of plasma fibronectin. J Cell Sci. 1981 Apr;48:19–34. [PubMed]
  • Harris A. Location of cellular adhesions to solid substrata. Dev Biol. 1973 Nov;35(1):97–114. [PubMed]
  • Heath JP, Dunn GA. Cell to substratum contacts of chick fibroblasts and their relation to the microfilament system. A correlated interference-reflexion and high-voltage electron-microscope study. J Cell Sci. 1978 Feb;29:197–212. [PubMed]
  • Heaysman JE, Pegrum SM. Early contacts between fibroblasts. An ultrastructural study. Exp Cell Res. 1973 Mar 30;78(1):71–78. [PubMed]
  • Heaysman JE, Pegrum SM. Early contacts between normal fibroblasts and mouse sarcoma cells. An ultrastructural study. Exp Cell Res. 1973 Apr;78(2):479–481. [PubMed]
  • Hedman K, Vaheri A, Wartiovaara J. External fibronectin of cultured human fibroblasts is predominantly a matrix protein. J Cell Biol. 1978 Mar;76(3):748–760. [PMC free article] [PubMed]
  • Heggeness MH, Ash JF, Singer SJ. Transmembrane linkage of fibronectin to intracellular actin-containing filaments in cultured human fibroblasts. Ann N Y Acad Sci. 1978 Jun 20;312:414–417. [PubMed]
  • Hynes RO, Bye JM. Density and cell cycle dependence of cell surface proteins in hamster fibroblasts. Cell. 1974 Oct;3(2):113–120. [PubMed]
  • Hynes RO, Destree AT. Relationships between fibronectin (LETS protein) and actin. Cell. 1978 Nov;15(3):875–886. [PubMed]
  • Hynes RO, Destree AT, Wagner DD. Relationships between microfilaments, cell-substratum adhesion, and fibronectin. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):659–670. [PubMed]
  • Izzard CS, Lochner LR. Cell-to-substrate contacts in living fibroblasts: an interference reflexion study with an evaluation of the technique. J Cell Sci. 1976 Jun;21(1):129–159. [PubMed]
  • Izzard CS, Lochner LR. Formation of cell-to-substrate contacts during fibroblast motility: an interference-reflexion study. J Cell Sci. 1980 Apr;42:81–116. [PubMed]
  • Kishida Y, Olsen BR, Berg RA, Prockop DJ. Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J Cell Biol. 1975 Feb;64(2):331–339. [PMC free article] [PubMed]
  • Knudsen KA, Rao PE, Damsky CH, Buck CA. Membrane glycoproteins involved in cell--substratum adhesion. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6071–6075. [PMC free article] [PubMed]
  • Lazarides E, Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. [PubMed]
  • Little CD, Chen WT. 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. [PubMed]
  • Pinto da Silva P, Gilula NB. Gap junctions in normal and transformed fibroblasts in culture. Exp Cell Res. 1972;71(2):393–401. [PubMed]
  • Polinger IS. Separation of cell types in embryonic heart cell cultures. Exp Cell Res. 1970 Nov;63(1):78–82. [PubMed]
  • Revel JP, Wolken K. Electronmicroscope investigations of the underside of cells in culture. Exp Cell Res. 1973 Mar 30;78(1):1–14. [PubMed]
  • Singer II. 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. [PubMed]
  • Singer II, Paradiso PR. 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. [PubMed]
  • Geuze HJ, Slot JW, van der Ley PA, Scheffer RC. Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol. 1981 Jun;89(3):653–665. [PMC free article] [PubMed]
  • Small JV, Isenberg G, Celis JE. Polarity of actin at the leading edge of cultured cells. Nature. 1978 Apr 13;272(5654):638–639. [PubMed]
  • Terry AH, Culp LA. Substrate-attached glycoproteins from normal and virus-transformed cells. Biochemistry. 1974 Jan 29;13(3):414–425. [PubMed]
  • Tokuyasu KT. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol. 1973 May;57(2):551–565. [PMC free article] [PubMed]
  • Tokuyasu KT, Singer SJ. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol. 1976 Dec;71(3):894–906. [PMC free article] [PubMed]
  • Tokuyasu KT, Dutton AH, Geiger B, Singer SJ. Ultrastructure of chicken cardiac muscle as studied by double immunolabeling in electron microscopy. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7619–7623. [PMC free article] [PubMed]
  • Vaheri A, Kurkinen M, Lehto VP, 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. [PMC free article] [PubMed]
  • Wehland J, Osborn M, Weber K. Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin. J Cell Sci. 1979 Jun;37:257–273. [PubMed]
  • Wilkins JA, Lin S. High-affinity interaction of vinculin with actin filaments in vitro. Cell. 1982 Jan;28(1):83–90. [PubMed]
  • Willingham MC, Yamada KM, Yamada SS, 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. [PubMed]
  • Yamada KM, Olden K. Fibronectins--adhesive glycoproteins of cell surface and blood. Nature. 1978 Sep 21;275(5677):179–184. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...