Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Oct 1;105(4):1483–1491. doi: 10.1083/jcb.105.4.1483

Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells

PMCID: PMC2114661  PMID: 3312231

Abstract

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.

Full Text

The Full Text of this article is available as a PDF (3.6 MB).

Selected References

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

  1. Ansorge W. Improved system for capillary microinjection into living cells. Exp Cell Res. 1982 Jul;140(1):31–37. doi: 10.1016/0014-4827(82)90152-5. [DOI] [PubMed] [Google Scholar]
  2. BROWN D. D., GURDON J. B. ABSENCE OF RIBOSOMAL RNA SYNTHESIS IN THE ANUCLEOLATE MUTANT OF XENOPUS LAEVIS. Proc Natl Acad Sci U S A. 1964 Jan;51:139–146. doi: 10.1073/pnas.51.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benavente R., Krohne G., Franke W. W. Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis. Cell. 1985 May;41(1):177–190. doi: 10.1016/0092-8674(85)90072-8. [DOI] [PubMed] [Google Scholar]
  4. Benavente R., Krohne G. Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies. J Cell Biol. 1986 Nov;103(5):1847–1854. doi: 10.1083/jcb.103.5.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benavente R., Krohne G., Stick R., Franke W. W. Electron microscopic immunolocalization of a karyoskeletal protein of molecular weight 145 000 in nucleoli and perinucleolar bodies of Xenopus laevis. Exp Cell Res. 1984 Mar;151(1):224–235. doi: 10.1016/0014-4827(84)90370-7. [DOI] [PubMed] [Google Scholar]
  6. Benavente R., KrohneG Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6176–6180. doi: 10.1073/pnas.82.18.6176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. ELSDALE T. R., FISCHBERG M., SMITH S. A mutation that reduces nucleolar number in Xenopus laevis. Exp Cell Res. 1958 Jun;14(3):642–643. doi: 10.1016/0014-4827(58)90175-7. [DOI] [PubMed] [Google Scholar]
  8. Eckert W. A., Franke W. W., Scheer U. Nucleocytoplasmic translocation of RNA in Tetrahymena pyriformis and its inhibition by actinomycin D and cycloheximide. Exp Cell Res. 1975 Aug;94(1):31–46. doi: 10.1016/0014-4827(75)90528-5. [DOI] [PubMed] [Google Scholar]
  9. Einck L., Bustin M. Functional histone antibody fragments traverse the nuclear envelope. J Cell Biol. 1984 Jan;98(1):205–213. doi: 10.1083/jcb.98.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fan H., Penman S. Regulation of synthesis and processing of nucleolar components in metaphase-arrested cells. J Mol Biol. 1971 Jul 14;59(1):27–42. doi: 10.1016/0022-2836(71)90411-6. [DOI] [PubMed] [Google Scholar]
  11. Franke W. W., Grund C., Osborn M., Weber K. The intermediate-sized filaments in rat kangaroo PtK2 cells. I. Morphology in situ. Cytobiologie. 1978 Aug;17(2):365–391. [PubMed] [Google Scholar]
  12. Franke W. W., Kleinschmidt J. A., Spring H., Krohne G., Grund C., Trendelenburg M. F., Stoehr M., Scheer U. A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis. J Cell Biol. 1981 Aug;90(2):289–299. doi: 10.1083/jcb.90.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gas N., Escande M. L., Stevens B. J. Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells. Biol Cell. 1985;53(3):209–218. doi: 10.1111/j.1768-322x.1985.tb00369.x. [DOI] [PubMed] [Google Scholar]
  14. Giménez-Martín G., De la Torre C., Fernández-Gómez M. E., González-Fernández A. Experimental analysis of nucleolar reorganization. J Cell Biol. 1974 Feb;60(2):502–507. doi: 10.1083/jcb.60.2.502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goessens G. Nucleolar structure. Int Rev Cytol. 1984;87:107–158. doi: 10.1016/s0074-7696(08)62441-9. [DOI] [PubMed] [Google Scholar]
  16. Guldner H. H., Szostecki C., Vosberg H. P., Lakomek H. J., Penner E., Bautz F. A. Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I. Chromosoma. 1986;94(2):132–138. doi: 10.1007/BF00286991. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hernandez-Verdun D., Bouteille M., Ege T., Ringertz N. R. Fine structure of nucleoli in micronucleated cells. Exp Cell Res. 1979 Dec;124(2):223–235. doi: 10.1016/0014-4827(79)90198-8. [DOI] [PubMed] [Google Scholar]
  19. Hügle B., Hazan R., Scheer U., Franke W. W. Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis. J Cell Biol. 1985 Mar;100(3):873–886. doi: 10.1083/jcb.100.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hügle B., Scheer U., Franke W. W. Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit. Cell. 1985 Jun;41(2):615–627. doi: 10.1016/s0092-8674(85)80034-9. [DOI] [PubMed] [Google Scholar]
  21. Kostura M., Craig N. Treatment of Chinese hamster ovary cells with the transcriptional inhibitor actinomycin D inhibits binding of messenger RNA to ribosomes. Biochemistry. 1986 Oct 21;25(21):6384–6391. doi: 10.1021/bi00369a007. [DOI] [PubMed] [Google Scholar]
  22. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  23. Krohne G., Stick R., Kleinschmidt J. A., Moll R., Franke W. W., Hausen P. Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis. J Cell Biol. 1982 Sep;94(3):749–754. doi: 10.1083/jcb.94.3.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lanford R. E., Kanda P., Kennedy R. C. Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell. 1986 Aug 15;46(4):575–582. doi: 10.1016/0092-8674(86)90883-4. [DOI] [PubMed] [Google Scholar]
  25. Lepoint A., Goessens G. Nucleologenesis in Ehrlich tumour cells. Exp Cell Res. 1978 Nov;117(1):89–94. doi: 10.1016/0014-4827(78)90430-5. [DOI] [PubMed] [Google Scholar]
  26. Madsen P., Nielsen S., Celis J. E. Monoclonal antibody specific for human nuclear proteins IEF 8Z30 and 8Z31 accumulates in the nucleus a few hours after cytoplasmic microinjection of cells expressing these proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2083–2089. doi: 10.1083/jcb.103.6.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mercer W. E., Avignolo C., Galanti N., Rose K. M., Hyland J. K., Jacob S. T., Baserga R. Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA. Exp Cell Res. 1984 Jan;150(1):118–130. doi: 10.1016/0014-4827(84)90707-9. [DOI] [PubMed] [Google Scholar]
  28. Moreno Diaz de la Espina S., Franke W. W., Krohne G., Trendelenburg M. F., Grund C., Scheer U. Medusoid fibril bodies: a novel type of nuclear filament of diameter 8 to 12 nm with periodic ultrastructure demonstrated in oocytes of Xenopus laevis. Eur J Cell Biol. 1982 Jun;27(2):141–150. [PubMed] [Google Scholar]
  29. Ochs R. L., Lischwe M. A., Shen E., Carroll R. E., Busch H. Nucleologenesis: composition and fate of prenucleolar bodies. Chromosoma. 1985;92(5):330–336. doi: 10.1007/BF00327463. [DOI] [PubMed] [Google Scholar]
  30. Ochs R., Lischwe M., O'Leary P., Busch H. Localization of nucleolar phosphoproteins B23 and C23 during mitosis. Exp Cell Res. 1983 Jun;146(1):139–149. doi: 10.1016/0014-4827(83)90332-4. [DOI] [PubMed] [Google Scholar]
  31. Phillips D. M., Phillips S. G. Repopulation of postmitotic nucleoli by preformed RNA. II. Ultrastructure. J Cell Biol. 1973 Jul;58(1):54–63. doi: 10.1083/jcb.58.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Phillips S. G. Repopulation of the postmitotic nucleolus by preformed RNA. J Cell Biol. 1972 Jun;53(3):611–623. doi: 10.1083/jcb.53.3.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Reimer G., Pollard K. M., Penning C. A., Ochs R. L., Lischwe M. A., Busch H., Tan E. M. Monoclonal autoantibody from a (New Zealand black x New Zealand white)F1 mouse and some human scleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNP particle. Arthritis Rheum. 1987 Jul;30(7):793–800. doi: 10.1002/art.1780300709. [DOI] [PubMed] [Google Scholar]
  35. Reimer G., Rose K. M., Scheer U., Tan E. M. Autoantibody to RNA polymerase I in scleroderma sera. J Clin Invest. 1987 Jan;79(1):65–72. doi: 10.1172/JCI112809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rose K. M., Stetler D. A., Jacob S. T. Protein kinase activity of RNA polymerase I purified from a rat hepatoma: probable function of Mr 42,000 and 24,600 polypeptides. Proc Natl Acad Sci U S A. 1981 May;78(5):2833–2837. doi: 10.1073/pnas.78.5.2833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scheer U., Rose K. M. Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1431–1435. doi: 10.1073/pnas.81.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scheer U., Trendelenburg F., Franke W. W. Effects of actinomycin D on the association of newly formed ribonucleoproteins with the cistrons of ribosomal RNA in Triturus oocytes. J Cell Biol. 1975 Apr;65(1):163–179. doi: 10.1083/jcb.65.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schlegel R. A., Miller L. S., Rose K. M. Reduction in RNA synthesis following red cell-mediated microinjection of antibodies to RNA polymerase I. Cell Biol Int Rep. 1985 Apr;9(4):341–350. doi: 10.1016/0309-1651(85)90029-3. [DOI] [PubMed] [Google Scholar]
  40. Schmidt-Zachmann M. S., Hügle-Dörr B., Franke W. W. A constitutive nucleolar protein identified as a member of the nucleoplasmin family. EMBO J. 1987 Jul;6(7):1881–1890. doi: 10.1002/j.1460-2075.1987.tb02447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schmidt-Zachmann M. S., Hügle B., Scheer U., Franke W. W. Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody. Exp Cell Res. 1984 Aug;153(2):327–346. doi: 10.1016/0014-4827(84)90604-9. [DOI] [PubMed] [Google Scholar]
  42. Semeshin V. F., Sherudilo A. I., Belyaeva E. S. Nucleoli formation under inhibited RNA synthesis. Exp Cell Res. 1975 Jul;93(2):458–467. doi: 10.1016/0014-4827(75)90472-3. [DOI] [PubMed] [Google Scholar]
  43. Steele R. E., Thomas P. S., Reeder R. H. Anucleolate frog embryos contain ribosomal DNA sequences and a nucleolar antigen. Dev Biol. 1984 Apr;102(2):409–416. doi: 10.1016/0012-1606(84)90205-7. [DOI] [PubMed] [Google Scholar]
  44. Tashiro K., Shiokawa K., Yamana K., Sakaki Y. Structural analysis of ribosomal DNA homologues in nucleolus-less mutant of Xenopus laevis. Gene. 1986;44(2-3):299–306. doi: 10.1016/0378-1119(86)90194-0. [DOI] [PubMed] [Google Scholar]
  45. Wallace H., Birnstiel M. L. Ribosomal cistrons and the nucleolar organizer. Biochim Biophys Acta. 1966 Feb 21;114(2):296–310. doi: 10.1016/0005-2787(66)90311-x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES