Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Jan 1;124(1):19–31. doi: 10.1083/jcb.124.1.19

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

PMCID: PMC2119895  PMID: 8294501

Abstract

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.

Full Text

The Full Text of this article is available as a PDF (3.6 MB).

Selected References

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

  1. Bacallao R., Antony C., Dotti C., Karsenti E., Stelzer E. H., Simons K. The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J Cell Biol. 1989 Dec;109(6 Pt 1):2817–2832. doi: 10.1083/jcb.109.6.2817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailly E., Dorée M., Nurse P., Bornens M. p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. EMBO J. 1989 Dec 20;8(13):3985–3995. doi: 10.1002/j.1460-2075.1989.tb08581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blow J. J., Laskey R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
  4. Bornens M., Paintrand M., Berges J., Marty M. C., Karsenti E. Structural and chemical characterization of isolated centrosomes. Cell Motil Cytoskeleton. 1987;8(3):238–249. doi: 10.1002/cm.970080305. [DOI] [PubMed] [Google Scholar]
  5. Bré M. H., Karsenti E. Effects of brain microtubule-associated proteins on microtubule dynamics and the nucleating activity of centrosomes. Cell Motil Cytoskeleton. 1990;15(2):88–98. doi: 10.1002/cm.970150205. [DOI] [PubMed] [Google Scholar]
  6. Bré M. H., Pepperkok R., Hill A. M., Levilliers N., Ansorge W., Stelzer E. H., Karsenti E. Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells. J Cell Biol. 1990 Dec;111(6 Pt 2):3013–3021. doi: 10.1083/jcb.111.6.3013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buendia B., Antony C., Verde F., Bornens M., Karsenti E. A centrosomal antigen localized on intermediate filaments and mitotic spindle poles. J Cell Sci. 1990 Oct;97(Pt 2):259–271. doi: 10.1242/jcs.97.2.259. [DOI] [PubMed] [Google Scholar]
  8. Buendia B., Draetta G., Karsenti E. Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase. J Cell Biol. 1992 Mar;116(6):1431–1442. doi: 10.1083/jcb.116.6.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burns R. G. Alpha-, beta-, and gamma-tubulins: sequence comparisons and structural constraints. Cell Motil Cytoskeleton. 1991;20(3):181–189. doi: 10.1002/cm.970200302. [DOI] [PubMed] [Google Scholar]
  10. Calarco-Gillam P. D., Siebert M. C., Hubble R., Mitchison T., Kirschner M. Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell. 1983 Dec;35(3 Pt 2):621–629. doi: 10.1016/0092-8674(83)90094-6. [DOI] [PubMed] [Google Scholar]
  11. Carlier M. F., Pantaloni D. Kinetic analysis of cooperativity in tubulin polymerization in the presence of guanosine di- or triphosphate nucleotides. Biochemistry. 1978 May 16;17(10):1908–1915. doi: 10.1021/bi00603a017. [DOI] [PubMed] [Google Scholar]
  12. Centonze V. E., Borisy G. G. Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope. J Cell Sci. 1990 Mar;95(Pt 3):405–411. doi: 10.1242/jcs.95.3.405. [DOI] [PubMed] [Google Scholar]
  13. Davis F. M., Tsao T. Y., Fowler S. K., Rao P. N. Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A. 1983 May;80(10):2926–2930. doi: 10.1073/pnas.80.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Erickson H. P., Pantaloni D. The role of subunit entropy in cooperative assembly. Nucleation of microtubules and other two-dimensional polymers. Biophys J. 1981 May;34(2):293–309. doi: 10.1016/S0006-3495(81)84850-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Evans L., Mitchison T., Kirschner M. Influence of the centrosome on the structure of nucleated microtubules. J Cell Biol. 1985 Apr;100(4):1185–1191. doi: 10.1083/jcb.100.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Felix M. A., Pines J., Hunt T., Karsenti E. A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. EMBO J. 1989 Oct;8(10):3059–3069. doi: 10.1002/j.1460-2075.1989.tb08457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gard D. L., Hafezi S., Zhang T., Doxsey S. J. Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. J Cell Biol. 1990 Jun;110(6):2033–2042. doi: 10.1083/jcb.110.6.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gerhart J., Wu M., Kirschner M. Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J Cell Biol. 1984 Apr;98(4):1247–1255. doi: 10.1083/jcb.98.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gueth-Hallonet C., Antony C., Aghion J., Santa-Maria A., Lajoie-Mazenc I., Wright M., Maro B. gamma-Tubulin is present in acentriolar MTOCs during early mouse development. J Cell Sci. 1993 May;105(Pt 1):157–166. doi: 10.1242/jcs.105.1.157. [DOI] [PubMed] [Google Scholar]
  20. Horio T., Uzawa S., Jung M. K., Oakley B. R., Tanaka K., Yanagida M. The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. J Cell Sci. 1991 Aug;99(Pt 4):693–700. doi: 10.1242/jcs.99.4.693. [DOI] [PubMed] [Google Scholar]
  21. Hyman A., Drechsel D., Kellogg D., Salser S., Sawin K., Steffen P., Wordeman L., Mitchison T. Preparation of modified tubulins. Methods Enzymol. 1991;196:478–485. doi: 10.1016/0076-6879(91)96041-o. [DOI] [PubMed] [Google Scholar]
  22. Joshi H. C., Palacios M. J., McNamara L., Cleveland D. W. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature. 1992 Mar 5;356(6364):80–83. doi: 10.1038/356080a0. [DOI] [PubMed] [Google Scholar]
  23. Julian M., Tollon Y., Lajoie-Mazenc I., Moisand A., Mazarguil H., Puget A., Wright M. gamma-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. J Cell Sci. 1993 May;105(Pt 1):145–156. doi: 10.1242/jcs.105.1.145. [DOI] [PubMed] [Google Scholar]
  24. Kalt A., Schliwa M. Molecular components of the centrosome. Trends Cell Biol. 1993 Apr;3(4):118–128. doi: 10.1016/0962-8924(93)90174-y. [DOI] [PubMed] [Google Scholar]
  25. Keryer G., Rios R. M., Landmark B. F., Skalhegg B., Lohmann S. M., Bornens M. A high-affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II in the centrosome of human cells. Exp Cell Res. 1993 Feb;204(2):230–240. doi: 10.1006/excr.1993.1029. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Kuriyama R. Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro. J Cell Sci. 1984 Mar;66:277–295. doi: 10.1242/jcs.66.1.277. [DOI] [PubMed] [Google Scholar]
  28. Kuriyama R., Borisy G. G. Cytasters induced within unfertilized sea-urchin eggs. J Cell Sci. 1983 May;61:175–189. doi: 10.1242/jcs.61.1.175. [DOI] [PubMed] [Google Scholar]
  29. Kuriyama R., Kanatani H. The centriolar complex isolated from starfish spermatozoa. J Cell Sci. 1981 Jun;49:33–49. doi: 10.1242/jcs.49.1.33. [DOI] [PubMed] [Google Scholar]
  30. Leiss D., Félix M. A., Karsenti E. Association of cyclin-bound p34cdc2 with subcellular structures in xenopus eggs. J Cell Sci. 1992 Jun;102(Pt 2):285–297. doi: 10.1242/jcs.102.2.285. [DOI] [PubMed] [Google Scholar]
  31. Liu B., Marc J., Joshi H. C., Palevitz B. A. A gamma-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner. J Cell Sci. 1993 Apr;104(Pt 4):1217–1228. doi: 10.1242/jcs.104.4.1217. [DOI] [PubMed] [Google Scholar]
  32. Maller J., Poccia D., Nishioka D., Kidd P., Gerhart J., Hartman H. Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm. Exp Cell Res. 1976 May;99(2):285–294. doi: 10.1016/0014-4827(76)90585-1. [DOI] [PubMed] [Google Scholar]
  33. Manes M. E., Barbieri F. D. On the possibility of sperm aster involvement in dorso-ventral polarization and pronuclear migration in the amphibian egg. J Embryol Exp Morphol. 1977 Aug;40:187–197. [PubMed] [Google Scholar]
  34. Maro B., Howlett S. K., Webb M. Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. J Cell Biol. 1985 Nov;101(5 Pt 1):1665–1672. doi: 10.1083/jcb.101.5.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Masuda H., Sevik M., Cande W. Z. In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts. J Cell Biol. 1992 Jun;117(5):1055–1066. doi: 10.1083/jcb.117.5.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mitchison T., Kirschner M. Microtubule assembly nucleated by isolated centrosomes. Nature. 1984 Nov 15;312(5991):232–237. doi: 10.1038/312232a0. [DOI] [PubMed] [Google Scholar]
  37. Muresan V., Joshi H. C., Besharse J. C. Gamma-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia. J Cell Sci. 1993 Apr;104(Pt 4):1229–1237. doi: 10.1242/jcs.104.4.1229. [DOI] [PubMed] [Google Scholar]
  38. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell. 1982 Oct;30(3):687–696. doi: 10.1016/0092-8674(82)90273-2. [DOI] [PubMed] [Google Scholar]
  39. Oakley B. R. Gamma-tubulin: the microtubule organizer? Trends Cell Biol. 1992 Jan;2(1):1–5. doi: 10.1016/0962-8924(92)90125-7. [DOI] [PubMed] [Google Scholar]
  40. Oakley B. R., Oakley C. E., Yoon Y., Jung M. K. Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell. 1990 Jun 29;61(7):1289–1301. doi: 10.1016/0092-8674(90)90693-9. [DOI] [PubMed] [Google Scholar]
  41. Palacios M. J., Joshi H. C., Simerly C., Schatten G. Gamma-tubulin reorganization during mouse fertilization and early development. J Cell Sci. 1993 Feb;104(Pt 2):383–389. doi: 10.1242/jcs.104.2.383. [DOI] [PubMed] [Google Scholar]
  42. Price C. M., Pettijohn D. E. Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly. Properties of purified NuMA protein. Exp Cell Res. 1986 Oct;166(2):295–311. doi: 10.1016/0014-4827(86)90478-7. [DOI] [PubMed] [Google Scholar]
  43. Raff J. W., Kellogg D. R., Alberts B. M. Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs. J Cell Biol. 1993 May;121(4):823–835. doi: 10.1083/jcb.121.4.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sager P. R., Rothfield N. L., Oliver J. M., Berlin R. D. A novel mitotic spindle pole component that originates from the cytoplasm during prophase. J Cell Biol. 1986 Nov;103(5):1863–1872. doi: 10.1083/jcb.103.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stearns T., Evans L., Kirschner M. Gamma-tubulin is a highly conserved component of the centrosome. Cell. 1991 May 31;65(5):825–836. doi: 10.1016/0092-8674(91)90390-k. [DOI] [PubMed] [Google Scholar]
  47. Tucker J. B., Paton C. C., Richardson G. P., Mogensen M. M., Russell I. J. A cell surface-associated centrosomal layer of microtubule-organizing material in the inner pillar cell of the mouse cochlea. J Cell Sci. 1992 Jun;102(Pt 2):215–226. doi: 10.1242/jcs.102.2.215. [DOI] [PubMed] [Google Scholar]
  48. Ulloa L., Avila J., Díaz-Nido J. Heterogeneity in the phosphorylation of microtubule-associated protein MAP1B during rat brain development. J Neurochem. 1993 Sep;61(3):961–972. doi: 10.1111/j.1471-4159.1993.tb03609.x. [DOI] [PubMed] [Google Scholar]
  49. Ulloa L., Díaz-Nido J., Avila J. Depletion of casein kinase II by antisense oligonucleotide prevents neuritogenesis in neuroblastoma cells. EMBO J. 1993 Apr;12(4):1633–1640. doi: 10.1002/j.1460-2075.1993.tb05808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4439–4443. doi: 10.1073/pnas.81.14.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Verde F., Labbé J. C., Dorée M., Karsenti E. Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature. 1990 Jan 18;343(6255):233–238. doi: 10.1038/343233a0. [DOI] [PubMed] [Google Scholar]
  52. Verlhac M. H., de Pennart H., Maro B., Cobb M. H., Clarke H. J. MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev Biol. 1993 Aug;158(2):330–340. doi: 10.1006/dbio.1993.1192. [DOI] [PubMed] [Google Scholar]
  53. Voter W. A., Erickson H. P. The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J Biol Chem. 1984 Aug 25;259(16):10430–10438. [PubMed] [Google Scholar]
  54. Wade R. H., Chrétien D. Cryoelectron microscopy of microtubules. J Struct Biol. 1993 Jan-Feb;110(1):1–27. doi: 10.1006/jsbi.1993.1001. [DOI] [PubMed] [Google Scholar]
  55. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zheng Y., Jung M. K., Oakley B. R. Gamma-tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome. Cell. 1991 May 31;65(5):817–823. doi: 10.1016/0092-8674(91)90389-g. [DOI] [PubMed] [Google Scholar]

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

RESOURCES