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. 1994 Jun 2;125(6):1371–1384. doi: 10.1083/jcb.125.6.1371

Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members

PMCID: PMC2290919  PMID: 8207064

Abstract

To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.

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Selected References

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  1. Algrain M., Turunen O., Vaheri A., Louvard D., Arpin M. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Biol. 1993 Jan;120(1):129–139. doi: 10.1083/jcb.120.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. A., Lovrien R. E. Glycophorin is linked by band 4.1 protein to the human erythrocyte membrane skeleton. Nature. 1984 Feb 16;307(5952):655–658. doi: 10.1038/307655a0. [DOI] [PubMed] [Google Scholar]
  3. Anderson R. A., Marchesi V. T. Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide. Nature. 1985 Nov 21;318(6043):295–298. doi: 10.1038/318295a0. [DOI] [PubMed] [Google Scholar]
  4. Berryman M., Franck Z., Bretscher A. Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J Cell Sci. 1993 Aug;105(Pt 4):1025–1043. doi: 10.1242/jcs.105.4.1025. [DOI] [PubMed] [Google Scholar]
  5. Bretscher A. Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J Cell Biol. 1983 Aug;97(2):425–432. doi: 10.1083/jcb.97.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bretscher A. Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Biol. 1989 Mar;108(3):921–930. doi: 10.1083/jcb.108.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Conboy J., Kan Y. W., Shohet S. B., Mohandas N. Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9512–9516. doi: 10.1073/pnas.83.24.9512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franck Z., Gary R., Bretscher A. Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable. J Cell Sci. 1993 May;105(Pt 1):219–231. doi: 10.1242/jcs.105.1.219. [DOI] [PubMed] [Google Scholar]
  9. Funayama N., Nagafuchi A., Sato N., Tsukita S., Tsukita S. Radixin is a novel member of the band 4.1 family. J Cell Biol. 1991 Nov;115(4):1039–1048. doi: 10.1083/jcb.115.4.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gould K. L., Bretscher A., Esch F. S., Hunter T. cDNA cloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1. EMBO J. 1989 Dec 20;8(13):4133–4142. doi: 10.1002/j.1460-2075.1989.tb08598.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gould K. L., Cooper J. A., Bretscher A., Hunter T. The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein. J Cell Biol. 1986 Feb;102(2):660–669. doi: 10.1083/jcb.102.2.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gu M. X., York J. D., Warshawsky I., Majerus P. W. Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5867–5871. doi: 10.1073/pnas.88.13.5867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter T., Cooper J. A. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell. 1981 Jun;24(3):741–752. doi: 10.1016/0092-8674(81)90100-8. [DOI] [PubMed] [Google Scholar]
  14. Hunter T., Cooper J. A. Role of tyrosine phosphorylation in malignant transformation by viruses and in cellular growth control. Prog Nucleic Acid Res Mol Biol. 1983;29:221–232. doi: 10.1016/s0079-6603(08)60450-x. [DOI] [PubMed] [Google Scholar]
  15. Itoh M., Yonemura S., Nagafuchi A., Tsukita S., Tsukita S. A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites. J Cell Biol. 1991 Dec;115(5):1449–1462. doi: 10.1083/jcb.115.5.1449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lankes W. T., Furthmayr H. Moesin: a member of the protein 4.1-talin-ezrin family of proteins. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8297–8301. doi: 10.1073/pnas.88.19.8297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lankes W., Griesmacher A., Grünwald J., Schwartz-Albiez R., Keller R. A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. Biochem J. 1988 May 1;251(3):831–842. doi: 10.1042/bj2510831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matsukura M., Shinozuka K., Zon G., Mitsuya H., Reitz M., Cohen J. S., Broder S. Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7706–7710. doi: 10.1073/pnas.84.21.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Osen-Sand A., Catsicas M., Staple J. K., Jones K. A., Ayala G., Knowles J., Grenningloh G., Catsicas S. Inhibition of axonal growth by SNAP-25 antisense oligonucleotides in vitro and in vivo. Nature. 1993 Jul 29;364(6436):445–448. doi: 10.1038/364445a0. [DOI] [PubMed] [Google Scholar]
  21. Pakkanen R., Hedman K., Turunen O., Wahlström T., Vaheri A. Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells. J Histochem Cytochem. 1987 Aug;35(8):809–816. doi: 10.1177/35.8.3298422. [DOI] [PubMed] [Google Scholar]
  22. Paria B. C., Dey S. K., Andrews G. K. Antisense c-myc effects on preimplantation mouse embryo development. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10051–10055. doi: 10.1073/pnas.89.21.10051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ratajczak M. Z., Kant J. A., Luger S. M., Hijiya N., Zhang J., Zon G., Gewirtz A. M. In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11823–11827. doi: 10.1073/pnas.89.24.11823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rees D. J., Ades S. E., Singer S. J., Hynes R. O. Sequence and domain structure of talin. Nature. 1990 Oct 18;347(6294):685–689. doi: 10.1038/347685a0. [DOI] [PubMed] [Google Scholar]
  25. Sato N., Funayama N., Nagafuchi A., Yonemura S., Tsukita S., Tsukita S. A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J Cell Sci. 1992 Sep;103(Pt 1):131–143. doi: 10.1242/jcs.103.1.131. [DOI] [PubMed] [Google Scholar]
  26. Sato N., Yonemura S., Obinata T., Tsukita S., Tsukita S. Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis. J Cell Biol. 1991 Apr;113(2):321–330. doi: 10.1083/jcb.113.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsukita S., Hieda Y., Tsukita S. A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization. J Cell Biol. 1989 Jun;108(6):2369–2382. doi: 10.1083/jcb.108.6.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsukita S., Tsukita S., Nagafuchi A., Yonemura S. Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions. Curr Opin Cell Biol. 1992 Oct;4(5):834–839. doi: 10.1016/0955-0674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  29. Turunen O., Winqvist R., Pakkanen R., Grzeschik K. H., Wahlström T., Vaheri A. Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization. J Biol Chem. 1989 Oct 5;264(28):16727–16732. [PubMed] [Google Scholar]
  30. Yang Q., Tonks N. K. Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5949–5953. doi: 10.1073/pnas.88.14.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yokozaki H., Budillon A., Tortora G., Meissner S., Beaucage S. L., Miki K., Cho-Chung Y. S. An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells. Cancer Res. 1993 Feb 15;53(4):868–872. [PubMed] [Google Scholar]
  32. Yonemura S., Nagafuchi A., Sato N., Tsukita S. Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons. J Cell Biol. 1993 Jan;120(2):437–449. doi: 10.1083/jcb.120.2.437. [DOI] [PMC free article] [PubMed] [Google Scholar]

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