Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Aug 1;90(2):289–299. doi: 10.1083/jcb.90.2.289

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis

PMCID: PMC2111883  PMID: 6169728

Abstract

The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Adolph K. W., Cheng S. M., Laemmli U. K. Role of nonhistone proteins in metaphase chromosome structure. Cell. 1977 Nov;12(3):805–816. doi: 10.1016/0092-8674(77)90279-3. [DOI] [PubMed] [Google Scholar]
  3. Adolph K. W. Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds. J Cell Sci. 1980 Apr;42:291–304. doi: 10.1242/jcs.42.1.291. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Berezney R. Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction. J Cell Biol. 1980 Jun;85(3):641–650. doi: 10.1083/jcb.85.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buongiorno-Nardelli M., Amaldi F., Lava-Sanchez P. A. Amplification as a rectification mechanism for the redundant rRNA genes. Nat New Biol. 1972 Aug 2;238(83):134–137. doi: 10.1038/newbio238134a0. [DOI] [PubMed] [Google Scholar]
  8. Clark T. G., Rosenbaum J. L. An actin filament matrix in hand-isolated nuclei of X. laevis oocytes. Cell. 1979 Dec;18(4):1101–1108. doi: 10.1016/0092-8674(79)90223-x. [DOI] [PubMed] [Google Scholar]
  9. Comings D. E., Okada T. A. Nuclear proteins. III. The fibrillar nature of the nuclear matrix. Exp Cell Res. 1976 Dec;103(2):341–360. doi: 10.1016/0014-4827(76)90271-8. [DOI] [PubMed] [Google Scholar]
  10. Dessev G. N., Grancharov K. Precipitation of RNA, DNA, and nucleoprotein particles from very dilute solutions. Anal Biochem. 1973 May;53(1):269–271. doi: 10.1016/0003-2697(73)90428-4. [DOI] [PubMed] [Google Scholar]
  11. Dwyer N., Blobel G. A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J Cell Biol. 1976 Sep;70(3):581–591. doi: 10.1083/jcb.70.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eckert W. A., Kaffenberger W., Krohne G., Franke W. W. Introduction of hidden breaks during rRNA maturation and ageing in Tetrahymena pyriformis. Eur J Biochem. 1978 Jul 3;87(3):607–616. doi: 10.1111/j.1432-1033.1978.tb12413.x. [DOI] [PubMed] [Google Scholar]
  13. Ely S., D'Arcy A., Jost E. Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells. Exp Cell Res. 1978 Oct 15;116(2):325–331. doi: 10.1016/0014-4827(78)90455-x. [DOI] [PubMed] [Google Scholar]
  14. Franke W. W., Scheer U. Pathways of nucleocytoplasmic translocation of ribonucleoproteins. Symp Soc Exp Biol. 1974;(28):249–282. [PubMed] [Google Scholar]
  15. Gerace L., Blobel G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell. 1980 Jan;19(1):277–287. doi: 10.1016/0092-8674(80)90409-2. [DOI] [PubMed] [Google Scholar]
  16. Gerace L., Blum A., Blobel G. Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. J Cell Biol. 1978 Nov;79(2 Pt 1):546–566. doi: 10.1083/jcb.79.2.546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glisin V., Crkvenjakov R., Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 1974 Jun 4;13(12):2633–2637. doi: 10.1021/bi00709a025. [DOI] [PubMed] [Google Scholar]
  18. Hay E. D., Gurdon J. B. Fine structure of the nucleolus in normal and mutant Xenopus embryos. J Cell Sci. 1967 Jun;2(2):151–162. doi: 10.1242/jcs.2.2.151. [DOI] [PubMed] [Google Scholar]
  19. Herlan G., Eckert W. A., Kaffenberger W., Wunderlich F. Isolation and characterization of an RNA-containing nuclear matrix from Tetrahymena macronuclei. Biochemistry. 1979 May 1;18(9):1782–1788. doi: 10.1021/bi00576a023. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Higashinakagawa T., Wahn H., Reeder R. H. Isolation of ribosomal gene chromatin. Dev Biol. 1977 Feb;55(2):375–386. doi: 10.1016/0012-1606(77)90180-4. [DOI] [PubMed] [Google Scholar]
  22. Hodge L. D., Mancini P., Davis F. M., Heywood P. Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle. J Cell Biol. 1977 Jan;72(1):194–208. doi: 10.1083/jcb.72.1.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jackson R. C. On the identity of nuclear membrane and non-histone nuclear proteins. Biochemistry. 1976 Dec 14;15(25):5652–5656. doi: 10.1021/bi00670a034. [DOI] [PubMed] [Google Scholar]
  24. Kelly P. T., Cotman C. W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 1978 Oct;79(1):173–183. doi: 10.1083/jcb.79.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krohne G., Franke W. W. A major soluble acidic protein located in nuclei of diverse vertebrate species. Exp Cell Res. 1980 Sep;129(1):167–189. doi: 10.1016/0014-4827(80)90341-9. [DOI] [PubMed] [Google Scholar]
  26. Krohne G., Franke W. W., Ely S., D'Arcy A., Jost E. Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material. Cytobiologie. 1978 Oct;18(1):22–38. [PubMed] [Google Scholar]
  27. Krohne G., Franke W. W. Immunological identification and localization of the predominant nuclear protein of the amphibian oocyte nucleus. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1034–1038. doi: 10.1073/pnas.77.2.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Krohne G., Franke W. W., Scheer U. The major polypeptides of the nuclear pore complex. Exp Cell Res. 1978 Oct 1;116(1):85–102. doi: 10.1016/0014-4827(78)90067-8. [DOI] [PubMed] [Google Scholar]
  29. Kumar A. Ribosome synthesis in Tetrahymena pyriformis. J Cell Biol. 1970 Jun;45(3):623–634. doi: 10.1083/jcb.45.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kumar A., Warner J. R. Characterization of ribosomal precursor particles from HeLa cell nucleoli. J Mol Biol. 1972 Jan 28;63(2):233–246. doi: 10.1016/0022-2836(72)90372-5. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Lam K. S., Kasper C. B. Electrophoretic analysis of three major nuclear envelope polypeptides. Topological relationship and sequence homology. J Biol Chem. 1979 Nov 25;254(22):11713–11720. [PubMed] [Google Scholar]
  33. 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]
  34. Matsuura S., Morimoto T., Tashiro Y., Higashinakagawa T., Muramatsu M. Ultrastructural and biochemical studies on the precursor ribosomal particles isolated from rat liver nucleoli. J Cell Biol. 1974 Nov;63(2 Pt 1):629–640. doi: 10.1083/jcb.63.2.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Merriam R. W., Clark T. G. Actin in Xenopus oocytes. II. Intracellular distribution and polymerizability. J Cell Biol. 1978 May;77(2):439–447. doi: 10.1083/jcb.77.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller O. L., Jr, Bakken A. H. Morphological studies of transcription. Acta Endocrinol Suppl (Copenh) 1972;168:155–177. doi: 10.1530/acta.0.071s155. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Mills A. D., Laskey R. A., Black P., De Robertis E. M. An acidic protein which assembles nucleosomes in vitro is the most abundant protein in Xenopus oocyte nuclei. J Mol Biol. 1980 May 25;139(3):561–568. doi: 10.1016/0022-2836(80)90148-5. [DOI] [PubMed] [Google Scholar]
  39. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  40. Penman S. RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966 May;17(1):117–130. doi: 10.1016/s0022-2836(66)80098-0. [DOI] [PubMed] [Google Scholar]
  41. Peters K. E., Commings D. E. Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins. J Cell Biol. 1980 Jul;86(1):135–155. doi: 10.1083/jcb.86.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rae M. M., Franke W. W. The interphase distribution of satellite DNA-containing heterochromatin in mouse nuclei. Chromosoma. 1972;39(4):443–456. doi: 10.1007/BF00326177. [DOI] [PubMed] [Google Scholar]
  43. Reeder R. H., Wahn H. L., Botchan P., Hipskind R., Sollner-Webb B. Ribosomal genes and their proteins from Xenopus. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):1167–1177. doi: 10.1101/sqb.1978.042.01.117. [DOI] [PubMed] [Google Scholar]
  44. Rodrigues-Pousada C., Cyrne M. L., Hayes D. Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis. Eur J Biochem. 1979 Dec 17;102(2):389–397. doi: 10.1111/j.1432-1033.1979.tb04254.x. [DOI] [PubMed] [Google Scholar]
  45. Scalenghe F., Buscaglia M., Steinheil C., Crippa M. Large scale isolation of nuclei and nucleoli from vitellogenic oocytes of Xenopus laevis. Chromosoma. 1978 May 16;66(4):299–308. doi: 10.1007/BF00328531. [DOI] [PubMed] [Google Scholar]
  46. Scheer U., Kartenbeck J., Trendelenburg M. F., Stadler J., Franke W. W. Experimental disintegration of the nuclear envelope. Evidence for pore-connecting fibrils. J Cell Biol. 1976 Apr;69(1):1–18. doi: 10.1083/jcb.69.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scheer U. Ultrastructure of the nuclear envelope of amphibian oocytes. IV. Chemical nature of the nuclear pore complex material. Z Zellforsch Mikrosk Anat. 1972;127(1):127–148. doi: 10.1007/BF00582762. [DOI] [PubMed] [Google Scholar]
  48. Stick R., Hausen P. Immunological analysis of nuclear lamina proteins. Chromosoma. 1980;80(2):219–236. doi: 10.1007/BF00286301. [DOI] [PubMed] [Google Scholar]
  49. Switzer R. C., 3rd, Merril C. R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem. 1979 Sep 15;98(1):231–237. doi: 10.1016/0003-2697(79)90732-2. [DOI] [PubMed] [Google Scholar]
  50. Todorov I. T., Hadjiolov A. A. A comparison of nuclear and nucleolar matrix proteins from rat liver. Cell Biol Int Rep. 1979 Dec;3(9):753–757. doi: 10.1016/0309-1651(79)90081-x. [DOI] [PubMed] [Google Scholar]
  51. Wunderlich F., Herlan G. Reversibly contractile nuclear matrix. Its isolation, structure, and composition. J Cell Biol. 1977 May;73(2):271–278. doi: 10.1083/jcb.73.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES