Abstract
Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the ‘ball and cup’ structure that had been presented for ‘prekinetochore’. We propose here that CENP-C constitutes a ‘kinetochore organizing center’ tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation.
Similar content being viewed by others
References
Antonacci R, Rocchi M, Archidiacono N, Baldini A (1995) Ordered mapping of three alpha satellite DNA subsets on human chromosome 22. Chrom Res 3: 124-127.
Brenner S, Pepper D, Berns MW, Tan E, Brinkley BR (1981) Kinetochore structure, duplication, and distribution in mammalian cells: Analysis by human autoantibodies from scleroderma patients. J Cell Biol 91: 95-102.
Choo KHA (1997) Centromere proteins of higher eukaryotes. In: The Centromere. Oxford University Press, pp 143-253.
Cooke CA, Bernat L, Earnshaw WC (1990) CENP-B: A major human centromere protein located beneath the kinetochore. J Cell Biol 110: 1475-1488.
Cooke CA, Bazett-Jones DP, Earnshaw WC, Rattner JB (1993) Mapping DNA within the mammalian kinetochore. J Cell Biol 120: 1083-1091.
Cooke CA, Schaar B, Yen TJ, Earnshaw WC (1997) Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase. Chromosoma 106: 446-455.
Depinet TW, Zackowski JL, Earnshaw WC, Kaffe S, Sekhon GS, et al. (1997) Characterization of neo-centromeres in marker chromosomes lacking detectable alpha-satellite DNA. Hum Mol Genet 6: 1195-1204.
Earnshaw WC, Ratrie H, Stetten G (1989) Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma 98: 1-12.
Haaf T, Ward DC (1994) Structural analysis of alpha-satellite DNA and centromere proteins using extended chromatin and chromosomes. Hum Mol Genet 3: 697-709.
He D and Brinkley BR (1996) Structure and dynamic organization of centromeres/prekinetochores in the nucleus of mammalian cells. J Cell Sci 109: 2693-2704.
Heneen WK (1975) Ultrastructure of the prophase kinetochore in cultured cells of rat-kangaroo (Potorous tridactylis). Hereditas 79: 209-220.
Knehr M, Poppe M, Schroeter D, Eickelbaum W et al. (1996) Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase. Proc Natl Acad Sci USA 93: 10234-10239.
Liao H, Winkfein RJ, Mack G, Rattner JB, Yen TJ (1995) CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis. J Cell Biol 130: 507-518.
Masumoto H, Sugimoto K, Okazaki T (1989) Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle. Exp Cell Res 181: 181-196.
Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM (1980) Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci USA 77: 1627-1631.
Muro Y, Sugimoto K, Okazaki T, Ohashi M (1990) The heterogeneity of anticentromere antibodies in immunoblotting analysis. J Rheumatol 17: 1042-1047.
O'Keefe CL, Warburton PE, Matera AG (1996) Oligonucleotide probes for alpha satellite DNA variants can distinguish homologous chromosomes by FISH. Hum Mol Genet 5: 1793-1799.
Parra I, Windle B (1993) High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet 5: 17-21.
Rieder CL (1982) The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol 79: 1-58.
Ris H, Witt PL (1981) Structure of the mammalian kinetochore. Chromosoma 82: 153-170.
Roos U-P (1973) Light and electron microscopy of rat kangaroo cells in mitosis. II. Kinetochore structure and function. Chromosoma 41: 195-220.
Saitoh H, Tomkiel J, Cooke CA, Ratrie H III et al. (1992) CENPC, an autoantigen in scleroderma, is a component of the human inner kinetochore plate. Cell 70: 115-125.
Shelby RD, Hahn KM, Sullivan KF (1996) Dynamic elastic behavior of α-satellite DNA domains visualized in situ in living human cells. J Cell Biol 135: 545-557.
Sugimoto K, Migita H, Hagishita Y, Yata H, Himeno M (1992a) An antigenic determinant on human centromere protein B (CENP-B) available for production of human-specific anticentromere antibodies in mouse. Cell Struct Funct 17: 129-138.
Sugimoto K, Muro Y, Himeno M (1992b) Anti-helix-loop-helix domain antibodies: Discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B). J Biochem 111: 478-483.
Sugimoto K, Yata H, Muro Y, Himeno M (1994a) Human centromere protein C (CENP-C) is a DNA-binding protein which possesses a novel DNA-binding motif. J Biochem 116: 877-881.
Sugimoto K, Furukawa K, Himeno M (1994b) Functional cloning of centromere protein B (CENP-B) box-enriched alphoid DNA repeats utilizing the sequence-specific DNA binding activity of human CENP-B in vitro. Chrom Res 2: 453-459.
Sugimoto K, Hagishita Y, Himeno M (1994c) Functional domain structure of human centromere protein B: implication of the internal and C-terminal self-association domains in centromeric heterochromatin condensation. J Biol Chem 269: 24271-24276.
Sugimoto K, Yamada T, Muro Y, Himeno M (1996) Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C). J Biochem 120: 153-159.
Sugimoto K, Kuriyama K, Shibata A, Himeno M (1997a) Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization. Chrom Res 5: 132-141.
Sugimoto K, Furukawa K, Kusumi K, Himeno M (1997b) The distribution of binding sites for centromere protein B (CENP-B) is partly conserved among diverged higher order repeating units of human chromosome 6-specific alphoid DNA. Chrom Res 5: 395-405.
Sugimoto K, Shibata A, Himeno M (1998) Nucleotide specificity at the boundary and size requirement of the target sites recognized by human centromere protein B (CENP-B) in vitro. Chrom Res 6: 133-140.
Sullivan BA, Schwartz S (1995) Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum Mol Genet 4: 2189-2197.
Tomkiel J, Cooke CA, Saitoh H, Bernat RL, Earnshaw WC (1994) CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J Cell Biol 125: 531-545.
Vig BK, Latour D, Brown M (1996) Localization of anti-CENP antibodies and alphoid sequences in acentric heterochromatin in a breast cancer cell line. Cancer Genet Cytogenet 88: 118-125.
Warburton PE, Cooke CA, Bourassa S, Vafa O et al. (1997) Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr Biol 7: 901-904.
Willard HF, Wayes JS (1987) Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 3: 192-198.
Wood KW, Sakowicz R, Goldstein LSB, Cleveland DW (1997) CENP-E is a plus end-directed kinetochore motor required for metaphase chromosome alignment. Cell 91: 357-366.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sugimoto, K., Tsutsui, M., AuCoin, D. et al. Visualization of Prekinetochore Locus on the Centromeric Region of Highly Extended Chromatin Fibers: Does Kinetochore Autoantigen CENP-C Constitute a Kinetochore Organizing Center?. Chromosome Res 7, 9–19 (1999). https://doi.org/10.1023/A:1009267010071
Issue Date:
DOI: https://doi.org/10.1023/A:1009267010071