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J Cell Biol. 1994 May 1; 125(3): 531–545.
PMCID: PMC2119987

CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase


The human autoantigen CENP-C has been demonstrated by immunoelectron microscopy to be a component of the inner kinetochore plate. Here we have used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti- CENP-C antibodies during interphase causes a transient arrest at the following metaphase. Injection of the same antibodies after the initiation of prophase, however, does not disrupt mitosis. Correspondingly, indirect immunofluorescence using affinity-purified human anti-CENP-C antibodies reveals that levels of CENP-C staining are reduced at centromeres in cells that were injected during interphase, but appear unaffected in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-arrested cells by electron microscopy reveals that the number of trilaminar structures is reduced. More surprisingly, the few remaining kinetochores in these cells retain a normal trilaminar morphology but are significantly reduced in diameter. In cells arrested for extended periods, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involvement of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetochore size and stabilizing microtubule attachments. These findings also support the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.

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Selected References

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  • Bernat RL, Borisy GG, Rothfield NF, Earnshaw WC. Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis. J Cell Biol. 1990 Oct;111(4):1519–1533. [PMC free article] [PubMed]
  • Bernat RL, Delannoy MR, Rothfield NF, Earnshaw WC. Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis. Cell. 1991 Sep 20;66(6):1229–1238. [PubMed]
  • Bloom K. The centromere frontier: kinetochore components, microtubule-based motility, and the CEN-value paradox. Cell. 1993 May 21;73(4):621–624. [PubMed]
  • Brinkley BR, Zinkowski RP, Mollon WL, Davis FM, Pisegna MA, Pershouse M, Rao PN. Movement and segregation of kinetochores experimentally detached from mammalian chromosomes. Nature. 1988 Nov 17;336(6196):251–254. [PubMed]
  • Chikashige Y, Kinoshita N, Nakaseko Y, Matsumoto T, Murakami S, Niwa O, Yanagida M. Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites. Cell. 1989 Jun 2;57(5):739–751. [PubMed]
  • Clarke L, Baum MP. 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. [PMC free article] [PubMed]
  • Comings DE, Okada TA. Fine structure of kinetochore in Indian muntjac. Exp Cell Res. 1971 Jul;67(1):97–110. [PubMed]
  • Compton DA, Yen TJ, Cleveland DW. Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds. J Cell Biol. 1991 Mar;112(6):1083–1097. [PMC free article] [PubMed]
  • Compton DA, Szilak I, Cleveland DW. Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis. J Cell Biol. 1992 Mar;116(6):1395–1408. [PMC free article] [PubMed]
  • Cooke CA, Bernat RL, Earnshaw WC. CENP-B: a major human centromere protein located beneath the kinetochore. J Cell Biol. 1990 May;110(5):1475–1488. [PMC free article] [PubMed]
  • Cooke CA, Bazett-Jones DP, Earnshaw WC, Rattner JB. Mapping DNA within the mammalian kinetochore. J Cell Biol. 1993 Mar;120(5):1083–1091. [PMC free article] [PubMed]
  • Doheny KF, Sorger PK, Hyman AA, Tugendreich S, Spencer F, Hieter P. Identification of essential components of the S. cerevisiae kinetochore. Cell. 1993 May 21;73(4):761–774. [PubMed]
  • Earnshaw WC, Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma. 1985;91(3-4):313–321. [PubMed]
  • Earnshaw WC, Ratrie H, 3rd, Stetten G. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma. 1989 Jun;98(1):1–12. [PubMed]
  • Goh PY, Kilmartin JV. NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae. J Cell Biol. 1993 May;121(3):503–512. [PMC free article] [PubMed]
  • Gorbsky GJ, Ricketts WA. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J Cell Biol. 1993 Sep;122(6):1311–1321. [PMC free article] [PubMed]
  • Guldner HH, Lakomek HJ, Bautz FA. Human anti-centromere sera recognise a 19.5 kD non-histone chromosomal protein from HeLa cells. Clin Exp Immunol. 1984 Oct;58(1):13–20. [PubMed]
  • Haaf T, Warburton PE, Willard HF. Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation. Cell. 1992 Aug 21;70(4):681–696. [PubMed]
  • Hildebrandt S, Weiner E, Senécal JL, Noell S, Daniels L, Earnshaw WC, Rothfield NF. The IgG, IgM, and IgA isotypes of anti-topoisomerase I and anticentromere autoantibodies. Arthritis Rheum. 1990 May;33(5):724–727. [PubMed]
  • Hoyt MA, Totis L, Roberts BT. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell. 1991 Aug 9;66(3):507–517. [PubMed]
  • Hyman AA, Middleton K, Centola M, Mitchison TJ, Carbon J. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature. 1992 Oct 8;359(6395):533–536. [PubMed]
  • Jiang W, Lechner J, Carbon J. Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore. J Cell Biol. 1993 May;121(3):513–519. [PMC free article] [PubMed]
  • Jokelainen PT. The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J Ultrastruct Res. 1967 Jul;19(1):19–44. [PubMed]
  • Kingsbury J, Koshland D. Centromere-dependent binding of yeast minichromosomes to microtubules in vitro. Cell. 1991 Aug 9;66(3):483–495. [PubMed]
  • Kingwell B, Rattner JB. Mammalian kinetochore/centromere composition: a 50 kDa antigen is present in the mammalian kinetochore/centromere. Chromosoma. 1987;95(6):403–407. [PubMed]
  • Lechner J, Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. [PubMed]
  • Li R, Murray AW. Feedback control of mitosis in budding yeast. Cell. 1991 Aug 9;66(3):519–531. [PubMed]
  • Lydersen BK, Pettijohn DE. Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980 Nov;22(2 Pt 2):489–499. [PubMed]
  • Madara PJ, Banghart LR, Jack LJ, Neira LM, Mather IH. Affinity purification of polyclonal antibodies from antigen immobilized in situ in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1990 Jun;187(2):246–250. [PubMed]
  • 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. [PMC free article] [PubMed]
  • McClintock B. The Production of Homozygous Deficient Tissues with Mutant Characteristics by Means of the Aberrant Mitotic Behavior of Ring-Shaped Chromosomes. Genetics. 1938 Jul;23(4):315–376. [PubMed]
  • McEwen BF, Arena JT, Frank J, Rieder CL. Structure of the colcemid-treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic tomography. J Cell Biol. 1993 Jan;120(2):301–312. [PMC free article] [PubMed]
  • Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1627–1631. [PubMed]
  • Nakaseko Y, Adachi Y, Funahashi S, Niwa O, Yanagida M. Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast. EMBO J. 1986 May;5(5):1011–1021. [PubMed]
  • Palmer DK, O'Day K, Wener MH, Andrews BS, Margolis RL. A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones. J Cell Biol. 1987 Apr;104(4):805–815. [PMC free article] [PubMed]
  • Palmer DK, O'Day K, Trong HL, Charbonneau H, Margolis RL. Purification of the centromere-specific protein CENP-A and demonstration that it is a distinctive histone. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3734–3738. [PubMed]
  • Rattner JB. The organization of the mammalian kinetochore: a scanning electron microscope study. Chromosoma. 1987;95(3):175–181. [PubMed]
  • Rieder CL, Alexander SP. The attachment of chromosomes to the mitotic spindle and the production of aneuploidy in newt lung cells. Prog Clin Biol Res. 1989;318:185–194. [PubMed]
  • Ris H, Witt PL. Structure of the mammalian kinetochore. Chromosoma. 1981;82(2):153–170. [PubMed]
  • Saitoh H, Tomkiel J, Cooke CA, Ratrie H, 3rd, Maurer M, Rothfield NF, Earnshaw WC. CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell. 1992 Jul 10;70(1):115–125. [PubMed]
  • SEARS ER. Misdivision of univalents in common wheat. Chromosoma. 1952;4(6):535–550. [PubMed]
  • Simerly C, Balczon R, Brinkley BR, Schatten G. Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes. J Cell Biol. 1990 Oct;111(4):1491–1504. [PMC free article] [PubMed]
  • Spencer F, Hieter P. Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8908–8912. [PubMed]
  • Tousson A, Zeng C, Brinkley BR, Valdivia MM. Centrophilin: a novel mitotic spindle protein involved in microtubule nucleation. J Cell Biol. 1991 Feb;112(3):427–440. [PMC free article] [PubMed]
  • Witt PL, Ris H, Borisy GG. Origin of kinetochore microtubules in Chinese hamster ovary cells. Chromosoma. 1980;81(3):483–505. [PubMed]
  • Yang CH, Snyder M. The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus. Mol Biol Cell. 1992 Nov;3(11):1259–1267. [PMC free article] [PubMed]
  • Yang CH, Lambie EJ, Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol. 1992 Mar;116(6):1303–1317. [PMC free article] [PubMed]
  • Yen TJ, Compton DA, Wise D, Zinkowski RP, Brinkley BR, Earnshaw WC, Cleveland DW. CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J. 1991 May;10(5):1245–1254. [PubMed]
  • Yen TJ, Li G, Schaar BT, Szilak I, Cleveland DW. CENP-E is a putative kinetochore motor that accumulates just before mitosis. Nature. 1992 Oct 8;359(6395):536–539. [PubMed]
  • Zinkowski RP, Meyne J, Brinkley BR. The centromere-kinetochore complex: a repeat subunit model. J Cell Biol. 1991 Jun;113(5):1091–1110. [PMC free article] [PubMed]
  • Zirkle RE. Ultraviolet-microbeam irradiation of newt-cell cytoplasm: spindle destruction, false anaphase, and delay of true anaphase. Radiat Res. 1970 Mar;41(3):516–537. [PubMed]

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