Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Oct 1;105(4):1707–1720. doi: 10.1083/jcb.105.4.1707

In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens

PMCID: PMC2114651  PMID: 3312237

Abstract

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta- tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

Full Text

The Full Text of this article is available as a PDF (5.0 MB).

Selected References

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

  1. Bhattacharyya B., Sackett D. L., Wolff J. Tubulin, hybrid dimers, and tubulin S. Stepwise charge reduction and polymerization. J Biol Chem. 1985 Aug 25;260(18):10208–10216. [PubMed] [Google Scholar]
  2. Binder L. I., Frankfurter A., Rebhun L. I. The distribution of tau in the mammalian central nervous system. J Cell Biol. 1985 Oct;101(4):1371–1378. doi: 10.1083/jcb.101.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  5. Brinkley B. R., Cartwright J., Jr Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann N Y Acad Sci. 1975 Jun 30;253:428–439. doi: 10.1111/j.1749-6632.1975.tb19218.x. [DOI] [PubMed] [Google Scholar]
  6. Bulinski J. C., Borisy G. G. Widespread distribution of a 210,000 mol wt microtubule-associated protein in cells and tissues of primates. J Cell Biol. 1980 Dec;87(3 Pt 1):802–808. doi: 10.1083/jcb.87.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caron J. M., Jones A. L., Kirschner M. W. Autoregulation of tubulin synthesis in hepatocytes and fibroblasts. J Cell Biol. 1985 Nov;101(5 Pt 1):1763–1772. doi: 10.1083/jcb.101.5.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleveland D. W., Lopata M. A., Sherline P., Kirschner M. W. Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell. 1981 Aug;25(2):537–546. doi: 10.1016/0092-8674(81)90072-6. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W. The multitubulin hypothesis revisited: what have we learned? J Cell Biol. 1987 Mar;104(3):381–383. doi: 10.1083/jcb.104.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gundersen G. G., Kalnoski M. H., Bulinski J. C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. 1984 Oct;38(3):779–789. doi: 10.1016/0092-8674(84)90273-3. [DOI] [PubMed] [Google Scholar]
  11. Hall J. L., Dudley L., Dobner P. R., Lewis S. A., Cowan N. J. Identification of two human beta-tubulin isotypes. Mol Cell Biol. 1983 May;3(5):854–862. doi: 10.1128/mcb.3.5.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hayden J. H., Allen R. D. Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule. J Cell Biol. 1984 Nov;99(5):1785–1793. doi: 10.1083/jcb.99.5.1785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heuser J. E., Kirschner M. W. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J Cell Biol. 1980 Jul;86(1):212–234. doi: 10.1083/jcb.86.1.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hiller G., Weber K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell. 1978 Aug;14(4):795–804. doi: 10.1016/0092-8674(78)90335-5. [DOI] [PubMed] [Google Scholar]
  15. Huber G., Matus A. Differences in the cellular distributions of two microtubule-associated proteins, MAP1 and MAP2, in rat brain. J Neurosci. 1984 Jan;4(1):151–160. doi: 10.1523/JNEUROSCI.04-01-00151.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kemphues K. J., Kaufman T. C., Raff R. A., Raff E. C. The testis-specific beta-tubulin subunit in Drosophila melanogaster has multiple functions in spermatogenesis. Cell. 1982 Dec;31(3 Pt 2):655–670. doi: 10.1016/0092-8674(82)90321-x. [DOI] [PubMed] [Google Scholar]
  17. Kirschner M., Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell. 1986 May 9;45(3):329–342. doi: 10.1016/0092-8674(86)90318-1. [DOI] [PubMed] [Google Scholar]
  18. L'Hernault S. W., Rosenbaum J. L. Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry. 1985 Jan 15;24(2):473–478. doi: 10.1021/bi00323a034. [DOI] [PubMed] [Google Scholar]
  19. Lee M. G., Lewis S. A., Wilde C. D., Cowan N. J. Evolutionary history of a multigene family: an expressed human beta-tubulin gene and three processed pseudogenes. Cell. 1983 Jun;33(2):477–487. doi: 10.1016/0092-8674(83)90429-4. [DOI] [PubMed] [Google Scholar]
  20. Lewis S. A., Gilmartin M. E., Hall J. L., Cowan N. J. Three expressed sequences within the human beta-tubulin multigene family each define a distinct isotype. J Mol Biol. 1985 Mar 5;182(1):11–20. doi: 10.1016/0022-2836(85)90023-3. [DOI] [PubMed] [Google Scholar]
  21. Lewis S. A., Gu W., Cowan N. J. Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules. Cell. 1987 May 22;49(4):539–548. doi: 10.1016/0092-8674(87)90456-9. [DOI] [PubMed] [Google Scholar]
  22. Lewis S. A., Lee M. G., Cowan N. J. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol. 1985 Sep;101(3):852–861. doi: 10.1083/jcb.101.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lopata M. A., Havercroft J. C., Chow L. T., Cleveland D. W. Four unique genes required for beta tubulin expression in vertebrates. Cell. 1983 Mar;32(3):713–724. doi: 10.1016/0092-8674(83)90057-0. [DOI] [PubMed] [Google Scholar]
  24. Murphy D. B., Wallis K. T. Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides. J Biol Chem. 1983 Jun 25;258(12):7870–7875. [PubMed] [Google Scholar]
  25. Murphy D. B., Wallis K. T. Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly. J Biol Chem. 1985 Oct 5;260(22):12293–12301. [PubMed] [Google Scholar]
  26. 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]
  27. Piperno G., Fuller M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985 Dec;101(6):2085–2094. doi: 10.1083/jcb.101.6.2085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Sackett D. L., Bhattacharyya B., Wolff J. Tubulin subunit carboxyl termini determine polymerization efficiency. J Biol Chem. 1985 Jan 10;260(1):43–45. [PubMed] [Google Scholar]
  30. Schulze E., Kirschner M. Dynamic and stable populations of microtubules in cells. J Cell Biol. 1987 Feb;104(2):277–288. doi: 10.1083/jcb.104.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Serrano L., de la Torre J., Maccioni R. B., Avila J. Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5989–5993. doi: 10.1073/pnas.81.19.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Sullivan K. F., Cleveland D. W. Identification of conserved isotype-defining variable region sequences for four vertebrate beta tubulin polypeptide classes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4327–4331. doi: 10.1073/pnas.83.12.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sullivan K. F., Cleveland D. W. Sequence of a highly divergent beta tubulin gene reveals regional heterogeneity in the beta tubulin polypeptide. J Cell Biol. 1984 Nov;99(5):1754–1760. doi: 10.1083/jcb.99.5.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sullivan K. F., Havercroft J. C., Machlin P. S., Cleveland D. W. Sequence and expression of the chicken beta 5- and beta 4-tubulin genes define a pair of divergent beta-tubulins with complementary patterns of expression. Mol Cell Biol. 1986 Dec;6(12):4409–4418. doi: 10.1128/mcb.6.12.4409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sullivan K. F., Machlin P. S., Ratrie H., 3rd, Cleveland D. W. Sequence and expression of the chicken beta 3 tubulin gene. A vertebrate testis beta-tubulin isotype. J Biol Chem. 1986 Oct 5;261(28):13317–13322. [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. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon. Cell. 1985 Feb;40(2):449–454. doi: 10.1016/0092-8674(85)90159-x. [DOI] [PubMed] [Google Scholar]
  40. Villasante A., Wang D., Dobner P., Dolph P., Lewis S. A., Cowan N. J. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes. Mol Cell Biol. 1986 Jul;6(7):2409–2419. doi: 10.1128/mcb.6.7.2409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang D., Villasante A., Lewis S. A., Cowan N. J. The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype. J Cell Biol. 1986 Nov;103(5):1903–1910. doi: 10.1083/jcb.103.5.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES