Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):2275–2288. doi: 10.1083/jcb.109.5.2275

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation

PMCID: PMC2115884  PMID: 2681230

Abstract

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.

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. Antin P. B., Forry-Schaudies S., Friedman T. M., Tapscott S. J., Holtzer H. Taxol induces postmitotic myoblasts to assemble interdigitating microtubule-myosin arrays that exclude actin filaments. J Cell Biol. 1981 Aug;90(2):300–308. doi: 10.1083/jcb.90.2.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Argarana C. E., Barra H. S., Caputto R. Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification. J Neurochem. 1980 Jan;34(1):114–118. doi: 10.1111/j.1471-4159.1980.tb04628.x. [DOI] [PubMed] [Google Scholar]
  3. Argaraña C. E., Barra H. S., Caputto R. Release of [14C]tyrosine from tubulinyl-[14C]tyrosine by brain extract. Separation of a carboxypeptidase from tubulin-tyrosine ligase. Mol Cell Biochem. 1978 Feb 24;19(1):17–21. doi: 10.1007/BF00231230. [DOI] [PubMed] [Google Scholar]
  4. Bader D., Masaki T., Fischman D. A. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982 Dec;95(3):763–770. doi: 10.1083/jcb.95.3.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Bergmann J. E., Kupfer A., Singer S. J. Membrane insertion at the leading edge of motile fibroblasts. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1367–1371. doi: 10.1073/pnas.80.5.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Black M. M., Keyser P. Acetylation of alpha-tubulin in cultured neurons and the induction of alpha-tubulin acetylation in PC12 cells by treatment with nerve growth factor. J Neurosci. 1987 Jun;7(6):1833–1842. doi: 10.1523/JNEUROSCI.07-06-01833.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bulinski J. C., Richards J. E., Piperno G. Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells. J Cell Biol. 1988 Apr;106(4):1213–1220. doi: 10.1083/jcb.106.4.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clegg C. H., Linkhart T. A., Olwin B. B., Hauschka S. D. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol. 1987 Aug;105(2):949–956. doi: 10.1083/jcb.105.2.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Devlin R. B., Emerson C. P., Jr Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell. 1978 Apr;13(4):599–611. doi: 10.1016/0092-8674(78)90211-8. [DOI] [PubMed] [Google Scholar]
  13. Fischman D. A. The synthesis and assembly of myofibrils in embryonic muscle. Curr Top Dev Biol. 1970;5:235–280. doi: 10.1016/s0070-2153(08)60057-5. [DOI] [PubMed] [Google Scholar]
  14. Flavin M., Murofushi H. Tyrosine incorporation in tubulin. Methods Enzymol. 1984;106:223–237. doi: 10.1016/0076-6879(84)06024-9. [DOI] [PubMed] [Google Scholar]
  15. Geiger P. J., Bessman S. P. Protein determination by Lowry's method in the presence of sulfhydryl reagents. Anal Biochem. 1972 Oct;49(2):467–473. doi: 10.1016/0003-2697(72)90450-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gu W., Lewis S. A., Cowan N. J. Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function. J Cell Biol. 1988 Jun;106(6):2011–2022. doi: 10.1083/jcb.106.6.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Gundersen G. G., Bulinski J. C. Selective stabilization of microtubules oriented toward the direction of cell migration. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5946–5950. doi: 10.1073/pnas.85.16.5946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Gundersen G. G., Khawaja S., Bulinski J. C. Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules. J Cell Biol. 1987 Jul;105(1):251–264. doi: 10.1083/jcb.105.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ishikawa H., Bischoff R., Holtzer H. Mitosis and intermediate-sized filaments in developing skeletal muscle. J Cell Biol. 1968 Sep;38(3):538–555. doi: 10.1083/jcb.38.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kalderon N., Gilula N. B. Membrane events involved in myoblast fusion. J Cell Biol. 1979 May;81(2):411–425. doi: 10.1083/jcb.81.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khawaja S., Gundersen G. G., Bulinski J. C. Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level. J Cell Biol. 1988 Jan;106(1):141–149. doi: 10.1083/jcb.106.