Summary
The roles of the microfilament-associated proteins vinculin, α-actinin, myosin and filamin have been studied by immunofluorescence and double fluorescence in conjunction with interference reflection microscopy (IRM), during the development of focal contacts and focal adhesions in a chick fibroblast system which initially has no such adhesion specializations but then develops them sequentially over a 48 h period. Without exception, all focal contacts and focal adhesions contain both vinculin and α-actinin at every stage that we can detect by IRM or by double staining to reveal the associated microfilament bundles. Indeed the appearance of small bodies containing α-actinin and vinculin is shown to precede focal contact formation in our model system and such structures (not visible by IRM) are proposed to be the precursors of focal contacts and adhesions.
Myosin and filamin are distributed generally with some reticular patterning in the early motile cells which lack the focal specializations, but as focal contacts and adhesions form these proteins become progressively recruited into the associated microfilament bundles. Only then do we see the marked depletion that has been reported earlier of diffusely distributed myosin and filamin in the leading lamella. Although this is not initially associated with any change in the motile status of the cells, the recruitment of these microfilament-associated proteins into stress fibres is proposed to occur in preparation for anchorage and bracing of cells to the substratum when they later become stationary.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
ABERCROMBIE, M. & DUNN, G. A. (1975) Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy.Expl Cell Res. 92, 57–62.
ABERCROMBIE, M., DUNN, G. A. & HEATH, J. P. (1976) Locomotion and contraction in non-muscle cells. InCell and Tissue Interactions. (edited by PERRY, S. V., MARGRETH, A. and ADELSTEIN, R. S.), pp. 3–11. Amsterdam: North Holland.
ABERCROMBIE, M., HEAYSMAN, J. E. M. & PEGRUM, S. M. (1971) The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella.Expl Cell Res. 67, 359–67.
BADLEY, R. A., COUCHMAN, J. R. & REES, D. A. (1980a) Comparison of the cell cytoskeleton in migratory and stationary chick fibroblasts.J. Musc. Res. Cell Motility 1, 5–14.
BADLEY, R. A., LLOYD, C. W., WOODS, A., CARRUTHERS, L., ALLCOCK, C. & REES, D. A. (1978) Mechanisms of cellular adhesion. III. Preparation and preliminary characterisation of adhesions.Expl Cell Res. 117, 231–44.
BADLEY, R. A., WOODS, A., SMITH, C. G. & REES, D. A. (1980b) Actomyosin relationships with surface features in fibroblast adhesion.Expl Cell Res. 126, 263–72.
BECK, K. & BEREITER-HAHN, J. (1981) Evaluation of reflection interference contrast microscope images of living cells.Microscopica Acta 84, 153–78.
BURRIDGE, K. & FERAMISCO, J. R. (1980) Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin.Cell 19, 587–95.
BURRIDGE, K. & FERAMISCO, J. R. (1981) Non-muscle α-actinins are calcium-sensitive actin-binding proteins.Nature 294, 565–67.
COUCHMAN, J. R. & REES, D. A. (1979a) Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts.Cell Biol. int. Rep. 3, 431–9.
COUCHMAN, J. R. & REES, D. A. (1979b) 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. 39, 149–65.
COUCHMAN, J. R. & REES, D. A. (1982) Organelle-cytoskeleton relationships in fibroblasts: Mitochondria, Golgi apparatus and endoplasmic reticulum in phases of movement and growth.Eur. J. Cell Biol. 27, 47–54.
COUCHMAN, J. R., REES, D. A., GREEN, M. R. & SMITH, C. G. (1982) Fibronectin has a dual role in motility and anchorage of primary chick fibroblasts and can promote entry into the division cycle.J. Cell Biol. 93, 402–10.
CURTIS, A. S. G. (1964) The mechanisms of adhesion of cells to glass. A study by interference reflection microscopy.J. Cell Biol. 20, 199–215.
DAVID-PFEUTY, T. & SINGER, S. J. (1980) Altered distributions of the cytoskeletal proteins vinculin and α-actinin in cultured fibroblasts transformed by Rous sarcoma virus.Proc. natn. Acad. Sci. 77, 6687–91.
FERAMISCO, J. R. & BLOSE, S. H. (1980) Distribution of fluorescently labelled α-actinin in living and fixed fibroblasts.J. Cell Biol. 86, 608–15.
FERAMISCO, J. R. & BURRIDGE, K. (1980) A rapid purification of α-actinin, filamin and a 130,000 dalton protein from smooth muscle.J. biol. Chem. 255, 1194–9.
GEIGER, B. (1979) A 130K protein from chicken gizzard: Its localisation at the termini of microfilament bundles in cultured chicken cells.Cell 18, 193–205.
GEIGER, B. (1981) Transmembrane linkage and cell attachment: the role of vinculin. InInternational Cell Biology 1980–1981 (edited by SCHWEIGER, H. G.), pp. 761–773. Berlin, Heidelberg, New York: Springer-Verlag.
GINGELL, D. (1981) The interpretation of interference-reflection images of spread cells: Significant contributions from thin peripheral cytoplasm.J. Cell Sci. 49, 237–47.
GOTLIEB, A. I., HEGGENESS, M. H., ASH, J. F. & SINGER, S. J. (1979) Mechanochemical proteins, cell motility and cell-cell contacts: The localization of mechanochemical proteins inside cultured cells at the edge of an in vitro ‘wound’.J. Cell Physiol. 100, 563–78.
HARTWIG, J. H., TYLER, J. & STOSSELL, T. P. (1980) Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.J. Cell Biol. 87, 841–8.
HEATH, J. P. (1981) Arcs: Curved microfilament bundles beneath the dorsal surface of the leading lamellae of moving chick embryo fibroblasts.Cell Biol. int. Rep. 5, 975–80.
HEATH, J. P. & DUNN, G. A. (1978) 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. 29, 197–212.
HEGGENESS, M. H., WANG, K. & SINGER, S. J. (1977) Intracellular distributions of mechanochemical proteins in cultured fibroblasts.Proc. natn. Acad. Sci. 74, 3883–7.
HELLEWELL, S. B. & TAYLOR, D. L. (1979) The contractile basis of amoeboid movement. VI. The solation-contraction coupling hypothesis.J. Cell Biol. 83, 633–48.
HERMAN, I. M., CRISONA, N. J. & POLLARD, T. D. (1981) Relation between cell activity and the distribution of cytoplasmic actin and myosin.J. Cell Biol. 90, 84–91.
HUXLEY, H. E. (1973) Muscular contraction and cell motility.Nature 243, 445–9.
ISENBERG, G., RATHKE, P., HULSMANN, N., FRANKE, W. W. & WOHLFARTH-BOTTERMAN, K. E. (1976) Cytoplasmic actomyosin fibrils in tissue culture cells. Direct proof of contractility by visualisation of ATP-induced contraction in fibrils isolated by laser micro-beam dissection.Cell Tiss. Res. 166, 427–43.
IZZARD, C. S. & LOCHNER, L. R. (1976) Cell-to-substrate contacts in living fibroblasts: An interference reflexion study with an evaluation of the technique.J. Cell Sci. 21, 129–59.
IZZARD, C. S. & LOCHNER, L. R. (1980) Formation of cell-to-substrate contacts during fibroblast motility: An interference-reflexion study.J. Cell Sci. 42, 81–116.
JOCKUSCH, B. M. & ISENBERG, G. (1981) Interaction of α-actinin and vinculin with actin: Opposite effects on filament network formation.Proc. natn. Acad. Sci. 78, 3005–9.
KREIS, T. E. & BIRCHMEIER, W. (1980) Stress fiber sarcomeres of fibroblasts are contractile.Cell 22, 555–61.
LAZARIDES, E. & BURRIDGE, K. (1975) α-Actinin: Immunofluorescent localization of a muscle structural protein in non-muscle cells.Cell 6, 289–98.
REES, D. A., BADLEY, R. A., BAYLEY, S. A., COUCHMAN, J. R., SMITH, C. G. & WOODS, A. (1981) Surface components in fibroblast adhesion and movement. InCellular Interactions (edited by DINGLE, J. T. and GORDON, J. L.), pp. 67–80. Amsterdam: Elsevier/North Holland.
REES, D. A., LLOYD, C. W. & THOM, D. (1977) Control of grip and stick in cell adhesion through lateral relationships of membrane glycoproteins.Nature 267, 124–8.
SHIZUTA, Y., SHIZUTA, H., GALLO, M., DAVIES, P., PASTAN, I. & LEWIS, M. S. (1976) Purification and properties of filamin, an actin-binding protein from chicken gizzard.J. biol. Chem. 251, 6562–7.
SINGER, I. I. (1982) Association of fibronectin and vinculin with focal contacts and stress fibres in stationary hamster fibroblasts.J. Cell Biol. 92, 398–408.
SINGER, I. I. & PARADISO, P. R. (1981) A transmembrane relationship between fibronectin and vinculin (130 kd protein): Serum modulation in normal and transformed hamster fibroblasts.Cell 24, 481–92.
VIRTANEN, I., LEHTO, V.-P., VARTIO, T., HOVI, T. & BADLEY, R. A. (1981) Reorganization of cytoskeletal elements during transition of human monocytes into adherent macrophage-like cells.Cell Biol. int. Rep. 5A, 52.
WANG, K. & SINGER, S. J. (1977) Interaction of filamin with F-actin in solution.Proc. natn. Acad. Sci. 74, 2021–5.
WEHLAND, J., OSBORN, M. & WEBER, K. (1977) Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth.Proc. natn. Acad. Sci. 74, 5613–7.
WEHLAND, J., STOCKEM, W. & WEBER, K. (1978) Cytoplasmic streaming in Amoeba proteus is inhibited by the actin specific drug phalloidin.Expl Cell Res. 115, 451–9.
ZIGMOND, S. H., OTTO, J. J. & BRYAN, J. (1979) Organization of myosin in a submembranous sheath in well-spread human fibroblasts.Expl Cell Res. 119, 205–19.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Couchman, J.R., Badley, R.A. & Rees, D.A. Redistribution of microfilament-associated proteins during the formation of focal contacts and adhesions in chick fibroblasts. J Muscle Res Cell Motil 4, 647–661 (1983). https://doi.org/10.1007/BF00712158
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00712158