The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells (original) (raw)

Abstract

We have followed the behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. We show that the fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. We show that the anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C.

Full Text

The Full Text of this article is available as a PDF (454.8 KB).

Selected References

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

  1. Adams R. R., Tavares A. A., Salzberg A., Bellen H. J., Glover D. M. pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 1998 May 15;12(10):1483–1494. doi: 10.1101/gad.12.10.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alfa C. E., Ducommun B., Beach D., Hyams J. S. Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast. Nature. 1990 Oct 18;347(6294):680–682. doi: 10.1038/347680a0. [DOI] [PubMed] [Google Scholar]
  3. Brand A. GFP in Drosophila. Trends Genet. 1995 Aug;11(8):324–325. doi: 10.1016/s0168-9525(00)89091-5. [DOI] [PubMed] [Google Scholar]
  4. Charles J. F., Jaspersen S. L., Tinker-Kulberg R. L., Hwang L., Szidon A., Morgan D. O. The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr Biol. 1998 Apr 23;8(9):497–507. doi: 10.1016/s0960-9822(98)70201-5. [DOI] [PubMed] [Google Scholar]
  5. Cohen-Fix O., Peters J. M., Kirschner M. W., Koshland D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 1996 Dec 15;10(24):3081–3093. doi: 10.1101/gad.10.24.3081. [DOI] [PubMed] [Google Scholar]
  6. Dawson I. A., Roth S., Artavanis-Tsakonas S. The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae. J Cell Biol. 1995 May;129(3):725–737. doi: 10.1083/jcb.129.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Debec A., Montmory C. Cyclin B is associated with centrosomes in Drosophila mitotic cells. Biol Cell. 1992;75(2):121–126. doi: 10.1016/0248-4900(92)90131-j. [DOI] [PubMed] [Google Scholar]
  8. Descombes P., Nigg E. A. The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J. 1998 Mar 2;17(5):1328–1335. doi: 10.1093/emboj/17.5.1328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edgar B. A., Sprenger F., Duronio R. J., Leopold P., O'Farrell P. H. Distinct molecular mechanism regulate cell cycle timing at successive stages of Drosophila embryogenesis. Genes Dev. 1994 Feb 15;8(4):440–452. doi: 10.1101/gad.8.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fang G., Yu H., Kirschner M. W. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev. 1998 Jun 15;12(12):1871–1883. doi: 10.1101/gad.12.12.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foe V. E. Mitotic domains reveal early commitment of cells in Drosophila embryos. Development. 1989 Sep;107(1):1–22. [PubMed] [Google Scholar]
  12. Funabiki H., Kumada K., Yanagida M. Fission yeast Cut1 and Cut2 are essential for sister chromatid separation, concentrate along the metaphase spindle and form large complexes. EMBO J. 1996 Dec 2;15(23):6617–6628. [PMC free article] [PubMed] [Google Scholar]
  13. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  14. Glover D. M., Hagan I. M., Tavares A. A. Polo-like kinases: a team that plays throughout mitosis. Genes Dev. 1998 Dec 15;12(24):3777–3787. doi: 10.1101/gad.12.24.3777. [DOI] [PubMed] [Google Scholar]
  15. Golsteyn R. M., Lane H. A., Mundt K. E., Arnaud L., Nigg E. A. The family of polo-like kinases. Prog Cell Cycle Res. 1996;2:107–114. doi: 10.1007/978-1-4615-5873-6_11. [DOI] [PubMed] [Google Scholar]
  16. Golsteyn R. M., Mundt K. E., Fry A. M., Nigg E. A. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J Cell Biol. 1995 Jun;129(6):1617–1628. doi: 10.1083/jcb.129.6.1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorbsky G. J., Chen R. H., Murray A. W. Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase. J Cell Biol. 1998 Jun 1;141(5):1193–1205. doi: 10.1083/jcb.141.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hagting A., Karlsson C., Clute P., Jackman M., Pines J. MPF localization is controlled by nuclear export. EMBO J. 1998 Jul 15;17(14):4127–4138. doi: 10.1093/emboj/17.14.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Harper J. W., Elledge S. J. Cdk inhibitors in development and cancer. Curr Opin Genet Dev. 1996 Feb;6(1):56–64. doi: 10.1016/s0959-437x(96)90011-8. [DOI] [PubMed] [Google Scholar]
  20. Hazelrigg T., Liu N., Hong Y., Wang S. GFP expression in Drosophila tissues: time requirements for formation of a fluorescent product. Dev Biol. 1998 Jul 15;199(2):245–249. doi: 10.1006/dbio.1998.8922. [DOI] [PubMed] [Google Scholar]
  21. Hershko A., Ganoth D., Sudakin V., Dahan A., Cohen L. H., Luca F. C., Ruderman J. V., Eytan E. Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2. J Biol Chem. 1994 Feb 18;269(7):4940–4946. [PubMed] [Google Scholar]
  22. Irniger S., Piatti S., Michaelis C., Nasmyth K. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell. 1995 Apr 21;81(2):269–278. doi: 10.1016/0092-8674(95)90337-2. [DOI] [PubMed] [Google Scholar]
  23. Jackman M., Firth M., Pines J. Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus. EMBO J. 1995 Apr 18;14(8):1646–1654. doi: 10.1002/j.1460-2075.1995.tb07153.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jacobs H. W., Knoblich J. A., Lehner C. F. Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B. Genes Dev. 1998 Dec 1;12(23):3741–3751. doi: 10.1101/gad.12.23.3741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Juang Y. L., Huang J., Peters J. M., McLaughlin M. E., Tai C. Y., Pellman D. APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle. Science. 1997 Feb 28;275(5304):1311–1314. doi: 10.1126/science.275.5304.1311. [DOI] [PubMed] [Google Scholar]
  26. Jörgensen P. M., Brundell E., Starborg M., Hög C. A subunit of the anaphase-promoting complex is a centromere-associated protein in mammalian cells. Mol Cell Biol. 1998 Jan;18(1):468–476. doi: 10.1128/mcb.18.1.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kallio M., Weinstein J., Daum J. R., Burke D. J., Gorbsky G. J. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J Cell Biol. 1998 Jun 15;141(6):1393–1406. doi: 10.1083/jcb.141.6.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Kidd D., Raff J. W. LK6, a short lived protein kinase in Drosophila that can associate with microtubules and centrosomes. J Cell Sci. 1997 Jan;110(Pt 2):209–219. doi: 10.1242/jcs.110.2.209. [DOI] [PubMed] [Google Scholar]
  30. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  31. King R. W., Peters J. M., Tugendreich S., Rolfe M., Hieter P., Kirschner M. W. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell. 1995 Apr 21;81(2):279–288. doi: 10.1016/0092-8674(95)90338-0. [DOI] [PubMed] [Google Scholar]
  32. Kotani S., Tugendreich S., Fujii M., Jorgensen P. M., Watanabe N., Hoog C., Hieter P., Todokoro K. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Mol Cell. 1998 Feb;1(3):371–380. doi: 10.1016/s1097-2765(00)80037-4. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Lee H. S., Simon J. A., Lis J. T. Structure and expression of ubiquitin genes of Drosophila melanogaster. Mol Cell Biol. 1988 Nov;8(11):4727–4735. doi: 10.1128/mcb.8.11.4727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee K. S., Yuan Y. L., Kuriyama R., Erikson R. L. Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1. Mol Cell Biol. 1995 Dec;15(12):7143–7151. doi: 10.1128/mcb.15.12.7143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lehner C. F., O'Farrell P. H. The roles of Drosophila cyclins A and B in mitotic control. Cell. 1990 May 4;61(3):535–547. doi: 10.1016/0092-8674(90)90535-m. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lew D. J., Kornbluth S. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 1996 Dec;8(6):795–804. doi: 10.1016/s0955-0674(96)80080-9. [DOI] [PubMed] [Google Scholar]
  38. Logarinho E., Sunkel C. E. The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. J Cell Sci. 1998 Oct;111(Pt 19):2897–2909. doi: 10.1242/jcs.111.19.2897. [DOI] [PubMed] [Google Scholar]
  39. Maldonado-Codina G., Glover D. M. Cyclins A and B associate with chromatin and the polar regions of spindles, respectively, and do not undergo complete degradation at anaphase in syncytial Drosophila embryos. J Cell Biol. 1992 Feb;116(4):967–976. doi: 10.1083/jcb.116.4.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mastronarde D. N., McDonald K. L., Ding R., McIntosh J. R. Interpolar spindle microtubules in PTK cells. J Cell Biol. 1993 Dec;123(6 Pt 1):1475–1489. doi: 10.1083/jcb.123.6.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Michaelis C., Ciosk R., Nasmyth K. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell. 1997 Oct 3;91(1):35–45. doi: 10.1016/s0092-8674(01)80007-6. [DOI] [PubMed] [Google Scholar]
  42. Micklem D. R., Dasgupta R., Elliott H., Gergely F., Davidson C., Brand A., González-Reyes A., St Johnston D. The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr Biol. 1997 Jul 1;7(7):468–478. doi: 10.1016/s0960-9822(06)00218-1. [DOI] [PubMed] [Google Scholar]
  43. Murray A. Cyclin ubiquitination: the destructive end of mitosis. Cell. 1995 Apr 21;81(2):149–152. doi: 10.1016/0092-8674(95)90322-4. [DOI] [PubMed] [Google Scholar]
  44. Pines J., Hunter T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol. 1991 Oct;115(1):1–17. doi: 10.1083/jcb.115.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Riabowol K., Draetta G., Brizuela L., Vandre D., Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 1989 May 5;57(3):393–401. doi: 10.1016/0092-8674(89)90914-8. [DOI] [PubMed] [Google Scholar]
  47. Rieder C. L., Khodjakov A., Paliulis L. V., Fortier T. M., Cole R. W., Sluder G. Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5107–5112. doi: 10.1073/pnas.94.10.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rieder C. L., Salmon E. D. The vertebrate cell kinetochore and its roles during mitosis. Trends Cell Biol. 1998 Aug;8(8):310–318. doi: 10.1016/s0962-8924(98)01299-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schuldt A. J., Adams J. H., Davidson C. M., Micklem D. R., Haseloff J., St Johnston D., Brand A. H. Miranda mediates asymmetric protein and RNA localization in the developing nervous system. Genes Dev. 1998 Jun 15;12(12):1847–1857. doi: 10.1101/gad.12.12.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shirayama M., Zachariae W., Ciosk R., Nasmyth K. The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J. 1998 Mar 2;17(5):1336–1349. doi: 10.1093/emboj/17.5.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sigrist S. J., Lehner C. F. Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell. 1997 Aug 22;90(4):671–681. doi: 10.1016/s0092-8674(00)80528-0. [DOI] [PubMed] [Google Scholar]
  52. Su T. T., Sprenger F., DiGregorio P. J., Campbell S. D., O'Farrell P. H. Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation. Genes Dev. 1998 May 15;12(10):1495–1503. doi: 10.1101/gad.12.10.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Theurkauf W. E., Hawley R. S. Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J Cell Biol. 1992 Mar;116(5):1167–1180. doi: 10.1083/jcb.116.5.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Townsley F. M., Ruderman J. V. Proteolytic ratchets that control progression through mitosis. Trends Cell Biol. 1998 Jun;8(6):238–244. doi: 10.1016/s0962-8924(98)01268-9. [DOI] [PubMed] [Google Scholar]
  56. Tugendreich S., Tomkiel J., Earnshaw W., Hieter P. CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition. Cell. 1995 Apr 21;81(2):261–268. doi: 10.1016/0092-8674(95)90336-4. [DOI] [PubMed] [Google Scholar]
  57. Wells W. A. The spindle-assembly checkpoint: aiming for a perfect mitosis, every time. Trends Cell Biol. 1996 Jun;6(6):228–234. doi: 10.1016/0962-8924(96)10018-0. [DOI] [PubMed] [Google Scholar]
  58. Wheatley S. P., Hinchcliffe E. H., Glotzer M., Hyman A. A., Sluder G., Wang Y. l. CDK1 inactivation regulates anaphase spindle dynamics and cytokinesis in vivo. J Cell Biol. 1997 Jul 28;138(2):385–393. doi: 10.1083/jcb.138.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Whitfield W. G., Gonzalez C., Maldonado-Codina G., Glover D. M. The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition. EMBO J. 1990 Aug;9(8):2563–2572. doi: 10.1002/j.1460-2075.1990.tb07437.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wianny F., Tavares A., Evans M. J., Glover D. M., Zernicka-Goetz M. Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation. Chromosoma. 1998 Dec;107(6-7):430–439. doi: 10.1007/s004120050327. [DOI] [PubMed] [Google Scholar]
  61. Yamada H., Kumada K., Yanagida M. Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast. J Cell Sci. 1997 Aug;110(Pt 15):1793–1804. doi: 10.1242/jcs.110.15.1793. [DOI] [PubMed] [Google Scholar]