Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Nov 1;103(5):1883–1893. doi: 10.1083/jcb.103.5.1883

Ultrastructural colocalization of tyrosinated and detyrosinated alpha- tubulin in interphase and mitotic cells

PMCID: PMC2114383  PMID: 3782287

Abstract

Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH- terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.

Full Text

The Full Text of this article is available as a PDF (5.1 MB).

Selected References

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

  1. Arce C. A., Hallak M. E., Rodriguez J. A., Barra H. S., Caputto R. Capability of tubulin and microtubules to incorporate and to release tyrosine and phenylalanine and the effect of the incorporation of these amino acids on tubulin assembly. J Neurochem. 1978 Jul;31(1):205–210. doi: 10.1111/j.1471-4159.1978.tb12449.x. [DOI] [PubMed] [Google Scholar]
  2. Barra H. S., Arce C. A., Rodríguez J. A., Caputto R. Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1384–1390. doi: 10.1016/0006-291x(74)90351-9. [DOI] [PubMed] [Google Scholar]
  3. Barra H. S., Rodriguez J. A., Arce C. A., Caputto R. A soluble preparation from rat brain that incorporates into its own proteins ( 14 C)arginine by a ribonuclease-sensitive system and ( 14 C)tyrosine by a ribonuclease-insensitive system. J Neurochem. 1973 Jan;20(1):97–108. doi: 10.1111/j.1471-4159.1973.tb12108.x. [DOI] [PubMed] [Google Scholar]
  4. De Brabander M., Bulinski J. C., Geuens G., De Mey J., Borisy G. G. Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates. J Cell Biol. 1981 Nov;91(2 Pt 1):438–445. doi: 10.1083/jcb.91.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Brabander M., Nuydens R., Geuens G., Moeremans M., De Mey J. The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell. Cell Motil Cytoskeleton. 1986;6(2):105–113. doi: 10.1002/cm.970060207. [DOI] [PubMed] [Google Scholar]
  6. Deanin G. G., Thompson W. C., Gordon M. W. Tyrosyltubulin ligase activity in brain, skeletal muscle, and liver of the developing chick. Dev Biol. 1977 May;57(1):230–233. doi: 10.1016/0012-1606(77)90370-0. [DOI] [PubMed] [Google Scholar]
  7. Gundersen G. G., Bulinski J. C. Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis. J Cell Biol. 1986 Mar;102(3):1118–1126. doi: 10.1083/jcb.102.3.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hamaguchi Y., Toriyama M., Sakai H., Hiramoto Y. Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage. J Cell Biol. 1985 Apr;100(4):1262–1272. doi: 10.1083/jcb.100.4.1262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kumar N., Flavin M. Modulation of some parameters of assembly of microtubules in vitro by tyrosinolation of tubulin. Eur J Biochem. 1982 Nov;128(1):215–222. doi: 10.1111/j.1432-1033.1982.tb06954.x. [DOI] [PubMed] [Google Scholar]
  12. Nath J., Flavin M. Tubulin tyrosylation in vivo and changes accompanying differentiation of cultured neuroblastoma-glioma hybrid cells. J Biol Chem. 1979 Nov 25;254(22):11505–11510. [PubMed] [Google Scholar]
  13. Preston S. F., Deanin G. G., Hanson R. K., Gordon M. W. Tubulin:tyrosine ligase in oocytes and embryos of Xenopus laevis. Dev Biol. 1981 Jan 15;81(1):36–42. doi: 10.1016/0012-1606(81)90345-6. [DOI] [PubMed] [Google Scholar]
  14. Raybin D., Flavin M. Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro. J Cell Biol. 1977 May;73(2):492–504. doi: 10.1083/jcb.73.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rodriguez J. A., Borisy G. G. Modification of the C-terminus of brain tubulin during development. Biochem Biophys Res Commun. 1978 Jul 28;83(2):579–586. doi: 10.1016/0006-291x(78)91029-x. [DOI] [PubMed] [Google Scholar]
  16. Rodriguez J. A., Borisy G. G. Tyrosination state of free tubulin subunits and tubulin disassembled from microtubules of rat brain tissue. Biochem Biophys Res Commun. 1979 Aug 13;89(3):893–899. doi: 10.1016/0006-291x(79)91862-x. [DOI] [PubMed] [Google Scholar]
  17. Rothwell S. W., Grasser W. A., Murphy D. B. End-to-end annealing of microtubules in vitro. J Cell Biol. 1986 Feb;102(2):619–627. doi: 10.1083/jcb.102.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Salmon E. D., Leslie R. J., Saxton W. M., Karow M. L., McIntosh J. R. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. doi: 10.1083/jcb.99.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wehland J., Willingham M. C., Sandoval I. V. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo. J Cell Biol. 1983 Nov;97(5 Pt 1):1467–1475. doi: 10.1083/jcb.97.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES