The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions

Abstract

Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: CID is localized to the inner kinetochore and the functional centromere.
Figure 2: Affinity-purified chicken anti-CID binds centromeres at all stages of the cell cycle in vivo, and induces several mitotic and cell-cycle defects.
Figure 3: CID RNAi results in several mitotic phenotypes in tissue culture cells.
Figure 4: The centromere region comprises several, spatially separable domains.
Figure 5: CID localization is unaffected by mutations in other centromere components and proteins involved in heterochromatin structure.
Figure 6: CID disruption results in mislocalization of transient kinetochore components and a sister cohesion protein.
Figure 7: CID RNAi results in mislocalization of many transient kinetochore components and a sister cohesion protein.
Figure 8: Structural and functional relationships within the Drosophila centromere region.

References

  1. 1

    Dobie, K. W., Hari, K. L., Maggert, K. A. & Karpen, G. H. Centromere proteins and chromosome inheritance: a complex affair. Curr. Opin. Genet. Dev. 9, 206–217 (1999).

  2. 2

    Palmer, D. K., O'Day, K., Trong, H. L., Charbonneau, H. & Margolis, R. L. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc. Natl Acad. Sci. USA 88, 3734–3738 (1991).

  3. 3

    Meluh, P. B., Yang, P., Glowczewski, L., Koshland, D. & Smith, M. M. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell 94, 607–613 (1998).

  4. 4

    Takahashi, K., Chen, E. S. & Yanagida, M. Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. Science 288, 2215–2219 (2000).

  5. 5

    Buchwitz, B. J., Ahmad, K., Moore, L. L., Roth, M. B. & Henikoff, S. A histone-H3-like protein in C. elegans. Nature 401, 547–548 (1999).

  6. 6

    Henikoff, S., Ahmad, K., Platero, J. S. & van Steensel, B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc. Natl Acad. Sci. USA 97, 716–721 (2000).

  7. 7

    Warburton, P. E. et al. Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr. Biol. 7, 901–904 (1997).

  8. 8

    Howman, E. V. et al. Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Proc. Natl Acad. Sci. USA 97, 1148–1153 (2000).

  9. 9

    Karpen, G. H. & Allshire, R. C. The case for epigenetic effects on centromere identity and function. Trends Genet. 13, 489–496 (1997).

  10. 10

    Torok, T., Harvie, P. D., Buratovich, M. & Bryant, P. J. The product of proliferation disrupter is concentrated at centromeres and required for mitotic chromosome condensation and cell proliferation in Drosophila. Genes Dev. 11, 213–225 (1997).

  11. 11

    Kerrebrock, A. W., Moore, D. P., Wu, J. S. & Orr-Weaver, T. L. Mei-S332, a Drosophila protein required for sister-chromatid cohesion, can localize to meiotic centromere regions. Cell 83, 247–256 (1995).

  12. 12

    Sunkel, C. E. & Glover, D. M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J. Cell Sci. 89, 25–38 (1988).

  13. 13

    Fanti, L., Giovinazzo, G., Berloco, M. & Pimpinelli, S. The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol. Cell 2, 527–538 (1998).

  14. 14

    Basu, J. et al. Mutations in the essential spindle checkpoint gene bub1 cause chromosome missegregation and fail to block apoptosis in Drosophila. J. Cell Biol. 146, 13–28 (1999).

  15. 15

    Williams, B. C., Murphy, T. D., Goldberg, M. L. & Karpen, G. H. Neocentromere activity of structurally acentric mini-chromosomes in Drosophila. Nature Genet. 18, 30–37 (1998).

  16. 16

    Scaerou, F. et al. The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila. J. Cell Sci. 112, 3757–3768 (1999).

  17. 17

    Wordeman, L., Steuer, E. R., Sheetz, M. P. & Mitchison, T. Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes. J. Cell Biol. 114, 285–294 (1991).

  18. 18

    Cooke, C. A., Schaar, B., Yen, T. J. & Earnshaw, W. C. Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase. Chromosoma 106, 446–455 (1997).

  19. 19

    Jablonski, S. A., Chan, G. K., Cooke, C. A., Earnshaw, W. C. & Yen, T. J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis. Chromosoma 107, 386–396 (1998).

  20. 20

    Starr, D. A., Williams, B. C., Hays, T. S. & Goldberg, M. L. ZW10 helps recruit dynactin and dynein to the kinetochore. J. Cell Biol. 142, 763–774 (1998).

  21. 21

    Murphy, T. D. & Karpen, G. H. Localization of centromere function in a Drosophila minichromosome. Cell 82, 599–609 (1995).

  22. 22

    Sun, X., Wahlstrom, J. & Karpen, G. Molecular structure of a functional Drosophila centromere. Cell 91, 1007–1019 (1997).

  23. 23

    Clarkson, M. & Saint, R. A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol. 18, 457–462 (1999).

  24. 24

    LeBlanc, H. N., Tang, T. T., Wu, J. S. & Orr-Weaver, T. L. The mitotic centromeric protein MEI-S332 and its role in sister- chromatid cohesion. Chromosoma 108, 401–411 (1999).

  25. 25

    Lopez, J., Karpen, G. & Orr-Weaver, T. Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere. Curr. Biol. 10, 997–1000 (2000).

  26. 26

    Platero, J. S., Csink, A. K., Quintanilla, A. & Henikoff, S. Changes in chromosomal localization of heterochromatin-binding proteins during the cell cycle in Drosophila. J. Cell Biol. 140, 1297–1306 (1998).

  27. 27

    Kellum, R. & Alberts, B. M. Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos. J. Cell Sci. 108, 1419–1431 (1995).

  28. 28

    Saffery, R. et al. Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins. Hum. Mol. Genet. 9, 175–185 (2000).

  29. 29

    Ekwall, K. et al. The chromodomain protein Swi6: a key component at fission yeast centromeres. Science 269, 1429–1431 (1995).

  30. 30

    Yucel, J. K. et al. CENP-meta, an essential kinetochore kinesin required for the maintenance of metaphase chromosome alignment in Drosophila. J. Cell Biol. 150, 1–11 (2000).

  31. 31

    Figueroa, J., Saffrich, R., Ansorge, W. & Valdivia, M. Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis. Chromosoma 107, 397–405 (1998).

  32. 32

    Zinkowski, R. P., Meyne, J. & Brinkley, B. R. The centromere–kinetochore complex: a repeat subunit model. J. Cell Biol. 113, 1091–1110 (1991).

  33. 33

    Adams, R. R. et al. INCENP binds the aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr. Biol. 10, 1075–1078 (2000).

  34. 34

    Karaiskou, A., Jessus, C., Brassac, T. & Ozon, R. Phosphatase 2A and polo kinase, two antagonistic regulators of cdc25 activation and MPF auto-amplification. J. Cell Sci. 112, 3747–3756. (1999).

  35. 35

    Pluta, A. F., Cooke, C. A. & Earnshaw, W. C. Structure of the human centromere at metaphase. Trends Biochem Sci. 15, 181–185 (1990).

  36. 36

    Partridge, J. F., Borgstrom, B. & Allshire, R. C. Distinct protein interaction domains and protein spreading in a complex centromere. Genes Dev. 14, 783–791 (2000).

  37. 37

    Hahnenberger, K. M., Carbon, J. & Clarke, L. Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I. Mol. Cell Biol. 11, 2206–2215 (1991).

  38. 38

    Rieder, C. L. & Cole, R. Chromatid cohesion during mitosis: lessons from meiosis. J. Cell Sci. 112, 2607–2613 (1999).

  39. 39

    Tanaka, T., Cosma, M. P., Wirth, K. & Nasmyth, K. Identification of cohesin association sites at centromeres and along chromosome arms. Cell 98, 847–858 (1999).

  40. 40

    Blat, Y. & Kleckner, N. Cohesins bind to preferential sites along yeast chromosome III, with differential regulation along arms versus the centric region. Cell 98, 249–259 (1999).

  41. 41

    Dernburg, A. F., Sedat, J. W. & Hawley, R. S. Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell 86, 135–146 (1996).

  42. 42

    Karpen, G. H., Le, M. H. & Le, H. Centric heterochromatin and the efficiency of achiasmate disjunction in Drosophila female meiosis. Science 273, 118–122 (1996).

  43. 43

    Murphy, T. D. & Karpen, G. H. Interactions between the nod+ kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome. Cell 81, 139–148 (1995).

  44. 44

    Shelby, R. D., Monier, K. & Sullivan, K. F. Chromatin assembly at kinetochores is uncoupled from DNA replication. J. Cell Biol. 151, 1113–1118 (2000).

  45. 45

    Casso, D., Ramirez-Weber, F. A. & Kornberg, T. B. GFP-tagged balancer chromosomes for Drosophila melanogaster. Mech. Dev. 88, 229–232 (1999); erratum ibid. 91, 451–454 (2000).

  46. 46

    Sharp, D. J. et al. Functional coordination of three mitotic motors in Drosophila embryos. Mol. Biol. Cell 11, 241–253 (2000).

  47. 47

    Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000).

  48. 48

    Bonaccorsi, S., Giansanti, M. G. & Gatti, M. Spindle assembly in Drosophila neuroblasts and ganglion mother cells. Nature Cell Biol. 2, 54–56 (2000).

  49. 49

    Theurkauf, W. E. Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. Methods Cell Biol 44, 489–505 (1994).

  50. 50

    Tang, T. T. L., Bickel, S. E., Young, L. M. & Orr-Weaver, T. L. Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein. Genes Dev. 12, 3843–3856 (1998).

  51. 51

    Llamazares, S. et al. polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 5, 2153–2165 (1991).

  52. 52

    Kellum, R., Raff, J. W. & Alberts, B. M. Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos. J. Cell Sci. 108, 1407–1418 (1995).

  53. 53

    Sullivan, B. A & Karpen, G. H. Centromere identity in Drosophila is not determiend in vivo by replication timing. J. Cell. Biol. (in the press).

  54. 54

    LeBlanc, H. N., Tang, T. T., Wu, J. S. & Orr-Weaver, T. L. The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion. Chromosoma 108, 401–411 (1999).

Download references

Acknowledgements

The authors thank K. Hari, J. Cordeiro and W. Sullivan for help with antibody injection, and K. Maggert, K. Sullivan and members of the Karpen lab for guidance and critical comments on the manuscript. We are grateful to B. Sullivan, B. Sullivan, S. Henikoff, C. Sunkel, T. Orr-Weaver, T. Torok, R. Karess and B. Williams for reagents, and M. Baker and The Salk Institute Sequencing Facility for analyses. M.B. is supported by an CMG training grant, and this project was funded by a grant from the NIH.

Author information

Correspondence to Gary H. Karpen.

Supplementary information

Supplementary figure

Figure S1 Structures and transmission frequencies of minichromosome derivatives used in these studies. (PDF 26 kb)

Supplementary movie

Movie 1 Anti-CID TMR: embryo injected with tetramethyl-rhodamine labelled anti-CID. Interphase injection: the antibody does not enter the nucleus until nuclear envelope breakdown at mitosis. The antibody specifically binds centromeres during all stages of the cell cycle, and binds in a gradient with decreasing antibody bound with increasing distance from the site of injection. (MOV 749 kb)

Supplementary movie

Movie 2 Anti-CID injection: embryo injected with anti-CID show a series of phenotypes. Embryos injected in interphase show phenotypes two cycles after injection. (MOV 2823 kb)

Supplementary movie

Movie 3 Anti-CID control: embryos injected with heat-killed anti-CID show few mitotic defects. (MOV 1247 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading