Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1455)

Abstract

Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller’s spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated “pipeline” for imaging.

Key words

Correlative light and electron microscopy (CLEM) Nucleolus RNA polymerase I (Pol I) Ribosomal RNA genes (rDNA) Pol I transcription Transmission electron microscopy (TEM) Fluorescence microscopy Yeast Saccharomyces cerevisiae 

References

  1. 1.
    Miller O, Beatty B (1969) Visualization of nucleolar genes. Science 164:955–957CrossRefPubMedGoogle Scholar
  2. 2.
    Engel C, Sainsbury S, Cheung AC, Kostrewa D, Cramer P (2013) RNA polymerase I structure and transcription regulation. Nature 502:650–655CrossRefPubMedGoogle Scholar
  3. 3.
    Merz K, Hondele M, Goetze H, Gmelch K, Stoeckl U, Griesenbeck J (2008) Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules. Genes Dev 22:1190–1204CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Léger-Silvestre I, Trumtel S, Noaillac-Depeyre J, Gas N (1999) Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae. Chromosoma 108:103–113CrossRefPubMedGoogle Scholar
  5. 5.
    Léger-Silvestre I, Noaillac-Depeyre J, Faubladier M, Gas N (1997) Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe. Eur J Cell Biol 72:13–23PubMedGoogle Scholar
  6. 6.
    Perkovic M, Kunz M, Endesfelder U, Bunse S, Wigge C, Yu Z, Hodirnau VV, Scheffer MP, Seybert A, Malkusch S, Schuman EM, Heilemann M, Frangakis AS (2014) Correlative Light- and Electron Microscopy with chemical tags. J Struct Biol 186:205–213CrossRefPubMedGoogle Scholar
  7. 7.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645CrossRefPubMedGoogle Scholar
  8. 8.
    Kanno H, Speedy RJ, Angell CA (1975) Supercooling of water to -92°C under pressure. Science 189:880–881CrossRefPubMedGoogle Scholar
  9. 9.
    Paez-Segala M, Sun MG, Shtengel G, Viswanathan S, Baird MA, Macklin JJ, Patel R, Allen JR, Howe ES, Piszczek G, Hess HF, Davidson MW, Wang Y, Looger LL (2015) Fixation-resistant photoactivatable fluorescent proteins for CLEM. Nat Methods 12:215–218CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Laboratoire de Biologie Moléculaire du CNRSUniversity of ToulouseToulouseFrance
  2. 2.CRCNA-UMR 892 INSERMNantesFrance
  3. 3.CNRSNantesFrance
  4. 4.University of NantesNantesFrance

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