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. 1986 Feb 1;102(2):576–586. doi: 10.1083/jcb.102.2.576

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

PMCID: PMC2114079  PMID: 3944191

Abstract

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A- horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.

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Selected References

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  1. Aguas A. P., Pinto da Silva P. High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells. J Cell Biol. 1984 Aug;99(2):655–660. doi: 10.1083/jcb.99.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aguas A. P., Pinto da Silva P. Regionalization of transmembrane glycoproteins in the plasma membrane of boar sperm head is revealed by fracture-label. J Cell Biol. 1983 Nov;97(5 Pt 1):1356–1364. doi: 10.1083/jcb.97.5.1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aguas A. P., Pinto da Silva P. The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates. J Cell Biol. 1985 Feb;100(2):528–534. doi: 10.1083/jcb.100.2.528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allen H. J., Johnson E. A., Matta K. L. A comparison of the binding specificities of lectins from Ulex europaeus and Lotus tetragonolobus. Immunol Commun. 1977;6(6):585–602. doi: 10.3109/08820137709093469. [DOI] [PubMed] [Google Scholar]
  5. Bakayev V. V., Schmatchenko V. V., Georgiev G. P. Subnucleosome particles containing high mobility group proteins HMG-E and HMG-G originate from transcriptionally active chromatin. Nucleic Acids Res. 1979 Nov 24;7(6):1525–1540. doi: 10.1093/nar/7.6.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barbosa M. L., Pinto da Silva P. Restriction of glycolipids to the outer half of a plasma membrane: concanavalin A labeling of membrane halves in Acanthamoeba castellanii. Cell. 1983 Jul;33(3):959–966. doi: 10.1016/0092-8674(83)90039-9. [DOI] [PubMed] [Google Scholar]
  7. Berezney R., Buchholtz L. A. Dynamic association of replicating DNA fragments with the nuclear matrix of regenerating liver. Exp Cell Res. 1981 Mar;132(1):1–13. doi: 10.1016/0014-4827(81)90076-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Debray H., Decout D., Strecker G., Spik G., Montreuil J. Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosylproteins. Eur J Biochem. 1981 Jun;117(1):41–55. doi: 10.1111/j.1432-1033.1981.tb06300.x. [DOI] [PubMed] [Google Scholar]
  12. Dijkwel P. A., Mullenders L. H., Wanka F. Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei. Nucleic Acids Res. 1979 Jan;6(1):219–230. doi: 10.1093/nar/6.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Egan P. A., Levy-Wilson B. Structure of transcriptionally active and inactive nucleosomes from butyrate-treated and control HeLa cells. Biochemistry. 1981 Jun 23;20(13):3695–3702. doi: 10.1021/bi00516a005. [DOI] [PubMed] [Google Scholar]
  14. Frenster J. H. Electron microscopic localization of acridine orange binding to DNA within human leukemic bone marrow cells. Cancer Res. 1971 Aug;31(8):1128–1133. [PubMed] [Google Scholar]
  15. Furukawa K., Terayama H. Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat. Biochim Biophys Acta. 1979 Jul 18;585(4):575–588. doi: 10.1016/0304-4165(79)90190-9. [DOI] [PubMed] [Google Scholar]
  16. Gazit B., Cedar H. Nuclease sensitivity of active chromatin. Nucleic Acids Res. 1980 Nov 25;8(22):5143–5155. doi: 10.1093/nar/8.22.5143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gazit B., Panet A., Cedar H. Reconstitution of a deoxyribonuclease I-sensitive structure on active genes. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1787–1790. doi: 10.1073/pnas.77.4.1787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geoghegan W. D., Ackerman G. A. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem. 1977 Nov;25(11):1187–1200. doi: 10.1177/25.11.21217. [DOI] [PubMed] [Google Scholar]
  19. Goldberg A. H., Yeoman L. C., Busch H. Chromatin-associated glycoproteins of normal rat liver and Novikoff hepatoma ascites cells. Cancer Res. 1978 Apr;38(4):1052–1056. [PubMed] [Google Scholar]
  20. Goodwin G. H., Mathew C. G., Wright C. A., Venkov C. D., Johns E. W. Analysis of the high mobility group proteins associated with salt-soluble nucleosomes. Nucleic Acids Res. 1979 Dec 11;7(7):1815–1835. doi: 10.1093/nar/7.7.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goodwin G. H., Wright C. A., Johns E. W. The characterisation of 1SF monomer nucleosomes from hen oviduct and the partial characterisation of a third HMG14/17-like in such nucleosomes. Nucleic Acids Res. 1981 Jun 25;9(12):2761–2775. doi: 10.1093/nar/9.12.2761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gros D., Bruce B., Challice C. E., Schrevel J. Ultrastructural localization of concanavalin A and wheat germ agglutinin binding sites in adult and embryonic mouse myocardium. J Histochem Cytochem. 1982 Mar;30(3):193–200. doi: 10.1177/30.3.7061821. [DOI] [PubMed] [Google Scholar]
  23. Herman R., Weymouth L., Penman S. Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei. J Cell Biol. 1978 Sep;78(3):663–674. doi: 10.1083/jcb.78.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunt B. F., Vogelstein B. Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum. Nucleic Acids Res. 1981 Jan 24;9(2):349–363. doi: 10.1093/nar/9.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jackson D. A., McCready S. J., Cook P. R. RNA is synthesized at the nuclear cage. Nature. 1981 Aug 6;292(5823):552–555. doi: 10.1038/292552a0. [DOI] [PubMed] [Google Scholar]
  26. Keshgegian A. A., Glick M. C. Glycoproteins associated with nuclei of cells before and after transformation by a ribonucleic acid virus. Biochemistry. 1973 Mar 13;12(6):1221–1226. doi: 10.1021/bi00730a032. [DOI] [PubMed] [Google Scholar]
  27. Levy W B., Wong N. C., Dixon G. H. Selective association of the trout-specific H6 protein with chromatin regions susceptible to DNase I and DNase II: possible location of HMG-T in the spacer region between core nucleosomes. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2810–2814. doi: 10.1073/pnas.74.7.2810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Levy-Wilson B., Dixon G. H. Limited action of micrococcal nuclease on trout testis nuclei generates two mononucleosome subsets enriched in transcribed DNA sequences. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1682–1686. doi: 10.1073/pnas.76.4.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levy-Wilson B. Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones. Biochemistry. 1983 Jan 18;22(2):484–489. doi: 10.1021/bi00271a035. [DOI] [PubMed] [Google Scholar]
  30. Levy B. W., Connor W., Dixon G. H. A subset of trout testis nucleosomes enriched in transcribed DNA sequences contains high mobility group proteins as major structural components. J Biol Chem. 1979 Feb 10;254(3):609–620. [PubMed] [Google Scholar]
  31. Long B. H., Huang C. Y., Pogo A. O. Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix. Cell. 1979 Dec;18(4):1079–1090. doi: 10.1016/0092-8674(79)90221-6. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Miller T. E., Huang C. Y., Pogo A. O. Rat liver nuclear skeleton and ribonucleoprotein complexes containing HnRNA. J Cell Biol. 1978 Mar;76(3):675–691. doi: 10.1083/jcb.76.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nicolson G. L. The interactions of lectins with animal cell surfaces. Int Rev Cytol. 1974;39:89–190. doi: 10.1016/s0074-7696(08)60939-0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Pardoll D. M., Vogelstein B. Sequence analysis of nuclear matrix associated DNA from rat liver. Exp Cell Res. 1980 Aug;128(2):466–470. doi: 10.1016/0014-4827(80)90083-x. [DOI] [PubMed] [Google Scholar]
  37. Pinto da Silva P., Branton D. Membrane splitting in freeze-ethching. Covalently bound ferritin as a membrane marker. J Cell Biol. 1970 Jun;45(3):598–605. doi: 10.1083/jcb.45.3.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pinto da Silva P., Parkison C., Dwyer N. Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane. Proc Natl Acad Sci U S A. 1981 Jan;78(1):343–347. doi: 10.1073/pnas.78.1.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pinto da Silva P., Torrisi M. R., Kachar B. Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells. J Cell Biol. 1981 Nov;91(2 Pt 1):361–372. doi: 10.1083/jcb.91.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reeves R., Chang D., Chung S. C. Carbohydrate modifications of the high mobility group proteins. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6704–6708. doi: 10.1073/pnas.78.11.6704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Reeves R., Chang D. Investigations of the possible functions for glycosylation in the high mobility group proteins. Evidence for a role in nuclear matrix association. J Biol Chem. 1983 Jan 10;258(1):679–687. [PubMed] [Google Scholar]
  42. Rizzo W. B., Bustin M. Lectins as probes of chromatin structure. Binding of concanavalin A to purified rat liver chromatin. J Biol Chem. 1977 Oct 25;252(20):7062–7067. [PubMed] [Google Scholar]
  43. 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]
  44. Roth J. Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections. J Histochem Cytochem. 1983 Aug;31(8):987–999. doi: 10.1177/31.8.6190857. [DOI] [PubMed] [Google Scholar]
  45. Roth J. Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes. J Histochem Cytochem. 1983 Apr;31(4):547–552. doi: 10.1177/31.4.6186737. [DOI] [PubMed] [Google Scholar]
  46. Seve A. P., Hubert J., Bouvier D., Masson C., Geraud G., Bouteille M. In situ distribution in different cell types of nuclear glycoconjugates detected by two lectins. J Submicrosc Cytol. 1984 Oct;16(4):631–641. [PubMed] [Google Scholar]
  47. Shannon L. M., Kay E., Lew J. Y. Peroxidase isozymes from horseradish roots. I. Isolation and physical properties. J Biol Chem. 1966 May 10;241(9):2166–2172. [PubMed] [Google Scholar]
  48. Stein G. S., Roberts R. M., Davis J. L., Head W. J., Stein J. L., Thrall C. L., Van Veen J., Welch D. W. Are glycoproteins and glycosaminoglycans components of the eukaryotic genome? Nature. 1975 Dec 18;258(5536):639–641. doi: 10.1038/258639a0. [DOI] [PubMed] [Google Scholar]
  49. Torrisi M. R., Da Silva P. P. T-lymphocyte heterogeneity: wheat germ agglutinin labeling of transmembrane glycoproteins. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5671–5674. doi: 10.1073/pnas.79.18.5671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Torrisi M. R., Pinto da Silva P. Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label. J Cell Biol. 1984 Jan;98(1):29–34. doi: 10.1083/jcb.98.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weisbrod S. T. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography. Nucleic Acids Res. 1982 Mar 25;10(6):2017–2042. doi: 10.1093/nar/10.6.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weisbrod S. Active chromatin. Nature. 1982 May 27;297(5864):289–295. doi: 10.1038/297289a0. [DOI] [PubMed] [Google Scholar]
  53. Weisbrod S., Groudine M., Weintraub H. Interaction of HMG 14 and 17 with actively transcribed genes. Cell. 1980 Jan;19(1):289–301. doi: 10.1016/0092-8674(80)90410-9. [DOI] [PubMed] [Google Scholar]
  54. Weisbrod S., Weintraub H. Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):630–634. doi: 10.1073/pnas.76.2.630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Westerink B. H., van der Heyden J. A., Korf J. Enhanced dopamine metabolism after small lesions in the midbrain of the rat. Life Sci. 1978 Mar;22(9):749–756. doi: 10.1016/0024-3205(78)90243-6. [DOI] [PubMed] [Google Scholar]
  56. Yeoman L. C., Jordan J. J., Busch R. K., Taylor C. W., Savage H. E., Busch H. A fetal protein in chromatin of Novikoff hepatoma and Walker 256 carcinosarcoma tumors that is absent from normal and regenerating rat liver. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3258–3262. doi: 10.1073/pnas.73.9.3258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. da Silva P. P., Kachar B., Torrisi M. R., Brown C., Parkison C. Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label. Science. 1981 Jul 10;213(4504):230–233. doi: 10.1126/science.7244630. [DOI] [PubMed] [Google Scholar]
  58. da Silva P. P., Parkison C., Dwyer N. Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fractures faces. J Histochem Cytochem. 1981 Aug;29(8):917–928. doi: 10.1177/29.8.7276536. [DOI] [PubMed] [Google Scholar]
  59. da Silva P. P., Torrisi M. R. Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes. J Cell Biol. 1982 May;93(2):463–469. doi: 10.1083/jcb.93.2.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. van Eekelen C. A., van Venrooij W. J. hnRNA and its attachment to a nuclear protein matrix. J Cell Biol. 1981 Mar;88(3):554–563. doi: 10.1083/jcb.88.3.554. [DOI] [PMC free article] [PubMed] [Google Scholar]

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