Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Sep 1;111(3):1009–1026. doi: 10.1083/jcb.111.3.1009

Two Drosophila beta tubulin isoforms are not functionally equivalent

PMCID: PMC2116267  PMID: 2118141

Abstract

We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.

Full Text

The Full Text of this article is available as a PDF (6.1 MB).

Selected References

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

  1. Bialojan S., Falkenburg D., Renkawitz-Pohl R. Characterization and developmental expression of beta tubulin genes in Drosophila melanogaster. EMBO J. 1984 Nov;3(11):2543–2548. doi: 10.1002/j.1460-2075.1984.tb02170.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Breitling F., Little M. Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL1/34, DM1A and DM1B. J Mol Biol. 1986 May 20;189(2):367–370. doi: 10.1016/0022-2836(86)90517-6. [DOI] [PubMed] [Google Scholar]
  5. Dabora S. L., Sheetz M. P. The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts. Cell. 1988 Jul 1;54(1):27–35. doi: 10.1016/0092-8674(88)90176-6. [DOI] [PubMed] [Google Scholar]
  6. Fridovich-Keil J. L., Bond J. F., Solomon F. Domains of beta-tubulin essential for conserved functions in vivo. Mol Cell Biol. 1987 Oct;7(10):3792–3798. doi: 10.1128/mcb.7.10.3792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuller M. T., Caulton J. H., Hutchens J. A., Kaufman T. C., Raff E. C. Genetic analysis of microtubule structure: a beta-tubulin mutation causes the formation of aberrant microtubules in vivo and in vitro. J Cell Biol. 1987 Mar;104(3):385–394. doi: 10.1083/jcb.104.3.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller M. T., Caulton J. H., Hutchens J. A., Kaufman T. C., Raff E. C. Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays. J Cell Biol. 1988 Jul;107(1):141–152. doi: 10.1083/jcb.107.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasch A., Hinz U., Leiss D., Renkawitz-Pohl R. The expression of beta 1 and beta 3 tubulin genes of Drosophila melanogaster is spatially regulated during embryogenesis. Mol Gen Genet. 1988 Jan;211(1):8–16. doi: 10.1007/BF00338387. [DOI] [PubMed] [Google Scholar]
  10. Gasch A., Hinz U., Renkawitz-Pohl R. Intron and upstream sequences regulate expression of the Drosophila beta 3-tubulin gene in the visceral and somatic musculature, respectively. Proc Natl Acad Sci U S A. 1989 May;86(9):3215–3218. doi: 10.1073/pnas.86.9.3215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hardy R. W. The influence of chromosome content on the size and shape of sperm heads in Drosophila melanogaster and the demonstration of chromosome loss during spermiogenesis. Genetics. 1975 Feb;79(2):231–264. doi: 10.1093/genetics/79.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hiraoka Y., Toda T., Yanagida M. The NDA3 gene of fission yeast encodes beta-tubulin: a cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis. Cell. 1984 Dec;39(2 Pt 1):349–358. doi: 10.1016/0092-8674(84)90013-8. [DOI] [PubMed] [Google Scholar]
  13. Keith C. H., Feramisco J. R., Shelanski M. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol. 1981 Jan;88(1):234–240. doi: 10.1083/jcb.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kellogg D. R., Mitchison T. J., Alberts B. M. Behaviour of microtubules and actin filaments in living Drosophila embryos. Development. 1988 Aug;103(4):675–686. doi: 10.1242/dev.103.4.675. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kemphues K. J., Raff E. C., Kaufman T. C. Genetic analysis of B2t, the structural gene for a testis-specific beta-tubulin subunit in Drosophila melanogaster. Genetics. 1983 Oct;105(2):345–356. doi: 10.1093/genetics/105.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kemphues K. J., Raff E. C., Raff R. A., Kaufman T. C. Mutation in a testis-specific beta-tubulin in Drosophila: analysis of its effects on meiosis and map location of the gene. Cell. 1980 Sep;21(2):445–451. doi: 10.1016/0092-8674(80)90481-x. [DOI] [PubMed] [Google Scholar]
  18. Kemphues K. J., Raff R. A., Kaufman T. C., Raff E. C. Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3991–3995. doi: 10.1073/pnas.76.8.3991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kimble M., Incardona J. P., Raff E. C. A variant beta-tubulin isoform of Drosophila melanogaster (beta 3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules. Dev Biol. 1989 Feb;131(2):415–429. doi: 10.1016/s0012-1606(89)80014-4. [DOI] [PubMed] [Google Scholar]
  20. Klemenz R., Weber U., Gehring W. J. The white gene as a marker in a new P-element vector for gene transfer in Drosophila. Nucleic Acids Res. 1987 May 26;15(10):3947–3959. doi: 10.1093/nar/15.10.3947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee C., Chen L. B. Dynamic behavior of endoplasmic reticulum in living cells. Cell. 1988 Jul 1;54(1):37–46. doi: 10.1016/0092-8674(88)90177-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Littauer U. Z., Giveon D., Thierauf M., Ginzburg I., Ponstingl H. Common and distinct tubulin binding sites for microtubule-associated proteins. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7162–7166. doi: 10.1073/pnas.83.19.7162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lopata M. A., Cleveland D. W. 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. J Cell Biol. 1987 Oct;105(4):1707–1720. doi: 10.1083/jcb.105.4.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maccioni R. B., Rivas C. I., Vera J. C. Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins. EMBO J. 1988 Jul;7(7):1957–1963. doi: 10.1002/j.1460-2075.1988.tb03033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maccioni R. B., Serrano L., Avila J., Cann J. R. Characterization and structural aspects of the enhanced assembly of tubulin after removal of its carboxyl-terminal domain. Eur J Biochem. 1986 Apr 15;156(2):375–381. doi: 10.1111/j.1432-1033.1986.tb09593.x. [DOI] [PubMed] [Google Scholar]
  27. May G. S. The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. J Cell Biol. 1989 Nov;109(5):2267–2274. doi: 10.1083/jcb.109.5.2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. May G. S., Tsang M. L., Smith H., Fidel S., Morris N. R. Aspergillus nidulans beta-tubulin genes are unusually divergent. Gene. 1987;55(2-3):231–243. doi: 10.1016/0378-1119(87)90283-6. [DOI] [PubMed] [Google Scholar]
  29. Michiels F., Falkenburg D., Müller A. M., Hinz U., Otto U., Bellmann R., Glätzer K. H., Brand R., Bialojan S., Renkawitz-Pohl R. Testis-specific beta 2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous beta 1 tubulin. Chromosoma. 1987;95(6):387–395. doi: 10.1007/BF00333989. [DOI] [PubMed] [Google Scholar]
  30. Michiels F., Gasch A., Kaltschmidt B., Renkawitz-Pohl R. A 14 bp promoter element directs the testis specificity of the Drosophila beta 2 tubulin gene. EMBO J. 1989 May;8(5):1559–1565. doi: 10.1002/j.1460-2075.1989.tb03540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Monteiro M. J., Cleveland D. W. Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes. J Mol Biol. 1988 Feb 5;199(3):439–446. doi: 10.1016/0022-2836(88)90616-x. [DOI] [PubMed] [Google Scholar]
  32. Natzle J. E., McCarthy B. J. Regulation of Drosophila alpha- and beta-tubulin genes during development. Dev Biol. 1984 Jul;104(1):187–198. doi: 10.1016/0012-1606(84)90047-2. [DOI] [PubMed] [Google Scholar]
  33. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  34. Paul E. C., Roobol A., Foster K. E., Gull K. Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum. Cell Motil Cytoskeleton. 1987;7(3):272–281. doi: 10.1002/cm.970070309. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Raff E. C., Fuller M. T., Kaufman T. C., Kemphues K. J., Rudolph J. E., Raff R. A. Regulation of tubulin gene expression during embryogenesis in Drosophila melanogaster. Cell. 1982 Jan;28(1):33–40. doi: 10.1016/0092-8674(82)90372-5. [DOI] [PubMed] [Google Scholar]
  37. Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rudolph J. E., Kimble M., Hoyle H. D., Subler M. A., Raff E. C. Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function. Mol Cell Biol. 1987 Jun;7(6):2231–2242. doi: 10.1128/mcb.7.6.2231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sackett D. L., Wolff J. Proteolysis of tubulin and the substructure of the tubulin dimer. J Biol Chem. 1986 Jul 5;261(19):9070–9076. [PubMed] [Google Scholar]
  40. 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]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Serrano L., Montejo de Garcini E., Hernández M. A., Avila J. Localization of the tubulin binding site for tau protein. Eur J Biochem. 1985 Dec 16;153(3):595–600. doi: 10.1111/j.1432-1033.1985.tb09342.x. [DOI] [PubMed] [Google Scholar]
  44. Spradling A. C., Rubin G. M. Transposition of cloned P elements into Drosophila germ line chromosomes. Science. 1982 Oct 22;218(4570):341–347. doi: 10.1126/science.6289435. [DOI] [PubMed] [Google Scholar]
  45. Steller H., Pirrotta V. A transposable P vector that confers selectable G418 resistance to Drosophila larvae. EMBO J. 1985 Jan;4(1):167–171. doi: 10.1002/j.1460-2075.1985.tb02332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Steller H., Pirrotta V. P transposons controlled by the heat shock promoter. Mol Cell Biol. 1986 May;6(5):1640–1649. doi: 10.1128/mcb.6.5.1640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Vale R. D., Hotani H. Formation of membrane networks in vitro by kinesin-driven microtubule movement. J Cell Biol. 1988 Dec;107(6 Pt 1):2233–2241. doi: 10.1083/jcb.107.6.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Wyss C., Bachmann G. Influence of amino acids, mammalian serum, and osmotic pressure on the proliferation of Drosophila cell lines. J Insect Physiol. 1976;22(12):1581–1586. doi: 10.1016/0022-1910(76)90049-4. [DOI] [PubMed] [Google Scholar]
  51. Youngblom J., Schloss J. A., Silflow C. D. The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins. Mol Cell Biol. 1984 Dec;4(12):2686–2696. doi: 10.1128/mcb.4.12.2686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES