Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Ultrastructural localization of epidermal growth factor (EGF)-receptor transcripts in the cell nucleus using pre-embedding in situ hybridization in combination with ultra-small gold probes and silver enhancement

  • 55 Accesses

  • 32 Citations

Abstract

A high-resolution in situ hybridization method is described for localizing epidermal growth factor (EGF)-receptor transcripts in nuclei of A431 epidermoid carcinoma cells. The method is based upon the use of ultra-small gold particles in combination with silver enhancement. The RNA of the EGF-receptor was detected mainly around the nucleoli. After removal of the DNA using nucleases and high salt extraction, the RNA of the EGF-receptor appears to be associated with the nuclear matrix. The RNA of the EGF-receptor was observed in close contact with the SC-35 splicing protein, but no exact colocalization was observed. These results demonstrate that high resolution pre-embedding in situ hybridization in combination with immunocytochemistry, both using ultra-small gold as a detection method, provides a powerful tool to unravel the organization of nuclear processes.

This is a preview of subscription content, log in to check access.

References

  1. Bassell GJ (1993) High resolution distribution of mRNA within the cytoskeleton. J Cell Biochem 52:127–133

  2. Berezney R (1991) The nuclear matrix: a heuristic model for investigating genomic organization and function in the cell nucleus. J Cell Biochem 47:109–123

  3. Bond VC, Wold B (1993) Nucleolar localization of myc transcripts. Mol Cell Biol 13:3221–3230

  4. Carmo-Fonseca M, Pepperkok R, Sproat BS, Ansorge W, Swanson MS, Lamond AI (1991a) In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells. EMBO J 10:1863–1873

  5. Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SML, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lamond AI (1991b) Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J 10:195–206

  6. Carter KC, Taneja KL, Lawrence JB (1991) Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol 115:1191–1202

  7. Cremers AFM, De Graaf A, Van Bergen en Henegouwen PMP, Humbel BM, Verkleij AJ (1990) The use of ultra small gold particles at the light and electron microscopical level. In: Elder HY (ed) Transaction of the Royal Microscopical Society. Adam Hilger, Bristol, pp 609–614

  8. Danscher G (1981) Localization of gold in biological tissue. A photochemical method for light and electron microscopy. Histochemistry 71:81–88

  9. De Graaf A, Van Bergen en Henegouwen PMP, Meijne AML, Van Driel R, Verkleij AJ (1991) Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver enhanced ultra small gold probes. J Histochem Cytochem 39:1035–1045

  10. De Graaf A, Humbel BM, Stuurman N, Van Bergen en Henegouwen PMP, Verkleij AJ (1992a) Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells. Cell Biol Int Rep 16:827–836

  11. De Graaf A, Meijne AML, Van Renswoude J, Humbel BM, Van Bergen en Henegouwen PMP, De Jong L, Van Driel R, Verkleij AJ (1992b) Heat shock induced redistribution of a 160 kDa nuclear matrix protein. Exp Cell Res 202:243–251

  12. Den Hertog JD, De Laat SW, Schlessinger J, Kruijer W (1991) Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors. Cell Growth Differ 2:155–164

  13. Dirks RW, Van Dorp AGM, Van Minnen J, Fransen JAM, Van Der Ploeg M, Raap AK (1993) Ultrastructural evidence for the axonal localization of caudodorsal cell hormone mRNA in the central nervous system of the mollusc Lymneae stagnalis. Microsc Res Tech 25:12–18

  14. Fey EG, Krochmalnic G, Penman S (1986) The non-chromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionating and resinless section electron microscopy. J Cell Biol 102:1654–1665

  15. Fu X-D, Maniatis T (1990) Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature 343:437–441

  16. Habets WJ, Hoet MH, De Jong BAW, Van der Kemp A, Van Venrooij WJ (1989) Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally induced mouse monoclonal antibodies. J Immunol 143:2560–2566

  17. Hozak P, Hassan AB, Jackson DA, Cook PR (1993) Visualization of replication factories attached to a nucleoskeleton. Cell 73:361–373

  18. Huang S, Spector DL (1991) Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev 5:2288–2302

  19. Huang S, Spector DL (1992) U1 and U2 snRNAs are present in nuclear speckles. Proc Natl Acad Sci USA 89:305–308

  20. Humbel BM, Biegelmann E (1992) A preparation protocol for postembedding immunoelectron microscopy of Dictyostelium discoideum cells with monoclonal antibodies. Scanning Microsc 6:817–825

  21. Jackson DA, Cook PR (1985) Replication occurs at a nucleoskeleton. EMBO J 4:919–925

  22. Jackson DA, Hassan AB, Errington RJ, Cook PR (1993) Visualization of focal sites of transcription within human nuclei. EMBO J 12:1059–1065

  23. Kellenberger E, Dürrenberger M, Villinger W, Carlemalm E, Wurtz M (1987) The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J Histochem Cytochem 35:959–969

  24. Kessler C, Höltke HJ, Seibl R, Burg J, Mühlegger K (1990) Non-radioactive labeling and detection of nuclei acids. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system). Biol Chem Hoppe-Seyler 371:917–927

  25. Krainer AR (1988) Pre-mRNA splicing complementation with purified human U1, U2, U4/U6, U5 snRNPs. Nucleic Acids Res 16:9415–9429

  26. Lawrence JB, Singer RH, Marselle LM (1989) Highly localized tracks of specific transcripts within the interphase nuclei visualized by in situ hybridization. Cell 57:493–502

  27. O'Keefe RT, Henderson SC, Spector DL (1992) Dynamic organization of DNA replication in mammalian cell nuclei: spatial and temporally defined replication of chromosome-specific α-satellite. DNA sequences. J Cell Biol 116:1095–1110

  28. Puvion-Dutilleul F (1993) Protocol of electron microscope in situ nucleic acid hybridization for the exclusive detection of double-stranded DNA sequences in cells containing large amounts of homologous stranded DNA and RNA sequences. Applications to adenovirus type 5 infected HeLa cells. Microsc Res Tech 25:2–11

  29. Puvion-Dutilleul F, Bachellerie JP, Puvion E (1991) Nucleolar organization of HeLe cells as studied by in situ hybridization. Chromosoma 100:395–409

  30. Raap AK, Van de Rijke FM, Dirks RW, Sol CJ, Boom R, Van der Ploeg M (1991) Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences. Exp Cell Res 197:319–322

  31. Raska I, Koberna K, Jarnik M, Petrasovicova V, Bednar J, Raska K, Bravo R (1989) Ultrastructural immunolocalization of cyclin/PCNA in synchronized 3T3 cells. Exp Cell Res 184:81–89

  32. Sibon OCM, Cremers AFM, Boonstra J, Humbel BM, Verkleij AJ (1993) Localization of EGF-receptor mRNA in the nucleus of A431 cells by light microscopy. Cell Biol Int 17:1–11

  33. Singer RH, Langevin GL, Lawrence JB (1989) Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization. J Cell Biol 108:2343–2353

  34. Smith HC, Harris SG, Zillmann M, Berget SM (1989) Evidence that a nuclear matrix protein participates in pre-messenger RNA splicing. Exp Cell Res 182:521–533

  35. Spector DL (1990) Higher order nucleolar organization: three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc Natl Acad Sci USA 87:147–151

  36. Spector DL, Fu X-D, Maniatis T (1991) Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J 10:3467–3481

  37. Stierhof YD, Schwarz H, Frank H (1986) Transverse sectioning of plastic embedded immunolabeled cryosections: morphology and permeability to protein A-colloidal gold complexes. J Ultrastruct Mol Struct Res 97:187–196

  38. Stierhof YD, Schwarz H, Dürrenberger M, Villiger W, Kellenberger E (1991) Yield of immunolabel compared to resin sections and thawed cryosections. In: Hayat MA (ed) Colloidal gold: principles, methods, and applications, vol 3. Academic Press, San Diego, Calif., USA, pp 88–113

  39. Thiry M, Thiry-Blaise L (1989) In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur J Cell Biol 50:235–243

  40. Verheijen R, Venrooij W, Ramaekers F (1988) Review. The nuclear matrix: structure and composition. J Cell Sci 90:11–36

  41. Wansink DG, Schul W, Van der Kraan I, Van Steensel B, Van Driel R, De Jong L (1993) Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol 122:283–293

  42. Xing Y, Lawrence JB (1991) Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol 112:1055–1063

  43. Xing Y, Johnson CV, Dobner PR, Lawrence JB (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259:1326–1330

Download references

Author information

Correspondence to O. C. M. Sibon.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sibon, O.C.M., Humbel, B.M., De Graaf, A. et al. Ultrastructural localization of epidermal growth factor (EGF)-receptor transcripts in the cell nucleus using pre-embedding in situ hybridization in combination with ultra-small gold probes and silver enhancement. Histochemistry 101, 223–232 (1994). https://doi.org/10.1007/BF00269548

Download citation

Keywords

  • Epidermal Growth Factor
  • Cell Nucleus
  • Close Contact
  • High Salt
  • Gold Particle