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. LeDizet M., Piperno G. Identification of an acetylation site of Chlamydomonas alpha-tubulin. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5720–5724. doi: 10.1073/pnas.84.16.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lewis S. A., Cowan N. J. Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells. J Cell Biol. 1988 Jun;106(6):2023–2033. doi: 10.1083/jcb.106.6.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minty A. J., Alonso S., Caravatti M., Buckingham M. E. A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA. Cell. 1982 Aug;30(1):185–192. doi: 10.1016/0092-8674(82)90024-1. [DOI] [PubMed] [Google Scholar]
  30. Okazaki K., Holtzer H. An analysis of myogenesis in vitro using fluorescein-labeled antimyosin. J Histochem Cytochem. 1965 Nov-Dec;13(8):726–739. doi: 10.1177/13.8.726. [DOI] [PubMed] [Google Scholar]
  31. Olmsted J. B. Microtubule-associated proteins. Annu Rev Cell Biol. 1986;2:421–457. doi: 10.1146/annurev.cb.02.110186.002225. [DOI] [PubMed] [Google Scholar]
  32. Piperno G., LeDizet M., Chang X. J. Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol. 1987 Feb;104(2):289–302. doi: 10.1083/jcb.104.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Sale W. S., Besharse J. C., Piperno G. Distribution of acetylated alpha-tubulin in retina and in vitro-assembled microtubules. Cell Motil Cytoskeleton. 1988;9(3):243–253. doi: 10.1002/cm.970090306. [DOI] [PubMed] [Google Scholar]
  36. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Senechal H., Schapira G., Wahrmann J. P. Changes in plasma membrane glycoproteins during differentiation of an established myoblast cell line and a non-fusing alpha-amanitin-resistant mutant. Exp Cell Res. 1982 Apr;138(2):355–365. doi: 10.1016/0014-4827(82)90184-7. [DOI] [PubMed] [Google Scholar]
  38. Tassin A. M., Maro B., Bornens M. Fate of microtubule-organizing centers during myogenesis in vitro. J Cell Biol. 1985 Jan;100(1):35–46. doi: 10.1083/jcb.100.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tornqvist H., Belfrage P. Determination of protein in adipose tissue extracts. J Lipid Res. 1976 Sep;17(5):542–545. [PubMed] [Google Scholar]
  40. Toyama Y., Forry-Schaudies S., Hoffman B., Holtzer H. Effects of taxol and Colcemid on myofibrillogenesis. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6556–6560. doi: 10.1073/pnas.79.21.6556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vallee R. B. MAP2 (microtubule-associated protein 2). Cell Muscle Motil. 1984;5:289–311. doi: 10.1007/978-1-4684-4592-3_8. [DOI] [PubMed] [Google Scholar]
  42. Vallee R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 1986;134:89–104. doi: 10.1016/0076-6879(86)34078-3. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Warren R. H. Microtubular organization in elongating myogenic cells. J Cell Biol. 1974 Nov;63(2 Pt 1):550–566. doi: 10.1083/jcb.63.2.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Webster D. R., Borisy G. G. Microtubules are acetylated in domains that turn over slowly. J Cell Sci. 1989 Jan;92(Pt 1):57–65. doi: 10.1242/jcs.92.1.57. [DOI] [PubMed] [Google Scholar]
  46. Webster D. R., Gundersen G. G., Bulinski J. C., Borisy G. G. Assembly and turnover of detyrosinated tubulin in vivo. J Cell Biol. 1987 Jul;105(1):265–276. doi: 10.1083/jcb.105.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Webster D. R., Gundersen G. G., Bulinski J. C., Borisy G. G. Differential turnover of tyrosinated and detyrosinated microtubules. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9040–9044. doi: 10.1073/pnas.84.24.9040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wehland J., Weber K. Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells. J Cell Sci. 1987 Sep;88(Pt 2):185–203. doi: 10.1242/jcs.88.2.185. [DOI] [PubMed] [Google Scholar]
  49. Yaffe D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc Natl Acad Sci U S A. 1968 Oct;61(2):477–483. doi: 10.1073/pnas.61.2.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zevin-Sonkin D., Yaffe D. Accumulation of muscle-specific RNA sequences during myogenesis. Dev Biol. 1980 Feb;74(2):326–334. doi: 10.1016/0012-1606(80)90434-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES