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. 1992 Feb 2;116(4):977–987. doi: 10.1083/jcb.116.4.977

A normally masked nuclear matrix antigen that appears at mitosis on cytoskeleton filaments adjoining chromosomes, centrioles, and midbodies

PMCID: PMC2289346  PMID: 1734026

Abstract

mAbs were generated against HeLa nuclear matrix proteins and one, HIB2, which selectively stained mitotic cells, was selected for further study. Western blot analysis showed H1B2 antibody detected a protein of 240 kD in the nuclear matrix fractions. The H1B2 antigen was completely masked in immunofluorescently stained interphase cells. However, removing chromatin with DNase I digestion and 0.25 M ammonium sulfate extraction exposed the protein epitope. The resulting fluorescence pattern was bright, highly punctate, and entirely nuclear. Further extraction of the nuclear matrix with 2 M NaCl uncovers an underlying, anastomosing network of 9-13 nm core filaments. Most of the H1B2 antigen was retained in the fibrogranular masses enmeshed in the core filament network and not in the filaments themselves. The H1B2 antigen showed remarkable behavior at mitosis. As cells approached prophase the antigen became unmasked to immunofluorescent staining without the removal of chromatin. First appearing as a bright spot, the antibody staining spread through the nucleus finally concentrating in the region around the condensed chromosomes. The antibody also brightly stained the spindle poles and, more weakly, in a punctate pattern in the cytoskeleton around the spindle. As the chromosomes separated at anaphase, H1B2 remained with the separating daughter sets of chromosomes. The H1B2 antigen returned to the reforming nucleus at telophase, but left a bright staining region in the midbody. Immunoelectron microscopy of resinless sections showed that, in the mitotic cell, the H1B2 antibody did not stain chromosomes and centrioles themselves, but decorated a fibrogranular network surrounding and connected to the chromosomes and a fibrogranular structure surrounding the centriole.

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Selected References

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  1. Barrack E. R., Coffey D. S. The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues. J Biol Chem. 1980 Aug 10;255(15):7265–7275. [PubMed] [Google Scholar]
  2. Barrack E. R. The nuclear matrix of the prostate contains acceptor sites for androgen receptors. Endocrinology. 1983 Jul;113(1):430–432. doi: 10.1210/endo-113-1-430. [DOI] [PubMed] [Google Scholar]
  3. Berezney R., Coffey D. S. Identification of a nuclear protein matrix. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1410–1417. doi: 10.1016/0006-291x(74)90355-6. [DOI] [PubMed] [Google Scholar]
  4. Berezney R., Coffey D. S. Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. J Cell Biol. 1977 Jun;73(3):616–637. doi: 10.1083/jcb.73.3.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berezney R., Coffey D. S. Nuclear protein matrix: association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291–293. doi: 10.1126/science.1145202. [DOI] [PubMed] [Google Scholar]
  6. Berrios M., Osheroff N., Fisher P. A. In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4142–4146. doi: 10.1073/pnas.82.12.4142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Capco D. G., Krochmalnic G., Penman S. A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks. J Cell Biol. 1984 May;98(5):1878–1885. doi: 10.1083/jcb.98.5.1878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Capco D. G., Penman S. Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm. J Cell Biol. 1983 Mar;96(3):896–906. doi: 10.1083/jcb.96.3.896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Capco D. G., Wan K. M., Penman S. The nuclear matrix: three-dimensional architecture and protein composition. Cell. 1982 Jul;29(3):847–858. doi: 10.1016/0092-8674(82)90446-9. [DOI] [PubMed] [Google Scholar]
  11. Chaly N., Bladon T., Setterfield G., Little J. E., Kaplan J. G., Brown D. L. Changes in distribution of nuclear matrix antigens during the mitotic cell cycle. J Cell Biol. 1984 Aug;99(2):661–671. doi: 10.1083/jcb.99.2.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chatterjee P. K., Flint S. J. Partition of E1A proteins between soluble and structural fractions of adenovirus-infected and -transformed cells. J Virol. 1986 Dec;60(3):1018–1026. doi: 10.1128/jvi.60.3.1018-1026.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ciejek E. M., Tsai M. J., O'Malley B. W. Actively transcribed genes are associated with the nuclear matrix. Nature. 1983 Dec 8;306(5943):607–609. doi: 10.1038/306607a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Dworetzky S. I., Fey E. G., Penman S., Lian J. B., Stein J. L., Stein G. S. Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4605–4609. doi: 10.1073/pnas.87.12.4605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fey E. G., Krochmalnic G., Penman S. The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J Cell Biol. 1986 May;102(5):1654–1665. doi: 10.1083/jcb.102.5.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fey E. G., Penman S. Nuclear matrix proteins reflect cell type of origin in cultured human cells. Proc Natl Acad Sci U S A. 1988 Jan;85(1):121–125. doi: 10.1073/pnas.85.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fisher D. Z., Chaudhary N., Blobel G. cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6450–6454. doi: 10.1073/pnas.83.17.6450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gerace L., Comeau C., Benson M. Organization and modulation of nuclear lamina structure. J Cell Sci Suppl. 1984;1:137–160. doi: 10.1242/jcs.1984.supplement_1.10. [DOI] [PubMed] [Google Scholar]
  20. He D. C., Nickerson J. A., Penman S. Core filaments of the nuclear matrix. J Cell Biol. 1990 Mar;110(3):569–580. doi: 10.1083/jcb.110.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hentzen P. C., Rho J. H., Bekhor I. Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences. Proc Natl Acad Sci U S A. 1984 Jan;81(2):304–307. doi: 10.1073/pnas.81.2.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krohne G., Wolin S. L., McKeon F. D., Franke W. W., Kirschner M. W. Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily. EMBO J. 1987 Dec 1;6(12):3801–3808. doi: 10.1002/j.1460-2075.1987.tb02716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. McCready S. J., Godwin J., Mason D. W., Brazell I. A., Cook P. R. DNA is replicated at the nuclear cage. J Cell Sci. 1980 Dec;46:365–386. doi: 10.1242/jcs.46.1.365. [DOI] [PubMed] [Google Scholar]
  26. Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
  27. Mullins J. M., McIntosh J. R. Isolation and initial characterization of the mammalian midbody. J Cell Biol. 1982 Sep;94(3):654–661. doi: 10.1083/jcb.94.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Müller W. E., Okamoto T., Reuter P., Ugarkovic D., Schröder H. C. Functional characterization of Tat protein from human immunodeficiency virus. Evidence that Tat links viral RNAs to nuclear matrix. J Biol Chem. 1990 Mar 5;265(7):3803–3808. [PubMed] [Google Scholar]
  29. Müller W. E., Wenger R., Reuter P., Renneisen K., Schröder H. C. Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells. Biochim Biophys Acta. 1989 Jul 7;1008(2):208–212. doi: 10.1016/0167-4781(80)90011-1. [DOI] [PubMed] [Google Scholar]
  30. Nelson W. G., Coffey D. S. Structural aspects of mammalian DNA replication: topoisomerase II. NCI Monogr. 1987;(4):23–29. [PubMed] [Google Scholar]
  31. Nickerson J. A., Krochmalnic G., Wan K. M., Penman S. Chromatin architecture and nuclear RNA. Proc Natl Acad Sci U S A. 1989 Jan;86(1):177–181. doi: 10.1073/pnas.86.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
  33. Rennie P. S., Bruchovsky N., Cheng H. Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion. J Biol Chem. 1983 Jun 25;258(12):7623–7630. [PubMed] [Google Scholar]
  34. Robinson S. I., Nelkin B. D., Vogelstein B. The ovalbumin gene is associated with the nuclear matrix of chicken oviduct cells. Cell. 1982 Jan;28(1):99–106. doi: 10.1016/0092-8674(82)90379-8. [DOI] [PubMed] [Google Scholar]
  35. Sahyoun N., LeVine H., 3rd, Cuatrecasas P. Ca2+/calmodulin-dependent protein kinases from the neuronal nuclear matrix and post-synaptic density are structurally related. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4311–4315. doi: 10.1073/pnas.81.14.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schirmbeck R., Deppert W. Nuclear subcompartmentalization of simian virus 40 large T antigen: evidence for in vivo regulation of biochemical activities. J Virol. 1989 May;63(5):2308–2316. doi: 10.1128/jvi.63.5.2308-2316.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sellitto C., Kuriyama R. Distribution of a matrix component of the midbody during the cell cycle in Chinese hamster ovary cells. J Cell Biol. 1988 Feb;106(2):431–439. doi: 10.1083/jcb.106.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simmen R. C., Means A. R., Clark J. H. Estrogen modulation of nuclear matrix-associated steroid hormone binding. Endocrinology. 1984 Sep;115(3):1197–1202. doi: 10.1210/endo-115-3-1197. [DOI] [PubMed] [Google Scholar]
  39. Slamon D. J., Boyle W. J., Keith D. E., Press M. F., Golde D. W., Souza L. M. Subnuclear localization of the trans-activating protein of human T-cell leukemia virus type I. J Virol. 1988 Mar;62(3):680–686. doi: 10.1128/jvi.62.3.680-686.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Small D., Nelkin B., Vogelstein B. The association of transcribed genes with the nuclear matrix of Drosophila cells during heat shock. Nucleic Acids Res. 1985 Apr 11;13(7):2413–2431. doi: 10.1093/nar/13.7.2413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stanley M. A., Browne H. M., Appleby M., Minson A. C. Properties of a non-tumorigenic human cervical keratinocyte cell line. Int J Cancer. 1989 Apr 15;43(4):672–676. doi: 10.1002/ijc.2910430422. [DOI] [PubMed] [Google Scholar]
  42. Stuurman N., Meijne A. M., van der Pol A. J., de Jong L., van Driel R., van Renswoude J. The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins. J Biol Chem. 1990 Apr 5;265(10):5460–5465. [PubMed] [Google Scholar]
  43. Stuurman N., Van Driel R., De Jong L., Meijne A. M., Van Renswoude J. The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent. Exp Cell Res. 1989 Feb;180(2):460–466. doi: 10.1016/0014-4827(89)90072-4. [DOI] [PubMed] [Google Scholar]
  44. Thorburn A., Moore R., Knowland J. Attachment of transcriptionally active DNA sequences to the nucleoskeleton under isotonic conditions. Nucleic Acids Res. 1988 Jul 25;16(14B):7183–7183. doi: 10.1093/nar/16.14.7183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wagner B., Krochmalnic G., Penman S. Resinless section electron microscopy of HeLa cell mitotic architecture. Proc Natl Acad Sci U S A. 1986 Dec;83(23):8996–9000. doi: 10.1073/pnas.83.23.8996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wolosewick J. J. The application of polyethylene glycol (PEG) to electron microscopy. J Cell Biol. 1980 Aug;86(2):675–661. doi: 10.1083/jcb.86.2.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zeitlin S., Parent A., Silverstein S., Efstratiadis A. Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol. 1987 Jan;7(1):111–120. doi: 10.1128/mcb.7.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zeitlin S., Wilson R. C., Efstratiadis A. Autonomous splicing and complementation of in vivo-assembled spliceosomes. J Cell Biol. 1989 Mar;108(3):765–777. doi: 10.1083/jcb.108.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]

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