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. 1991 Jun 1;113(5):1091–1110. doi: 10.1083/jcb.113.5.1091

The centromere-kinetochore complex: a repeat subunit model

PMCID: PMC2289018  PMID: 1828250

Abstract

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

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Selected References

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  1. Balczon R. D., Brinkley B. R. Tubulin interaction with kinetochore proteins: analysis by in vitro assembly and chemical cross-linking. J Cell Biol. 1987 Aug;105(2):855–862. doi: 10.1083/jcb.105.2.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloom K., Yeh E. Centromeres and telomeres: structural elements of eukaryotic chromosomes. Curr Opin Cell Biol. 1989 Jun;1(3):526–532. doi: 10.1016/0955-0674(89)90015-x. [DOI] [PubMed] [Google Scholar]
  3. Borland L., Harauz G., Bahr G., van Heel M. Packing of the 30 nm chromatin fiber in the human metaphase chromosome. Chromosoma. 1988;97(2):159–163. doi: 10.1007/BF00327373. [DOI] [PubMed] [Google Scholar]
  4. Brenner S., Pepper D., Berns M. W., Tan E., Brinkley B. R. Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients. J Cell Biol. 1981 Oct;91(1):95–102. doi: 10.1083/jcb.91.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brinkley B. R., Cox S. M., Pepper D. A. Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro. Cytogenet Cell Genet. 1980;26(2-4):165–174. doi: 10.1159/000131438. [DOI] [PubMed] [Google Scholar]
  6. Brinkley B. R., Stubblefield E. The fine structure of the kinetochore of a mammalian cell in vitro. Chromosoma. 1966;19(1):28–43. doi: 10.1007/BF00332792. [DOI] [PubMed] [Google Scholar]
  7. Brinkley B. R., Tousson A., Valdivia M. M. The kinetochore of mammalian chromosomes: structure and function in normal mitosis and aneuploidy. Basic Life Sci. 1985;36:243–267. doi: 10.1007/978-1-4613-2127-9_16. [DOI] [PubMed] [Google Scholar]
  8. Brinkley B. R., Valdivia M. M., Tousson A., Brenner S. L. Compound kinetochores of the Indian muntjac. Evolution by linear fusion of unit kinetochores. Chromosoma. 1984;91(1):1–11. doi: 10.1007/BF00286479. [DOI] [PubMed] [Google Scholar]
  9. Brinkley B. R., Zinkowski R. P., Mollon W. L., Davis F. M., Pisegna M. A., Pershouse M., Rao P. N. Movement and segregation of kinetochores experimentally detached from mammalian chromosomes. Nature. 1988 Nov 17;336(6196):251–254. doi: 10.1038/336251a0. [DOI] [PubMed] [Google Scholar]
  10. Cherry L. M., Faulkner A. J., Grossberg L. A., Balczon R. Kinetochore size variation in mammalian chromosomes: an image analysis study with evolutionary implications. J Cell Sci. 1989 Feb;92(Pt 2):281–289. doi: 10.1242/jcs.92.2.281. [DOI] [PubMed] [Google Scholar]
  11. Clarke L., Baum M. P. Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences. Mol Cell Biol. 1990 May;10(5):1863–1872. doi: 10.1128/mcb.10.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarke L. Centromeres of budding and fission yeasts. Trends Genet. 1990 May;6(5):150–154. doi: 10.1016/0168-9525(90)90149-z. [DOI] [PubMed] [Google Scholar]
  13. Comings D. E., Okada T. A. Fine structure of kinetochore in Indian muntjac. Exp Cell Res. 1971 Jul;67(1):97–110. doi: 10.1016/0014-4827(71)90625-2. [DOI] [PubMed] [Google Scholar]
  14. Cooke C. A., Bernat R. L., Earnshaw W. C. CENP-B: a major human centromere protein located beneath the kinetochore. J Cell Biol. 1990 May;110(5):1475–1488. doi: 10.1083/jcb.110.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cooke C. A., Heck M. M., Earnshaw W. C. The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J Cell Biol. 1987 Nov;105(5):2053–2067. doi: 10.1083/jcb.105.5.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Earnshaw W. C., Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma. 1985;91(3-4):313–321. doi: 10.1007/BF00328227. [DOI] [PubMed] [Google Scholar]
  17. Fitzgerald-Hayes M. Yeast centromeres. Yeast. 1987 Sep;3(3):187–200. doi: 10.1002/yea.320030306. [DOI] [PubMed] [Google Scholar]
  18. Haaf T., Schmid M. Centromeric association and non-random distribution of centromeres in human tumour cells. Hum Genet. 1989 Jan;81(2):137–143. doi: 10.1007/BF00293889. [DOI] [PubMed] [Google Scholar]
  19. Hayden J. H., Bowser S. S., Rieder C. L. Kinetochores capture astral microtubules during chromosome attachment to the mitotic spindle: direct visualization in live newt lung cells. J Cell Biol. 1990 Sep;111(3):1039–1045. doi: 10.1083/jcb.111.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heath I. B. Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax. J Cell Biol. 1980 Mar;84(3):531–546. doi: 10.1083/jcb.84.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ishida R., Kozaki M., Takahashi T. Caffeine alone causes DNA damage in Chinese hamster ovary cells. Cell Struct Funct. 1985 Dec;10(4):405–409. doi: 10.1247/csf.10.405. [DOI] [PubMed] [Google Scholar]
  22. Masumoto H., Masukata H., Muro Y., Nozaki N., Okazaki T. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite. J Cell Biol. 1989 Nov;109(5):1963–1973. doi: 10.1083/jcb.109.5.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Masumoto H., Sugimoto K., Okazaki T. Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle. Exp Cell Res. 1989 Mar;181(1):181–196. doi: 10.1016/0014-4827(89)90192-4. [DOI] [PubMed] [Google Scholar]
  24. Meyne J., Baker R. J., Hobart H. H., Hsu T. C., Ryder O. A., Ward O. G., Wiley J. E., Wurster-Hill D. H., Yates T. L., Moyzis R. K. Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma. 1990 Apr;99(1):3–10. doi: 10.1007/BF01737283. [DOI] [PubMed] [Google Scholar]
  25. Meyne J., Littlefield L. G., Moyzis R. K. Labeling of human centromeres using an alphoid DNA consensus sequence: application to the scoring of chromosome aberrations. Mutat Res. 1989 Jun;226(2):75–79. doi: 10.1016/0165-7992(89)90046-8. [DOI] [PubMed] [Google Scholar]
  26. Meyne J., Ratliff R. L., Moyzis R. K. Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7049–7053. doi: 10.1073/pnas.86.18.7049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller J. M., Wang W., Balczon R., Dentler W. L. Ciliary microtubule capping structures contain a mammalian kinetochore antigen. J Cell Biol. 1990 Mar;110(3):703–714. doi: 10.1083/jcb.110.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J Cell Biol. 1985 Sep;101(3):755–765. doi: 10.1083/jcb.101.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Mole-Bajer J., Bajer A. S., Zinkowski R. P., Balczon R. D., Brinkley B. R. Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus. Proc Natl Acad Sci U S A. 1990 May;87(9):3599–3603. doi: 10.1073/pnas.87.9.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moroi Y., Peebles C., Fritzler M. J., Steigerwald J., Tan E. M. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1627–1631. doi: 10.1073/pnas.77.3.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nicklas R. B. The motor for poleward chromosome movement in anaphase is in or near the kinetochore. J Cell Biol. 1989 Nov;109(5):2245–2255. doi: 10.1083/jcb.109.5.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pepper D. A., Brinkley B. R. Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody. J Cell Biol. 1979 Aug;82(2):585–591. doi: 10.1083/jcb.82.2.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pepper D. A., Brinkley B. R. Tubulin nucleation and assembly in mitotic cells: evidence for nucleic acids in kinetochores and centrosomes. Cell Motil. 1980;1(1):1–15. doi: 10.1002/cm.970010102. [DOI] [PubMed] [Google Scholar]
  36. Peterson J. B., Ris H. Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae. J Cell Sci. 1976 Nov;22(2):219–242. doi: 10.1242/jcs.22.2.219. [DOI] [PubMed] [Google Scholar]
  37. Pfarr C. M., Coue M., Grissom P. M., Hays T. S., Porter M. E., McIntosh J. R. Cytoplasmic dynein is localized to kinetochores during mitosis. Nature. 1990 May 17;345(6272):263–265. doi: 10.1038/345263a0. [DOI] [PubMed] [Google Scholar]
  38. Rattner J. B., Bazett-Jones D. P. Kinetochore structure: electron spectroscopic imaging of the kinetochore. J Cell Biol. 1989 Apr;108(4):1209–1219. doi: 10.1083/jcb.108.4.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rattner J. B. Organization within the mammalian kinetochore. Chromosoma. 1986;93(6):515–520. doi: 10.1007/BF00386793. [DOI] [PubMed] [Google Scholar]
  40. Rattner J. B. The organization of the mammalian kinetochore: a scanning electron microscope study. Chromosoma. 1987;95(3):175–181. doi: 10.1007/BF00330348. [DOI] [PubMed] [Google Scholar]
  41. Rieder C. L., Alexander S. P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol. 1990 Jan;110(1):81–95. doi: 10.1083/jcb.110.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rieder C. L. Localization of ribonucleoprotein in the trilaminar kinetochore of PtK1. J Ultrastruct Res. 1979 Feb;66(2):109–119. doi: 10.1016/s0022-5320(79)90128-x. [DOI] [PubMed] [Google Scholar]
  43. Rieder C. L. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol. 1982;79:1–58. doi: 10.1016/s0074-7696(08)61672-1. [DOI] [PubMed] [Google Scholar]
  44. Ris H., Witt P. L. Structure of the mammalian kinetochore. Chromosoma. 1981;82(2):153–170. doi: 10.1007/BF00286101. [DOI] [PubMed] [Google Scholar]
  45. Roos U. P. Light and electron microscopy of rat kangaroo cells in mitosis. II. Kinetochore structure and function. Chromosoma. 1973;41(2):195–220. doi: 10.1007/BF00319696. [DOI] [PubMed] [Google Scholar]
  46. Sawecka J., Gołos B., Malec J. Modification by caffeine of acute cytotoxic response of cultured L5178Y cells to hydroxyurea treatment. Neoplasma. 1987;34(4):369–377. [PubMed] [Google Scholar]
  47. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  48. Schlegel R., Pardee A. B. Periodic mitotic events induced in the absence of DNA replication. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9025–9029. doi: 10.1073/pnas.84.24.9025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Steuer E. R., Wordeman L., Schroer T. A., Sheetz M. P. Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature. 1990 May 17;345(6272):266–268. doi: 10.1038/345266a0. [DOI] [PubMed] [Google Scholar]
  50. Valdivia M. M., Brinkley B. R. Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes. J Cell Biol. 1985 Sep;101(3):1124–1134. doi: 10.1083/jcb.101.3.1124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Valdivia M. M., Tousson A., Brinkley B. R. Human antibodies and their use for the study of chromosome organization. Methods Achiev Exp Pathol. 1986;12:200–223. [PubMed] [Google Scholar]
  52. Vig B. K., Athwal R. S. Sequence of centromere separation: separation in a quasi-stable mouse-human somatic cell hybrid. Chromosoma. 1989 Sep;98(3):167–173. doi: 10.1007/BF00329680. [DOI] [PubMed] [Google Scholar]
  53. Waye J. S., Willard H. F. Concerted evolution of alpha satellite DNA: evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates. Chromosoma. 1989 Oct;98(4):273–279. doi: 10.1007/BF00327313. [DOI] [PubMed] [Google Scholar]

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