Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Jun 1;96(6):1622–1630. doi: 10.1083/jcb.96.6.1622

Actin-independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas

PMCID: PMC2112438  PMID: 6406516

Abstract

We investigated the mode of association of vinculin with areas of contact between the termini of microfilament bundles and the cell membrane in sites of focal contact with the substrate by selective removal of actin from these areas. Opened-up substrate-attached membranes of chick fibroblasts as well as detergent-permeabilized cells were treated with fragmin from Physarum in the presence of Ca+2. This treatment removed actin filaments from the cytoplasmic faces of the membranes, along with several actin-associated proteins (alpha-actinin, tropomyosin, myosin, and filamin). Vinculin distribution was not affected by treatment. Moreover, rhodamine- or fluorescein-conjugated vinculin, when added to these preparations, became specifically associated with the focal contacts regardless of whether the latter were pretreated with fragmin or not. We conclude that the association of vinculin with focal contacts is largely actin-independent. We discuss the implications of these findings in the molecular mechanisms of microfilament membrane association in areas of cell contact.

Full Text

The Full Text of this article is available as a PDF (4.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abercrombie M., Heaysman J. E., Pegrum S. M. The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Exp Cell Res. 1971 Aug;67(2):359–367. doi: 10.1016/0014-4827(71)90420-4. [DOI] [PubMed] [Google Scholar]
  2. Avnur Z., Geiger B. Substrate-attached membranes of cultured cells isolation and characterization of ventral cell membranes and the associated cytoskeleton. J Mol Biol. 1981 Dec 5;153(2):361–379. doi: 10.1016/0022-2836(81)90283-7. [DOI] [PubMed] [Google Scholar]
  3. Badley R. A., Lloyd C. W., Woods A., Carruthers L., Allcock C., Rees D. A. Mechanisms of cellular adhesion. III. Preparation and preliminary characterisation of adhesions. Exp Cell Res. 1978 Dec;117(2):231–244. doi: 10.1016/0014-4827(78)90136-2. [DOI] [PubMed] [Google Scholar]
  4. Birchmeier W., Libermann T. A., Imhof B. A., Kreis T. E. Intracellular and extracellular components involved in the formation of ventral surfaces of fibroblasts. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):755–767. doi: 10.1101/sqb.1982.046.01.072. [DOI] [PubMed] [Google Scholar]
  5. Brandtzaeg P. Conjugates of immunoglobulin G with different fluorochromes. I. Characterization by anionic-exchange chromatography. Scand J Immunol. 1973;2(3):273–290. doi: 10.1111/j.1365-3083.1973.tb02037.x. [DOI] [PubMed] [Google Scholar]
  6. Burridge K., Feramisco J. R. Alpha-actinin and vinculin from nonmuscle cells: calcium-sensitive interactions with actin. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):587–597. doi: 10.1101/sqb.1982.046.01.055. [DOI] [PubMed] [Google Scholar]
  7. Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
  8. FARQUHAR M. G., PALADE G. E. Junctional complexes in various epithelia. J Cell Biol. 1963 May;17:375–412. doi: 10.1083/jcb.17.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Geiger B., Dutton A. H., Tokuyasu K. T., Singer S. J. 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. doi: 10.1083/jcb.91.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geiger B. Involvement of vinculin in contact-induced cytoskeletal interactions. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):671–682. doi: 10.1101/sqb.1982.046.01.063. [DOI] [PubMed] [Google Scholar]
  12. Geiger B., Singer S. J. The participation of alpha-actinin in the capping of cell membrane components. Cell. 1979 Jan;16(1):213–222. doi: 10.1016/0092-8674(79)90202-2. [DOI] [PubMed] [Google Scholar]
  13. Geiger B., Tokuyasu K. T., Dutton A. H., Singer S. J. 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. doi: 10.1073/pnas.77.7.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  15. Heath J. P., Dunn G. A. 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. doi: 10.1242/jcs.29.1.197. [DOI] [PubMed] [Google Scholar]
  16. Hinssen H. An actin-modulating protein from Physarum polycephalum. I. Isolation and purification. Eur J Cell Biol. 1981 Feb;23(2):225–233. [PubMed] [Google Scholar]
  17. Hinssen H. An actin-modulating protein from Physarum polycephalum. II. Ca++-dependence and other properties. Eur J Cell Biol. 1981 Feb;23(2):234–240. [PubMed] [Google Scholar]
  18. Isenberg G., Leonard K., Jockusch B. M. Structural aspects of vinculin-actin interactions. J Mol Biol. 1982 Jun 25;158(2):231–249. doi: 10.1016/0022-2836(82)90431-4. [DOI] [PubMed] [Google Scholar]
  19. Lazarides E. Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells. J Cell Biol. 1976 Feb;68(2):202–219. doi: 10.1083/jcb.68.2.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
  21. Lin S., Wilkins J. A., Cribbs D. H., Grumet M., Lin D. C. Proteins and complexes that affect actin-filament assembly and interactions. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 2):625–632. doi: 10.1101/sqb.1982.046.01.058. [DOI] [PubMed] [Google Scholar]
  22. Small J. V. Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J Cell Biol. 1981 Dec;91(3 Pt 1):695–705. doi: 10.1083/jcb.91.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Staehelin L. A. Structure and function of intercellular junctions. Int Rev Cytol. 1974;39:191–283. doi: 10.1016/s0074-7696(08)60940-7. [DOI] [PubMed] [Google Scholar]
  24. Tokuyasu K. T., Dutton A. H., Geiger B., Singer S. J. 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. doi: 10.1073/pnas.78.12.7619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weatherbee J. A. Membranes and cell movement: interactions of membranes with the proteins of the cytoskeleton. Int Rev Cytol Suppl. 1981;12:113–176. doi: 10.1016/b978-0-12-364373-5.50014-7. [DOI] [PubMed] [Google Scholar]
  26. 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. doi: 10.1242/jcs.37.1.257. [DOI] [PubMed] [Google Scholar]
  27. Wieland T., Faulstich H. Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms. CRC Crit Rev Biochem. 1978 Dec;5(3):185–260. doi: 10.3109/10409237809149870. [DOI] [PubMed] [Google Scholar]
  28. Wilkins J. A., Lin S. High-affinity interaction of vinculin with actin filaments in vitro. Cell. 1982 Jan;28(1):83–90. doi: 10.1016/0092-8674(82)90377-4. [DOI] [PubMed] [Google Scholar]
  29. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES