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. 1988 Dec 1;7(12):3667–3677. doi: 10.1002/j.1460-2075.1988.tb03248.x

Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat.

D A Jackson 1, P R Cook 1
PMCID: PMC454939  PMID: 3208744

Abstract

Whether nucleoskeletons seen after extracting cells are preparative artefacts is controversial. Using an extraction method that preserves vital nuclear functions, we have visualized part of a nucleoskeleton by electron microscopy of thick resinless sections. Cells encapsulated in agarose microbeads are lysed using Triton in a physiological buffer; the agarose coat prevents aggregation and protects fragile cell contents. These extracted cells are accessible to small molecules and transcribe and replicate at rates close to those in vivo. After electroeluting most chromatin after treatment with HaeIII, a skeleton is uncovered which ramifies throughout the nucleus. Individual filaments are approximately 10 nm wide with an axial repeat of 23 nm, characteristic of intermediate filaments.

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Selected References

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  1. Aaronson R. P., Blobel G. Isolation of nuclear pore complexes in association with a lamina. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1007–1011. doi: 10.1073/pnas.72.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aebi U., Cohn J., Buhle L., Gerace L. The nuclear lamina is a meshwork of intermediate-type filaments. Nature. 1986 Oct 9;323(6088):560–564. doi: 10.1038/323560a0. [DOI] [PubMed] [Google Scholar]
  3. Agutter P. S., Richardson J. C. Nuclear non-chromatin proteinaceous structures: their role in the organization and function of the interphase nucleus. J Cell Sci. 1980 Aug;44:395–435. doi: 10.1242/jcs.44.1.395. [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. Blobel G. Gene gating: a hypothesis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8527–8529. doi: 10.1073/pnas.82.24.8527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Cervera M., Dreyfuss G., Penman S. Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell. 1981 Jan;23(1):113–120. doi: 10.1016/0092-8674(81)90276-2. [DOI] [PubMed] [Google Scholar]
  8. Cook P. R. A general method for preparing intact nuclear DNA. EMBO J. 1984 Aug;3(8):1837–1842. doi: 10.1002/j.1460-2075.1984.tb02056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cook P. R., Brazell I. A. Supercoils in human DNA. J Cell Sci. 1975 Nov;19(2):261–279. doi: 10.1242/jcs.19.2.261. [DOI] [PubMed] [Google Scholar]
  10. Cook P. R. Hypothesis on differentiation and the inheritance of gene superstructure. Nature. 1973 Sep 7;245(5419):23–25. doi: 10.1038/245023a0. [DOI] [PubMed] [Google Scholar]
  11. Cook P. R. The nucleoskeleton: artefact, passive framework or active site? J Cell Sci. 1988 May;90(Pt 1):1–6. doi: 10.1242/jcs.90.1.1. [DOI] [PubMed] [Google Scholar]
  12. Evan G. I., Hancock D. C. Studies on the interaction of the human c-myc protein with cell nuclei: p62c-myc as a member of a discrete subset of nuclear proteins. Cell. 1985 Nov;43(1):253–261. doi: 10.1016/0092-8674(85)90030-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Franke W. W. Nuclear lamins and cytoplasmic intermediate filament proteins: a growing multigene family. Cell. 1987 Jan 16;48(1):3–4. doi: 10.1016/0092-8674(87)90345-x. [DOI] [PubMed] [Google Scholar]
  15. Georgatos S. D., Blobel G. Lamin B constitutes an intermediate filament attachment site at the nuclear envelope. J Cell Biol. 1987 Jul;105(1):117–125. doi: 10.1083/jcb.105.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Georgatos S. D., Blobel G. Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments. J Cell Biol. 1987 Jul;105(1):105–115. doi: 10.1083/jcb.105.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hitchcock S. E., Carisson L., Lindberg U. Depolymerization of F-actin by deoxyribonuclease I. Cell. 1976 Apr;7(4):531–542. doi: 10.1016/0092-8674(76)90203-8. [DOI] [PubMed] [Google Scholar]
  18. Jackson D. A., Caton A. J., McCready S. J., Cook P. R. Influenza virus RNA is synthesized at fixed sites in the nucleus. Nature. 1982 Mar 25;296(5855):366–368. doi: 10.1038/296366a0. [DOI] [PubMed] [Google Scholar]
  19. Jackson D. A., Cook P. R. A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin. J Mol Biol. 1986 Nov 5;192(1):65–76. doi: 10.1016/0022-2836(86)90464-x. [DOI] [PubMed] [Google Scholar]
  20. Jackson D. A., Cook P. R. A general method for preparing chromatin containing intact DNA. EMBO J. 1985 Apr;4(4):913–918. doi: 10.1002/j.1460-2075.1985.tb03718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jackson D. A., Cook P. R. Different populations of DNA polymerase alpha in HeLa cells. J Mol Biol. 1986 Nov 5;192(1):77–86. doi: 10.1016/0022-2836(86)90465-1. [DOI] [PubMed] [Google Scholar]
  22. Jackson D. A., Cook P. R. Replication occurs at a nucleoskeleton. EMBO J. 1986 Jun;5(6):1403–1410. doi: 10.1002/j.1460-2075.1986.tb04374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Jackson D. A., McCready S. J., Cook P. R. Replication and transcription depend on attachment of DNA to the nuclear cage. J Cell Sci Suppl. 1984;1:59–79. doi: 10.1242/jcs.1984.supplement_1.5. [DOI] [PubMed] [Google Scholar]
  26. Jackson D. A., Yuan J., Cook P. R. A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. J Cell Sci. 1988 Jul;90(Pt 3):365–378. doi: 10.1242/jcs.90.3.365. [DOI] [PubMed] [Google Scholar]
  27. Kaufmann S. H., Coffey D. S., Shaper J. H. Considerations in the isolation of rat liver nuclear matrix, nuclear envelope, and pore complex lamina. Exp Cell Res. 1981 Mar;132(1):105–123. doi: 10.1016/0014-4827(81)90088-4. [DOI] [PubMed] [Google Scholar]
  28. Lawrence J. B., Singer R. H. Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell. 1986 May 9;45(3):407–415. doi: 10.1016/0092-8674(86)90326-0. [DOI] [PubMed] [Google Scholar]
  29. Lebkowski J. S., Laemmli U. K. Evidence for two levels of DNA folding in histone-depleted HeLa interphase nuclei. J Mol Biol. 1982 Apr 5;156(2):309–324. doi: 10.1016/0022-2836(82)90331-x. [DOI] [PubMed] [Google Scholar]
  30. Littlewood T. D., Hancock D. C., Evan G. I. Characterization of a heat shock-induced insoluble complex in the nuclei of cells. J Cell Sci. 1987 Aug;88(Pt 1):65–72. doi: 10.1242/jcs.88.1.65. [DOI] [PubMed] [Google Scholar]
  31. Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McConnell M., Whalen A. M., Smith D. E., Fisher P. A. Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ. J Cell Biol. 1987 Sep;105(3):1087–1098. doi: 10.1083/jcb.105.3.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McKeon F. D., Kirschner M. W., Caput D. Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins. Nature. 1986 Feb 6;319(6053):463–468. doi: 10.1038/319463a0. [DOI] [PubMed] [Google Scholar]
  34. Milam L., Erickson H. P. Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments. J Cell Biol. 1982 Sep;94(3):592–596. doi: 10.1083/jcb.94.3.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miller O. L., Jr Some ultrastructural aspects of genetic activity in eukaryotes. J Cell Sci Suppl. 1984;1:81–93. doi: 10.1242/jcs.1984.supplement_1.6. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Paulson J. R., Laemmli U. K. The structure of histone-depleted metaphase chromosomes. Cell. 1977 Nov;12(3):817–828. doi: 10.1016/0092-8674(77)90280-x. [DOI] [PubMed] [Google Scholar]
  38. Ris H. The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections. J Cell Biol. 1985 May;100(5):1474–1487. doi: 10.1083/jcb.100.5.1474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Skaer R. J., Whytock S. The fixation of nuclei in glutaraldehyde. J Cell Sci. 1977;27:13–21. doi: 10.1242/jcs.27.1.13. [DOI] [PubMed] [Google Scholar]
  40. Steinert P. M., Parry D. A. Intermediate filaments: conformity and diversity of expression and structure. Annu Rev Cell Biol. 1985;1:41–65. doi: 10.1146/annurev.cb.01.110185.000353. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Ward W. S., Schmidt W. N., Schmidt C. A., Hnilica L. S. Association of cytokeratin p39 with DNA in intact Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1984 Jan;81(2):419–423. doi: 10.1073/pnas.81.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Welch W. J., Suhan J. P. Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol. 1985 Oct;101(4):1198–1211. doi: 10.1083/jcb.101.4.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wolosewick J. J., Porter K. R. Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality. J Cell Biol. 1979 Jul;82(1):114–139. doi: 10.1083/jcb.82.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wrigley N. G. The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J Ultrastruct Res. 1968 Sep;24(5):454–464. doi: 10.1016/s0022-5320(68)80048-6. [DOI] [PubMed] [Google Scholar]
  46. Zackroff R. V., Goldman R. D. In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6226–6230. doi: 10.1073/pnas.76.12.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]

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