Histochemistry and Cell Biology

, Volume 128, Issue 2, pp 97–104 | Cite as

Advances in imaging the interphase nucleus using thin cryosections

  • Ana Pombo


The mammalian genome is partitioned amongst various chromosomes and encodes for approximately 30,000 protein-coding genes. Gene expression occurs after exit from mitosis, when chromosomes partially decondense within the cell nucleus to allow the enzymatic activities that work on chromatin to access each gene in a regulated fashion. Differential patterns of gene expression evolve during cell differentiation to give rise to the over 200 cell types in higher eukaryotes. The architectural organisation of the genome inside the interphase cell nucleus, and associated enzymatic activities, reveals dynamic and functional compartmentalization of the genome. In this review, I highlight the advantages of Tokuyasu cryosectioning on the investigation of nuclear structure and function.


Nuclear compartments Gene expression Chromosome territories Transcription factories Confocal microscopy Correlative microscopy 



I would like to thank David J. Vaux for first suggesting the use of Tokuyasu cryosections, Mike Hollinshead for generously sharing his knowledge about cryosectioning and for many fruitful collaborations, my laboratory co-workers, especially Sonya Martin, Sheila Q. Xie, and Miguel R. Branco, for their contribution in furthering the development of high-resolution labelling strategies, Sheila Q. Xie and Emily Brookes for comments on the manuscript, and Miguel R. Branco for the kind gift of images in Figs. 2a and 3, and Sheila Xie for image 1b. The work in my laboratory is supported by the Medical Research Council (UK).


  1. Bisotto S, Lauriault P, Duval M, Vincent M (1995) Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes. J Cell Sci 108:1873–1882PubMedGoogle Scholar
  2. Branco MR, Pombo A (2006) Intermingling of chromosome territories in interphase suggests role in translocations and transcription-dependent associations. PLoS Biol 4:e138PubMedCrossRefGoogle Scholar
  3. Branco MR, Xie SQ, Martin S, Pombo A (2005) Correlative microscopy using Tokuyasu cryosections: applications for immunolabeling and in situ hybridization. In: Stephens D (ed) Cell imaging. Scion Publishing, pp 201–217Google Scholar
  4. Bregman DB, Du L, Li Y, Ribisi S, Warren SL (1994) Cytostellin distributes to nuclear regions enriched with splicing factors. J Cell Sci 107:387–396PubMedGoogle Scholar
  5. Bregman DB, Du L, Van Der Zee S, Warren SL (1995) Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. J Cell Biol 129:287–298PubMedCrossRefGoogle Scholar
  6. Brown JM, Leach J, Reittie JE, Atzberger A, Lee-Prudhoe J, Wood WG, Higgs DR, Iborra FJ, Buckle VJ (2006) Coregulated human globin genes are frequently in spatial proximity when active. J Cell Biol 172:177–187PubMedCrossRefGoogle Scholar
  7. Ching RW, Dellaire G, Eskiw CH, Bazett-Jones DP (2005) PML bodies: a meeting place for genomic loci? J Cell Sci 118:847–854PubMedCrossRefGoogle Scholar
  8. Cioce M, Lamond AI (2005) Cajal bodies: a long history of discovery. Annu Rev Cell Dev Biol 21:105–131PubMedCrossRefGoogle Scholar
  9. Cmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu XD, van Driel R, Fakan S (1999) Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol Biol Cell 10:211–223PubMedGoogle Scholar
  10. Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: RNA polymerase II at 2.8 Angstrom resolution. Science 292:1863–1876PubMedCrossRefGoogle Scholar
  11. Cremer T, Cremer C (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2:292–301PubMedCrossRefGoogle Scholar
  12. Doyle O, Corden JL, Murphy C, Gall JG (2002) The distribution of RNA polymerase II largest subunit (RPB1) in the Xenopus germinal vesicle. J Struct Biol 140:154–166PubMedCrossRefGoogle Scholar
  13. Esa A, Coleman AE, Edelmann P, Silva S, Cremer CG, Janz S (2001) Conformational differences in the 3-D nanostructure of the immunoglobulin heavy-chain locus, a hotspot of chromosomal translocations in B lymphocytes. Cancer Genet Cytogenet 127:168–173PubMedCrossRefGoogle Scholar
  14. Failla AV, Spoeri U, Albrecht B, Kroll A, Cremer C (2002) Nanosizing of fluorescent objects by spatially modulated illumination microscopy. Appl Opt 41:275–283Google Scholar
  15. Faro-Trindade I, Cook PR (2006) A conserved organization of transcription during embryonic stem cell differentiation and in cells with high C value. Mol Biol Cell 17:910–920CrossRefGoogle Scholar
  16. Fay FS, Taneja KL, Shenoy S, Lifshitz L, Singer RH (1997) Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A). Exp Cell Res 231:27–37PubMedCrossRefGoogle Scholar
  17. Frey MR, Matera AG (2001) RNA-mediated interaction of Cajal bodies and U2 snRNA genes. J Cell Biol 154:499–509PubMedCrossRefGoogle Scholar
  18. Fuchsova B, Novak P, Kafkova J, Hozak P (2002) Nuclear DNA helicase II is recruited to IFN-alpha-activated transcription sites at PML nuclear bodies. J Cell Biol 158:463–473PubMedCrossRefGoogle Scholar
  19. Gall JG (2001) A role for Cajal bodies in assembly of the nuclear transcription machinery. FEBS Lett 498:164–167PubMedCrossRefGoogle Scholar
  20. Gall JG, Bellini M, Wu Z, Murphy C (1999) Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. Mol Biol Cell 10:4385–4402PubMedGoogle Scholar
  21. Gao L, Frey MR, Matera AG (1997) Human genes encoding U3 snRNA associate with coiled bodies in interphase cells and are clustered on chromosome 17p11.2 in a complex inverted repeat structure. Nucleic Acid Res 25:4740–4747PubMedCrossRefGoogle Scholar
  22. Grande MA, van der Kraan I, van Steensel B, Schul W, de The H, van der Voort HT, de Jong L, van Driel R (1996) PML-containing nuclear bodies: Their spatial distribution in relation to other nuclear components. J Cell Biochem 63:280–291PubMedCrossRefGoogle Scholar
  23. Grande MA, van der Kraan I, de Jong L, van Driel R (1997) Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II. J Cell Sci 110:1781–1791PubMedGoogle Scholar
  24. Griffiths G, Simons K, Warren G, Tokuyasu KT (1983) Immunoelectron microscopy using thin, frozen sections: Application to studies of the intracellular transport of Semliki forest virus spike glycoproteins. Methods Enzymol 96:466–485PubMedCrossRefGoogle Scholar
  25. Guillot PV, Xie SQ, Hollinshead M, Pombo A (2004) Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells. Exp Cell Res 295:460–468PubMedCrossRefGoogle Scholar
  26. Gustafsson MG (2005) Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102:13081–13086PubMedCrossRefGoogle Scholar
  27. Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158PubMedCrossRefGoogle Scholar
  28. Iborra FJ, Cook PR (1998) The size of sites containing SR proteins in human nuclei: Problems associated with characterizing small structures by immunogold labelling. J Histochem Cytochem 46:985–992PubMedGoogle Scholar
  29. Iborra FJ, Pombo A, Jackson DA, Cook PR (1996) Active RNA polymerases are localized within discrete transcription “factories” in human nuclei. J Cell Sci 109:1427–1436PubMedGoogle Scholar
  30. Iborra FJ, Jackson DA, Cook PR (1998) The path of transcripts from extra-nucleolar synthetic sites to nuclear pores: Transcripts in transit are concentrated in discrete structures containing SR proteins. J Cell Sci 111:2269–2282PubMedGoogle Scholar
  31. Jackson DA, Hassan AB, Errington RJ, Cook PR (1993) Visualization of focal sites of transcription within human nuclei. EMBO J 12:1059–1065PubMedGoogle Scholar
  32. Jackson DA, Iborra FJ, Manders EM, Cook PR (1998) Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol Biol Cell 9:1523–1536PubMedGoogle Scholar
  33. Jacobs EY, Frey MR, Wu W, Ingledue TC, Gebuhr TC, Gao L, Marzluff WF, Matera AG (1999) Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase hela cells but not with U6 and U7 genes. Mol Biol Cell 10:1653–1663PubMedGoogle Scholar
  34. Johnson C, Primorac D, McKinstry M, McNeil J, Rowe D, Lawrence JB (2000) Tracking COL1A1 RNA in osteogenesis imperfecta: Splice-defective transcripts initiate transport from the gene but are retained within the SC35 domain. J Cell Biol 150:417–432PubMedCrossRefGoogle Scholar
  35. Kiesslich A, von Mikecz A, Hemmerich P (2002) Cell cycle-dependent association of PML bodies with sites of active transcription in nuclei of mammalian cells. J Struct Biol 140:167–179PubMedCrossRefGoogle Scholar
  36. Kimura H, Tao Y, Roeder RG, Cook PR (1999) Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: Little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure. Mol Cell Biol 19:5383–5392PubMedGoogle Scholar
  37. LaMorte VJ, Dyck JA, Ochs RL, Evans RM (1998) Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. Proc Natl Acad Sci USA 95:4991–4996PubMedCrossRefGoogle Scholar
  38. Martin S, Failla AV, Spori U, Cremer C, Pombo A (2004) Measuring the size of biological nanostructures with spatially modulated illumination microscopy. Mol Biol Cell 15:2449–2455PubMedCrossRefGoogle Scholar
  39. Matera AG (2003) Cajal bodies. Curr Biol 13:R503PubMedCrossRefGoogle Scholar
  40. McDowall A, Gruenberg J, Romisch K, Griffiths G (1989) The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells. Eur J Cell Biol 49:281–294PubMedGoogle Scholar
  41. Mintz PJ, Spector DL (2000) Compartmentalization of RNA processing factors within nuclear speckles. J Struct Biol 129:241–251PubMedCrossRefGoogle Scholar
  42. Misteli T, Spector DL (1999) RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. Mol Cell 3:697–705PubMedCrossRefGoogle Scholar
  43. Moen PT Jr, Johnson CV, Byron M, Shopland LS, de la Serna IL, Imbalzano AN, Lawrence JB (2004) Repositioning of muscle-specific genes relative to the periphery of SC-35 domains during skeletal myogenesis. Mol Biol Cell 15:197–206PubMedCrossRefGoogle Scholar
  44. Mortillaro MJ, Blencowe BJ, Wei X, Nakayasu H, Du L, Warren SL, Sharp PA, Berezney R (1996) A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix. Proc Natl Acad Sci USA 93:8253–8257PubMedCrossRefGoogle Scholar
  45. Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nat Genet 36:1065–1071PubMedCrossRefGoogle Scholar
  46. Patturajan M, Wei X, Berezney R, Corden JL (1998) A nuclear matrix protein interacts with the phosphorylated c-terminal domain of RNA polymerase II. Mol Cell Biol 18:2406–2415PubMedGoogle Scholar
  47. Pombo A, Cook PR (1996) The localization of sites containing nascent RNA and splicing factors. Exp Cell Res 229:201–203PubMedCrossRefGoogle Scholar
  48. Pombo A, Hollinshead M, Cook PR (1999a) Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy. J Histochem Cytochem 47:471–480PubMedGoogle Scholar
  49. Pombo A, Jackson DA, Hollinshead M, Wang Z, Roeder RG, Cook PR (1999b) Regional specialization in human nuclei: Visualization of discrete sites of transcription by RNA polymerase III. EMBO J 18:2241–2253PubMedCrossRefGoogle Scholar
  50. Puvion E, Puvion-Dutilleul F (1996) Ultrastructure of the nucleus in relation to transcription and splicing: Roles of perichromatin fibrils and interchromatin granules. Exp Cell Res 229:217–225PubMedCrossRefGoogle Scholar
  51. Schul W, Adelaar B, van Driel R, de Jong L (1999) Coiled bodies are predisposed to a spatial association with genes that contain snoRNA sequences in their introns. J Cell Biochem 75:393–403PubMedCrossRefGoogle Scholar
  52. Shopland LS, Byron M, Stein JL, Lian JB, Stein GS, Lawrence JB (2001) Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact. Mol Biol Cell 12:565–576PubMedGoogle Scholar
  53. Shopland LS, Johnson CV, Byron M, McNeil J, Lawrence JB (2003) Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: Evidence for local euchromatic neighborhoods. J Cell Biol 162:981–990PubMedCrossRefGoogle Scholar
  54. Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E, van Steensel B, de Laat W (2006) Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet 38:1348–1354PubMedCrossRefGoogle Scholar
  55. Smith KP, Carter KC, Johnson CV, Lawrence JB (1995) U2 and U1 snRNA gene loci associate with coiled bodies. J Cell Biochem 59:473–485PubMedCrossRefGoogle Scholar
  56. Smith KP, Lawrence JB (2000) Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: Evidence for preU2 within Cajal bodies. Mol Biol Cell 11:2987–2998PubMedGoogle Scholar
  57. Solovei I, Cavallo A, Schermelleh L, Jaunin F, Scasselati C, Cmarko D, Cremer C, Fakan S, Cremer T (2002) Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH). Exp Cell Res 276:10–23PubMedCrossRefGoogle Scholar
  58. Spector DL (2001) Nuclear domains. J Cell Sci 114:2891–2893PubMedGoogle Scholar
  59. Stierhof YD, Schwarz H (1989) Labeling properties of sucrose-infiltrated cryosections. Scanning Microsc Suppl 3:35–46PubMedGoogle Scholar
  60. 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 97:187–196PubMedCrossRefGoogle Scholar
  61. Sugaya K, Vigneron M, Cook PR (2000) Mammalian cell lines expressing functional RNA polymerase II tagged with the green fluorescent protein. J Cell Sci 113:2679–2683PubMedGoogle Scholar
  62. Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551–565PubMedCrossRefGoogle Scholar
  63. Tokuyasu KT, Slot JW, Singer R (1978) Simultaneous observations of immunolabelled frozen sections. In: LM and EM (eds) Ninth International Congress on Electron Microscopy, TorontoGoogle Scholar
  64. von Mikecz A, Zhang S, Montminy M, Tan EM, Hemmerich P (2000) Creb-binding protein (CBP)/p300 and RNA polymerase II colocalize in transcriptionally active domains in the nucleus. J Cell Biol 150:265–273CrossRefGoogle Scholar
  65. Wang J, Shiels C, Sasieni P, Wu PJ, Islam SA, Freemont PS, Sheer D (2004) Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions. J Cell Biol 164:515–526PubMedCrossRefGoogle Scholar
  66. 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 122:283–293PubMedCrossRefGoogle Scholar
  67. Wansink DG, Sibon OC, Cremers FF, van Driel R, de Jong L (1996) Ultrastructural localization of active genes in nuclei of A431 cells. J Cell Biochem 62:10–18PubMedCrossRefGoogle Scholar
  68. Wei X, Somanathan S, Samarabandu J, Berezney R (1999) Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles. J Cell Biol 146:543–558PubMedCrossRefGoogle Scholar
  69. Xie SQ, Martin S, Guillot PV, Bentley DL, Pombo A (2006) Splicing speckles are not reservoirs of RNA polymerase II, but contain an inactive form, phosphorylated on serine2 residues of the C-terminal domain. Mol Biol Cell 17:1723–1733PubMedCrossRefGoogle Scholar
  70. Xie SQ, Pombo A (2006) Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: An ultrastructural study. Histochem Cell Biol 125:21–31PubMedCrossRefGoogle Scholar
  71. Zeng C, Kim E, Warren SL, Berget SM (1997) Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity. EMBO J 16:1401–1412PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Nuclear Organisation Group, MRC Clinical Sciences CentreImperial College School of MedicineLondonUK

Personalised recommendations