Microtubule Architecture Specified by a β-Tubulin Isoform
10.1126/science.275.5296.70
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

In Drosophila melanogaster , a testis-specific β-tubulin (β2) is required for spermatogenesis. A sequence motif was identified in carboxyl termini of axonemal β-tubulins in diverse taxa. As a test of whether orthologous β-tubulins from different species are functionally equivalent, the moth Heliothis virescens β2 homolog was expressed in Drosophila testes. When coexpressed with β2, the moth isoform imposed the 16-protofilament structure characteristic of that found in the moth on the corresponding subset of Drosophila microtubules, which normally contain only 13-protofilament microtubules. Thus, the architecture of the microtubule cytoskeleton can be directed by a component β-tubulin.

Bibliography

Raff, E. C., Fackenthal, J. D., Hutchens, J. A., Hoyle, H. D., & Turner, F. R. (1997). Microtubule Architecture Specified by a β-Tubulin Isoform. Science, 275(5296), 70–73.

Authors 5
  1. Elizabeth C. Raff (first)
  2. James D. Fackenthal (additional)
  3. Jeffrey A. Hutchens (additional)
  4. Henry D. Hoyle (additional)
  5. F. Rudolf Turner (additional)
References 33 Referenced 119
  1. Sullivan K. F., Cleveland D. W., Proc. Natl. Acad. Sci. U.S.A. 83, 4327 (1986); (10.1073/pnas.83.12.4327) / Proc. Natl. Acad. Sci. U.S.A. by Sullivan K. F. (1986)
  2. Sullivan K. F. , Machlin P. S. , Ratrie H. , Cleveland D. W., J. Biol. Chem. 261, 13317 (1986); (10.1016/S0021-9258(18)69306-8) / J. Biol. Chem. by Sullivan K. F. (1986)
  3. Sullivan K. F., Annu. Rev. Cell Biol. 4, 687 (1988); (10.1146/annurev.cb.04.110188.003351) / Annu. Rev. Cell Biol. by Sullivan K. F. (1988)
  4. Burns R. G. and , Surridge C., FEBS Lett. 27, 1 (1990); (10.1016/0014-5793(90)80359-Q) / FEBS Lett. by Burns R. G. (1990)
  5. in Microtubules Hyams J. S. and Lloyd C. W. Eds. (Wiley New York 1994) pp. 3–31.
  6. Fackenthal J. D., Turner F. R., Raff E. C., Dev. Biol. 158, 213 (1993); (10.1006/dbio.1993.1180) / Dev. Biol. by Fackenthal J. D. (1993)
  7. Fackenthal J. D. , Hutchens J. A. , Turner F. R. , Raff E. C., Genetics 139, 267 (1995). (10.1093/genetics/139.1.267) / Genetics by Fackenthal J. D. (1995)
  8. Hoyle H. D., Raff E. C., J. Cell Biol. 111, 1009 (1990); (10.1083/jcb.111.3.1009) / J. Cell Biol. by Hoyle H. D. (1990)
  9. Hoyle H. D. , Hutchens J. A. , Turner F. R. , Raff E. C., Dev. Genet. 16, 148 (1995). (10.1002/dvg.1020160208) / Dev. Genet. by Hoyle H. D. (1995)
  10. Gasch A., Hinz U., Leiss D., Renkawitz-Pohl R., Mol. Gen. Genet. 211, 8 (1988); (10.1007/BF00338387) / Mol. Gen. Genet. by Gasch A. (1988)
  11. Kimble M. , Incardona J. P. , Raff E. C., Dev. Biol. 131, 415 (1989); (10.1016/S0012-1606(89)80014-4) / Dev. Biol. by Kimble M. (1989)
  12. Kimble M. , Dettman R. W. , Raff E. C., Genetics 126, 991 (1990); (10.1093/genetics/126.4.991) / Genetics by Kimble M. (1990)
  13. Dettman R. W. , Turner F. R. , Raff E. C., Dev. Biol. 177, 117 (1996). (10.1006/dbio.1996.0150) / Dev. Biol. by Dettman R. W. (1996)
  14. Davis M.-T. B., Miller S. G., Insect Biochem. 18, 389 (1988). (10.1016/0020-1790(88)90054-6) / Insect Biochem. by Davis M.-T. B. (1988)
  15. Silflow C. D., Protoplasma 164, 9 (1991). (10.1007/BF01320810) / Protoplasma by Silflow C. D. (1991)
  16. Raff E. C., in Microtubules, , Hyams J. S., Lloyd C. W., Eds. (Wiley, New York, 1994), pp. 85-109. / Microtubules by Raff E. C. (1994)
  17. Tates A. D., Cytodifferentiation During Spermatogenesis in Drosophila melanogaster: An Electron Microscope Study (Pasmans, The Hague, 1971); L. E. LaChance and G. Olstad, Ann. Entomol. Soc. Am.81, 292 (1988); J. D. Fackenthal, thesis, Indiana Univ. (1993). / Cytodifferentiation During Spermatogenesis in Drosophila melanogaster: An Electron Microscope Study by Tates A. D. (1971)
  18. Even when the synthesis rate was the same less Hvβtprotein accumulated than β2; the moth protein is thus less stable than the endogenous isoform. However testes of sterile males carrying multiple inserts of the Hvβt transgene accumulated Hvβt in amounts equivalent to those of β2 that we have shown to be sufficient to support microtubule function. Thus the functional deficit of the moth isoform is not attributable simply to its relative instability in Drosophila cells. The total failure of Hvβt to support microtubule function contrasts with ectopic expression of Drosophila β3 in the male germ cells; β3 can support wild-type function of one class of cytoplasmic microtubules even though it cannot assemble axonemes spindles or other microtubule arrays (3).
  19. In a given spermatid the threshold may be lower; we did not detect Hvβt in motile sperm in males in which the amount of Hvβt was sufficiently low as to allow fertility.
  20. Savage C., et al., Genes Dev. 3, 870 (1989). (10.1101/gad.3.6.870) / Genes Dev. by Savage C. (1989)
  21. Mogensen M. M., Tucker J. B., J. Cell Sci. 88, 95 (1987); (10.1242/jcs.88.1.95) / J. Cell Sci. by Mogensen M. M. (1987)
  22. ___, Stebbings H., J. Cell Biol. 108, 1445 (1989). (10.1083/jcb.108.4.1445) / J. Cell Biol. by Stebbings H. (1989)
  23. 10.1083/jcb.100.4.1185
  24. Vertebrate isoforms are designated by the organism followed by the isotype class to which the sequence shown belongs (1): class II major neuronal; class III minor neuronal; class IVb predominant testis; class VI hematopoetic. COOH-termini of isoforms of the same class in other species are identical or very similar to those shown. Widely expressed class I and V isotypes also lack the axoneme motif. β-Tubulins in other Drosophila species appear to be identical to those in D. melanogaster [(4); F. Michiels et al . Chromosoma 95 387 (1987)].
  25. Aspergillus nidulans has two β-tubulin genes; benA also lacks the axoneme motif.
  26. The Tetrahymena thermophila axoneme motif is identical to Paramecium. Giardia represents the deepest group in eukaryotic taxa and Chlamydomonas groups with plants [M. L. Sogin H. G. Morrison G. Hinkle J. D. Silberman Microbiol. Semin. 12 17 (1996)]. β-Tubulins in higher plants which do not have ciliated cells lack axoneme motifs.
  27. Diaz H. B., Conine M. A., Raff E. C., J. Cell Biol. 111, 412 (1990); / J. Cell Biol. by Diaz H. B. (1990)
  28. Diaz H. and Raff E. in preparation.
  29. To generate the Hvβt transgene we generated cloning sites 29 base pairs (bp) 5′ and 27 bp 3′ of the Hvβt coding sequence and the resulting fragment was inserted between 2.1 kb of the 5′ β2 genomic sequences and 1.5 kb of the 3′ β2 genomic sequences previously shown to be sufficient to drive expression of heterologous proteins in the postmitotic male germ cells with correct developmental specificity and at the same level of expression as wild-type β2 (2 3) (J. Hutchens H. Hoyle F. R. Turner E. C. Raff Mol. Biol. Cell in press). Intronless and intron-containing versions were constructed; in the latter an oligonucleotide matching the 59-bp β2 intron sequence was inserted between Hvβt codons 73 and 74. Transgenes were inserted into the CaSpeR vector [V. Pirrotta Biotechnology 10 437 (1988)] and introduced into the Drosophila genome by P element-mediated transformation (2 3). Multiple transgenic lines were obtained and testis tubulins analyzed on two-dimensional gels as described previously (2 3); the level of Hvβt expression depended on the site of insertion and presence of the intron. We obtained wild-type β2-like levels of expression (as in Fig. 2) only with an intron-containing insert suggesting that splicing may be important in normal β2 expression. All transgenic lines exhibited the same suite of defects in spermatogenesis; thus the phenotype is attributable solely to expression of the moth β-tubulin.
  30. Electron microscopy and tannic acid staining were done as previously described (2 3).
  31. The morphology of doublet microtubules and the central pair is the same in moths flies and transgenic flies. Doublets have a 13-pf A-tubule and a 10-pf shared-wall B-tubule; central pair microtubules are 13-pf. Accessory microtubules in fly axonemes are 13-pf but 16-pf in moth. Most accessory microtubules in transgenic males are 13-pf but the abnormal large-diameter accessory microtubules are 16-pf.
  32. Accessory microtubules begin as a projection of a protofilament sheet from the B-tubule of each doublet but completed accessory microtubules are no longer physically associated with the doublet. Completed accessory microtubules in immature axonemes of flies and moths were of a slightly larger diameter than in mature axonemes; thus adjacent protofilaments in the walls of the accessory microtubules appear to “tighten up” as they form.
  33. We thank M.-T. Davis and S. Miller for providing us with the H. virescens testis-specific β-tubulin cDNA clone; C.-S. Hong and M. Martin for their enthusiastic participation and contributions as undergraduate research students in the early parts of this study; and W. Saxton and R. Raff for critical reading of the manuscript. This work was supported by a grant from National Institute of Child Health and Human Development (of NIH) to E.C.R.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:45 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:36 p.m.)
Indexed 3 weeks, 3 days ago (Aug. 6, 2025, 8:05 a.m.)
Issued 28 years, 7 months ago (Jan. 3, 1997)
Published 28 years, 7 months ago (Jan. 3, 1997)
Published Print 28 years, 7 months ago (Jan. 3, 1997)
Funders 0

None

@article{Raff_1997, title={Microtubule Architecture Specified by a β-Tubulin Isoform}, volume={275}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.275.5296.70}, DOI={10.1126/science.275.5296.70}, number={5296}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Raff, Elizabeth C. and Fackenthal, James D. and Hutchens, Jeffrey A. and Hoyle, Henry D. and Turner, F. Rudolf}, year={1997}, month=jan, pages={70–73} }