Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues. Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative

Journal of Cell Science

Volumn 112, Issue 8, 1999, Pages 1149-1158

  Hayashi, Ken ^a   Yonemura, Shigenobu ^a   Matsui, Takeshi ^a   Tsukita, Sachiko ^a   Tsukita, Shoichiro ^a  

^a KYOTO UNIVERSITY   (Japan)

Author keywords

ERM; Ezrin; Moesin; Phosphorylation; Radixin; Trichloroacetic acid

Indexed keywords

CELL MEMBRANE PROTEIN; EZRIN; MEMBRANE PROTEIN; MOESIN; PHOSPHATASE; RADIXIN; THREONINE; TRICHLOROACETIC ACID;

ACTIN FILAMENT; ANIMAL CELL; ANOXIA; ARTICLE; CARBOXY TERMINAL SEQUENCE; CELL CULTURE; CELL MEMBRANE; CELL TYPE; CELLULAR DISTRIBUTION; CONFOCAL MICROSCOPY; CONTROLLED STUDY; CROSS LINKING; CYTOPLASM; CYTOSKELETON; ENZYME INACTIVATION; HUMAN; HUMAN CELL; IMMUNOBLOTTING; IMMUNOFLUORESCENCE MICROSCOPY; MOUSE; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; TISSUE FIXATION;

3T3 CELLS; ACETONE; ANIMALS; ANOXIA; BLOOD PROTEINS; BRAIN; CELLS, CULTURED; CYTOSKELETAL PROTEINS; DOGS; EPITHELIAL CELLS; ETHANOL; FEMALE; FORMALDEHYDE; HUMANS; IMMUNOBLOTTING; INTESTINE, SMALL; KIDNEY; LIVER; MEMBRANE PROTEINS; METHANOL; MICE; MICROFILAMENT PROTEINS; MICROSCOPY, CONFOCAL; MICROSCOPY, FLUORESCENCE; OVARY; PHOSPHOPROTEINS; PHOSPHORYLATION; RATS; SCIATIC NERVE; TONGUE; TRICHLOROACETIC ACID; TUMOR CELLS, CULTURED;

ANIMALIA;

EID: 0032950502     PISSN: 00219533     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Article

References (55)

1. Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J. Cell Biol. 120, 129-139.
2. Arpin, M., Algrain, M. and Louvard, D. (1994). Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. Curr. Opin. Cell Biol. 6, 136-141.
3. Berryman, M., Franck, Z. and Bretscher, A. (1993). 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. 105, 1025-1043.
4. Berryman, M., Gary, R. and Bretscher, A. (1995). Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J Cell Biol. 131, 1231-1242.
5. Bretscher, A. (1983). 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. 97, 425-432.
6. Bretscher, A. (1989). Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J. Cell Biol. 108, 921-930.
7. Bretscher, A., Gary, R. and Berryman, M. (1995). Soluble ezrin purified from placenta exists as stable monomers and elongated dimers with masked C-terminal ezrin-radixin-moesin association domains. Biochemistry 34, 16830-16837.
8. Bretscher, A., Reczek, D. and Berryman, M. (1997). Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J. Cell Sci. 110, 3011-3018.
9. Chen, J., Cohn, J. A. and Mandel, L. J. (1995). Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc. Nat. Acad. Sci. USA 92, 7495-7499.
10. Chen, J. and Mandel, L. J. (1997). Unopposed phosphatase action initiates ezrin dysfunction: a potential mechanism for anoxic injury. Am. J. Physiol. 273, C710-C716.
11. Clark, R. K. and Damjanov, I. (1986). Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives. J. Histochem Cytochem. 34, 679-682.
12. Earl, W. R. (1943). The mouse fibroblast cultures and changes seen in the living cells. J. Nat. Cancer Inst. 4, 165-212.
13. Edwards, K. A., Montague, R. A., Shepard, S., Edgar, B. A., Erikson, R. L. and Kiehart, D. P. (1994). Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. Proc. Nat. Acad. Sci. USA 91, 4589-4593.
14. Enami, J., Enami, S. and Koga, M. (1984). Isolation of an insulin-responsive preadipose cell line and a mammary tumor-producing, dome-forming epithelial cell line from a mouse mammary tumor. Dev. Growth Differ. 26, 223-234.
15. Fabricant, R. N., De Larco, J. E. and Todaro, G. J. (1977). Nerve growth factor receptors on human melanoma cells in culture. Proc. Nat. Acad. Sci. USA 74, 565-569.
16. Gary, R. and Bretscher, A. (1995). Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell 6, 1061-1075.
17. Gaush, C. R., Hard, W. L. and Smith, T. F. (1966). Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol. Med. 122, 931-935.
18. Henry, M. D., Gonzalez Agosti, C. and Solomon, F. (1995). Molecular dissection of radixin: distinct and interdependent functions of the amino-and carboxy-terminal domains. J. Cell Biol. 129, 1007-1022.
19. Herman, I. M., Crisona, N. J. and Pollard, T. D. (1981). Relation between cell activity and the distribution of cytoplasmic actin and myosin. J. Cell Biol. 90, 84-91.
20. Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takeichi, M. (1987). Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles. J. Cell Biol. 105, 2501-2510.
21. Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T., Takai, V., Tsukita, Sh. and Tsukita, Sa. (1996). Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway. J. Cell Biol. 135, 37-51.
22. Ikeda, K., Monden, T., Kanoh, T., Tsujie, M., Izawa, H., Haba, A., Ohnishi, T., Sekimoto, M., Tomita, N., Shiozaki, H. and Monden, M. (1998). Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J. Histochem. Cytochem. 46, 397-403.
23. Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakizuka, A., Morii, N. and Narumiya, S. (1996). The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 15, 1885-1893
24. Itoh, M., Yonemura, S., Nagafuchi, A., Tsukita, Sa. and Tsukita, Sh. (1991). A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites. J. Cell Biol. 115, 1449-1462.
25. Jainchill, J. L., Aaronson, S. A. and Todaro, G. J. (1969). Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4, 549-553.
26. Kimura, G., Itagaki, A. and Summers, J. (1975). Rat cell line 3yl and its virogenic polyoma-and sv40-transformed derivatives. Int. J. Cancer 15, 694-706.
27. Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S., Tsukita, Sh. and Tsukita, Sa. (1997). ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol. 139, 749-758.
28. Krieg, J. and Hunter, T. (1992). Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol Chem. 267, 19258-19265.
29. Lankes, W., Griesmacher, A., Grunwald, J., Schwartz-Albiez, R. and Keller, R. (1988). A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation. Biochem J. 251, 831-842.
30. Lankes, W. T. and Furthmayr, H. (1991). Moesin: a member of the protein 4.1-talin-ezrin family of proteins. Proc. Nat. Acad. Sci. USA 88, 8297-8301.
31. Leong, S. P. L., Cooperband, S. R., Deckers, P. J., Sutherland, C. M., Cesare, J. F. and Krementz, E. T. (1979). Effect of different fixatives on the localization of human melanoma antigens by immunofluorescence. Oncology 36, 202-207.
32. Leung, T., Manser, E., Tan, L. and Lim, L. (1995). A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol Chem. 270, 29051-29054.
33. Magendantz, M., Henry, M. D., Lander, A. and Solomon, F. (1995). Interdomain interactions of radixin in vitro. J. Biol Chem. 270, 25324-25327.
34. Martin, M., Andreoli, C., Sahuquet, A., Montcourrier, P., Algrain, M. and Mangeat, P. (1995). Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain. J. Cell Biol. 128, 1081-1093.
35. Matsui, T., Amano, M., Yamamoto, T., Chihara, K., Nakafuku, M., Ito, M., Nakano, T., Okawa, K., Iwamatsu, A. and Kaibuchi, K. (1996). Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15, 2208-2216.
36. Matsui, T., Maeda, M., Doi, Y., Yonemura, S., Amano, M., Kaibuchi, K., Tsukita, Sa. and Tsukita, Sh. (1998). Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J. Cell Biol. 140, 647-657.
37. Nakamura, F., Amieva, M. R. and Furthmayr, H. (1995). Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J. Biol Chem. 270, 31377-31385.
38. Nakamura, F., Amieva, M. R., Hirota, C., Mizuno, Y. and Furthmayr, H. (1996). Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages. Biochem. Biophys. Res. Commun 226, 650-656.
39. Oshiro, N., Fukata, Y. and Kaibuchi, K. (1998). Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol Chem. 273, 34663-34666.
40. Pakkanen, R., Hedman, K., Turunen, O., Wahlstrom, T. and Vaheri, A. (1987). Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells. J. Histochem. Cytochem. 35, 809-816.
41. Pestonjamasp, K., Amieva, M. R., Strassel, C. P., Nauseef, W. M., Furthmayr, H. and Luna, E. J. (1995). Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes. Mol. Biol Cell. 6, 247-259.
42. Pietromonaco, S. F., Simons, P. C., Altman, A. and Elias, L. (1998). Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J. Biol Chem. 273, 7594-7603.
43. Sato, N., Yonemura, S., Obinata, T., Tsukita, Sa. and Tsukita, Sh. (1991). Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis. J. Cell Biol. 113, 321-330.
44. Sato, N., Funayama, N., Nagafuchi, A., Yonemura, S., Tsukita, Sa. and Tsukita, Sh. (1992). A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites. J. Cell Sci. 103, 131-143.
45. Serrador, J. M., Alonso-Lebrero, J. L., del Pozo, M. A., Furthmayr, H., Schwartz-Albiez, R., Calvo, J., Lozano, F. and Sanchez-Madrid, F. (1997) Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J. Cell Biol. 138, 1409-1423.
46. Schwartz-Albiez, R., Merling, A., Spring, H., Moller, P. and Koretz, K. (1995). Differential expression of the microspike-associated protein moesin in human tissues. Eur. J. Cell Biol. 67, 189-198.
47. Shaw, R. J., Henry, M., Solomon, F. and Jacks, T. (1998). RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol. Biol. Cell 9, 403-419.
48. Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura, S., Tsukita, Sa. and Tsukita, Sh. (1994). Perturbation of cell adhesion and microvilli formation by antisense oligonucleolides to ERM family members. J. Cell Biol. 125, 1371-1384.
49. Tsukita, Sa., Hieda, Y. and Tsukita, Sh. (1989). A new 82-kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: purification and characterization. J. Cell Biol. 108, 2369-2382.
50. Tsukita, Sa., Oishi, K., Sato, N., Sagara, J., Kawai, A. and Tsukita, Sh. (1994). ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J. Cell Biol. 126, 391-401.
51. Tsukita, Sa., Yonemura, S. and Tsukita, Sh. (1997a). ERM proteins: head-to-tail regulation of actin-plasma membrane interaction. Trends Biochem Sci. 22, 53-58.
52. Tsukita, Sa., Yonemura, S. and Tsukita, Sh. (1997b). ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr. Opin. Cell Biol. 9, 70-75.
53. Turunen, O., Wahlstrom, T. and Vaheri, A. (1994). Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol. 126, 1445-1453.
54. Urushidani, T., Hanzel, D. K. and Forte, J. G. (1989). Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation. Am. J. Physiol. 256, G1070-G1081.
55. Yonemura, S., Hirao, M., Doi, Y., Takahashi, N., Kondo, T., Tsukita, Sa. and Tsukita, Sh. (1998). Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J. Cell Biol. 140, 885-895.