Assembly and turnover of detyrosinated tubulin in vivo (original) (raw)

Abstract

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.

Full Text

The Full Text of this article is available as a PDF (4.7 MB).

Selected References

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

  1. Arce C. A., Barra H. S. Association of tubulinyl-tyrosine carboxypeptidase with microtubules. FEBS Lett. 1983 Jun 27;157(1):75–78. doi: 10.1016/0014-5793(83)81119-3. [DOI] [PubMed] [Google Scholar]
  2. Arce C. A., Barra H. S. Release of C-terminal tyrosine from tubulin and microtubules at steady state. Biochem J. 1985 Feb 15;226(1):311–317. doi: 10.1042/bj2260311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bergen L. G., Borisy G. G. Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly. J Cell Biol. 1980 Jan;84(1):141–150. doi: 10.1083/jcb.84.1.141. [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. Brinkley B. R. Microtubule organizing centers. Annu Rev Cell Biol. 1985;1:145–172. doi: 10.1146/annurev.cb.01.110185.001045. [DOI] [PubMed] [Google Scholar]
  7. Cleveland D. W., Sullivan K. F. Molecular biology and genetics of tubulin. Annu Rev Biochem. 1985;54:331–365. doi: 10.1146/annurev.bi.54.070185.001555. [DOI] [PubMed] [Google Scholar]
  8. Cumming R., Burgoyne R. D., Lytton N. A. Immunocytochemical demonstration of alpha-tubulin modification during axonal maturation in the cerebellar cortex. J Cell Biol. 1984 Jan;98(1):347–351. doi: 10.1083/jcb.98.1.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Field D. J., Collins R. A., Lee J. C. Heterogeneity of vertebrate brain tubulins. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4041–4045. doi: 10.1073/pnas.81.13.4041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Geuens G., Gundersen G. G., Nuydens R., Cornelissen F., Bulinski J. C., DeBrabander M. Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells. J Cell Biol. 1986 Nov;103(5):1883–1893. doi: 10.1083/jcb.103.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gundersen G. G., Bulinski J. C. Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin. Eur J Cell Biol. 1986 Dec;42(2):288–294. [PubMed] [Google Scholar]
  13. 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]
  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. Horio T., Hotani H. Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature. 1986 Jun 5;321(6070):605–607. doi: 10.1038/321605a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Kumar N., Flavin M. Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin. J Biol Chem. 1981 Jul 25;256(14):7678–7686. [PubMed] [Google Scholar]
  19. Kuriyama R., Borisy G. G. Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle. J Cell Biol. 1981 Dec;91(3 Pt 1):822–826. doi: 10.1083/jcb.91.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. L'Hernault S. W., Rosenbaum J. L. Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly. J Cell Biol. 1983 Jul;97(1):258–263. doi: 10.1083/jcb.97.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. L'Hernault S. W., Rosenbaum J. L. Reversal of the posttranslational modification on Chlamydomonas flagellar alpha-tubulin occurs during flagellar resorption. J Cell Biol. 1985 Feb;100(2):457–462. doi: 10.1083/jcb.100.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. LeDizet M., Piperno G. Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties. J Cell Biol. 1986 Jul;103(1):13–22. doi: 10.1083/jcb.103.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Margolis R. L., Wilson L. Opposite end assembly and disassembly of microtubules at steady state in vitro. Cell. 1978 Jan;13(1):1–8. doi: 10.1016/0092-8674(78)90132-0. [DOI] [PubMed] [Google Scholar]
  25. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature. 1984 Nov 15;312(5991):237–242. doi: 10.1038/312237a0. [DOI] [PubMed] [Google Scholar]
  26. Murphy D. B. Assembly-disassembly purification and characterization of microtubule protein without glycerol. Methods Cell Biol. 1982;24:31–49. doi: 10.1016/s0091-679x(08)60646-9. [DOI] [PubMed] [Google Scholar]
  27. Olmsted J. B., Cox J. V., Asnes C. F., Parysek L. M., Lyon H. D. Cellular regulation of microtubule organization. J Cell Biol. 1984 Jul;99(1 Pt 2):28s–32s. doi: 10.1083/jcb.99.1.28s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Raybin D., Flavin M. An enzyme tyrosylating alpha-tubulin and its role in microtubule assembly. Biochem Biophys Res Commun. 1975 Aug 4;65(3):1088–1095. doi: 10.1016/s0006-291x(75)80497-9. [DOI] [PubMed] [Google Scholar]
  30. Raybin D., Flavin M. Enzyme which specifically adds tyrosine to the alpha chain of tubulin. Biochemistry. 1977 May 17;16(10):2189–2194. doi: 10.1021/bi00629a023. [DOI] [PubMed] [Google Scholar]
  31. Rothwell S. W., Grasser W. A., Murphy D. B. Direct observation of microtubule treadmilling by electron microscopy. J Cell Biol. 1985 Nov;101(5 Pt 1):1637–1642. doi: 10.1083/jcb.101.5.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Sammak P. J., Gorbsky G. J., Borisy G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. doi: 10.1083/jcb.104.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scherson T., Kreis T. E., Schlessinger J., Littauer U. Z., Borisy G. G., Geiger B. Dynamic interactions of fluorescently labeled microtubule-associated proteins in living cells. J Cell Biol. 1984 Aug;99(2):425–434. doi: 10.1083/jcb.99.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schliwa M., Euteneuer U., Bulinski J. C., Izant J. G. Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1037–1041. doi: 10.1073/pnas.78.2.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schröder H. C., Wehland J., Weber K. Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods. J Cell Biol. 1985 Jan;100(1):276–281. doi: 10.1083/jcb.100.1.276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Soltys B. J., Borisy G. G. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol. 1985 May;100(5):1682–1689. doi: 10.1083/jcb.100.5.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thompson W. C., Deanin G. G., Gordon M. W. Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1318–1322. doi: 10.1073/pnas.76.3.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wadsworth P., Salmon E. D. Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching. J Cell Biol. 1986 Mar;102(3):1032–1038. doi: 10.1083/jcb.102.3.1032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]