Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Oct 1;109(4):1609–1620. doi: 10.1083/jcb.109.4.1609

A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins

PMCID: PMC2115795  PMID: 2677023

Abstract

The marginal band of nucleated erythrocytes is a microtubule organelle under rigorous quantitative and spatial control, with properties quite different from those of the microtubule organelles of cultured cells. Previous results suggest that proteins other than tubulin may participate in organizing the marginal band, and may interact with elements of the erythrocyte cytoskeleton in addition to microtubules. To identify such species, we raised mAbs against the proteins that assemble from chicken brain homogenates with tubulin. One such antibody binds to a single protein in chicken erythrocytes, and produces an immunofluorescence pattern colocalizing with marginal band microtubules. Several properties of this protein are identical to those of ezrin, a protein isolated from brush border and localized to motile elements of cultured cells. A significant proportion of the antigen is not soluble in erythrocytes, as determined by extraction with nonionic detergent. This cytoskeleton-associated fraction is unaffected by treatments that solubilize the marginal band microtubules. The protein has properties of both microtubule- and microfilament-associated proteins. In the accompanying manuscript (Goslin, K., E. Birgbauer, G. Banker, and F. Solomon. 1989. J. Cell Biol. 109:1621-1631), we show that the same antibody recognizes a component of growth cones with a similar dual nature. In early embryonic red blood cells, the antigen is dispersed throughout the cell and does not colocalize with assembled tubulin. Its confinement to the marginal band during development follows rather than precedes that of microtubules. These results, along with previous work, suggest models for the formation of the marginal band.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

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

  1. Behnke O. A comparative study of microtubules of disk-shaped blood cells. J Ultrastruct Res. 1970 Apr;31(1):61–75. doi: 10.1016/s0022-5320(70)90145-0. [DOI] [PubMed] [Google Scholar]
  2. Behnke O. Microtubules in disk-shaped blood cells. Int Rev Exp Pathol. 1970;9:1–92. [PubMed] [Google Scholar]
  3. Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
  4. Bloom G. S., Luca F. C., Vallee R. B. Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules. J Cell Biol. 1984 Jan;98(1):331–340. doi: 10.1083/jcb.98.1.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borisy G. G., Marcum J. M., Olmsted J. B., Murphy D. B., Johnson K. A. Purification of tubulin and associated high molecular weight proteins from porcine brain and characterization of microtubule assembly in vitro. Ann N Y Acad Sci. 1975 Jun 30;253:107–132. doi: 10.1111/j.1749-6632.1975.tb19196.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Bulinski J. C., Borisy G. G. Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo. J Cell Biol. 1980 Dec;87(3 Pt 1):792–801. doi: 10.1083/jcb.87.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen W. D., Bartelt D., Jaeger R., Langford G., Nemhauser I. The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements. J Cell Biol. 1982 Jun;93(3):828–828. doi: 10.1083/jcb.93.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen W. D. Observations of the marginal band system of nucleated erythrocytes. J Cell Biol. 1978 Jul;78(1):260–273. doi: 10.1083/jcb.78.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2437–2440. doi: 10.1073/pnas.74.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goniakowska-Witalińska L., Witaliński W. Evidence for a correlation between the number of marginal band microtubules and the size of vertebrate erthrocytes. J Cell Sci. 1976 Nov;22(2):397–401. doi: 10.1242/jcs.22.2.397. [DOI] [PubMed] [Google Scholar]
  14. Goslin K., Birgbauer E., Banker G., Solomon F. The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization. J Cell Biol. 1989 Oct;109(4 Pt 1):1621–1631. doi: 10.1083/jcb.109.4.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Griffith L. M., Pollard T. D. Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins. J Cell Biol. 1978 Sep;78(3):958–965. doi: 10.1083/jcb.78.3.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huber G., Alaimo-Beuret D., Matus A. MAP3: characterization of a novel microtubule-associated protein. J Cell Biol. 1985 Feb;100(2):496–507. doi: 10.1083/jcb.100.2.496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joshi H. C., Chu D., Buxbaum R. E., Heidemann S. R. Tension and compression in the cytoskeleton of PC 12 neurites. J Cell Biol. 1985 Sep;101(3):697–705. doi: 10.1083/jcb.101.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim S., Magendantz M., Katz W., Solomon F. Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo. J Cell Biol. 1987 Jan;104(1):51–59. doi: 10.1083/jcb.104.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Lazarides E., Moon R. T. Assembly and topogenesis of the spectrin-based membrane skeleton in erythroid development. Cell. 1984 Jun;37(2):354–356. doi: 10.1016/0092-8674(84)90364-7. [DOI] [PubMed] [Google Scholar]
  23. Lee J. C., Tweedy N., Timasheff S. N. In vitro reconstitution of calf brain microtubules: effects of macromolecules. Biochemistry. 1978 Jul 11;17(14):2783–2790. doi: 10.1021/bi00607a013. [DOI] [PubMed] [Google Scholar]
  24. Magendantz M., Solomon F. Analyzing the components of microtubules: antibodies against chartins, associated proteins from cultured cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6581–6585. doi: 10.1073/pnas.82.19.6581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Manser T., Gefter M. L. Isolation of hybridomas expressing a specific heavy chain variable region gene segment by using a screening technique that detects mRNA sequences in whole cell lysates. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2470–2474. doi: 10.1073/pnas.81.8.2470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Murphy D. B., Grasser W. A., Wallis K. T. Immunofluorescence examination of beta tubulin expression and marginal band formation in developing chicken erythroblasts. J Cell Biol. 1986 Feb;102(2):628–635. doi: 10.1083/jcb.102.2.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nachmias V. T., Sullender J., Fallon J., Asch A. Observations on the "cytoskeleton" of human platelets. Thromb Haemost. 1980 Feb 29;42(5):1661–1666. [PubMed] [Google Scholar]
  28. Osborn M., Weber K. Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures. Methods Cell Biol. 1982;24:97–132. doi: 10.1016/s0091-679x(08)60650-0. [DOI] [PubMed] [Google Scholar]
  29. Pallas D., Solomon F. Cytoplasmic microtubule-associated proteins: phosphorylation at novel sites is correlated with their incorporation into assembled microtubules. Cell. 1982 Sep;30(2):407–414. doi: 10.1016/0092-8674(82)90238-0. [DOI] [PubMed] [Google Scholar]
  30. Parysek L. M., Asnes C. F., Olmsted J. B. MAP 4: occurrence in mouse tissues. J Cell Biol. 1984 Oct;99(4 Pt 1):1309–1315. doi: 10.1083/jcb.99.4.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Peng I., Binder L. I., Black M. M. Cultured neurons contain a variety of microtubule-associated proteins. Brain Res. 1985 Dec 30;361(1-2):200–211. doi: 10.1016/0006-8993(85)91290-9. [DOI] [PubMed] [Google Scholar]
  32. Rothwell S. W., Grasser W. A., Murphy D. B. Tubulin variants exhibit different assembly properties. Ann N Y Acad Sci. 1986;466:103–110. doi: 10.1111/j.1749-6632.1986.tb38387.x. [DOI] [PubMed] [Google Scholar]
  33. Sloboda R. D., Dickersin K. Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band. J Cell Biol. 1980 Oct;87(1):170–179. doi: 10.1083/jcb.87.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Solomon F., Magendantz M. Cytochalasin separates microtubule disassembly from loss of asymmetric morphology. J Cell Biol. 1981 Apr;89(1):157–161. doi: 10.1083/jcb.89.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  36. Solomon F. What might MAPs do? Results of an in situ analysis. Ann N Y Acad Sci. 1986;466:322–327. doi: 10.1111/j.1749-6632.1986.tb38403.x. [DOI] [PubMed] [Google Scholar]
  37. Swan J. A., Solomon F. Reformation of the marginal band of avian erythrocytes in vitro using calf-brain tubulin: peripheral determinants of microtubule form. J Cell Biol. 1984 Dec;99(6):2108–2113. doi: 10.1083/jcb.99.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Swan J. A., Solomon F. Reformation of the marginal band of avian erythrocytes in vitro using calf-brain tubulin: peripheral determinants of microtubule form. J Cell Biol. 1984 Dec;99(6):2108–2113. doi: 10.1083/jcb.99.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tablin F., Reeber M. J., Nachmias V. T. Platelets contain a 210K microtubule-associated protein related to a similar protein in HeLa cells. J Cell Sci. 1988 Jun;90(Pt 2):317–324. doi: 10.1242/jcs.90.2.317. [DOI] [PubMed] [Google Scholar]
  40. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES