High-Throughput Live-Cell Microscopy Analysis of Association Between Chromosome Domains and the Nucleolus in S. cerevisiae

  • Renjie Wang
  • Christophe Normand
  • Olivier GadalEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1455)


Spatial organization of the genome has important impacts on all aspects of chromosome biology, including transcription, replication, and DNA repair. Frequent interactions of some chromosome domains with specific nuclear compartments, such as the nucleolus, are now well documented using genome-scale methods. However, direct measurement of distance and interaction frequency between loci requires microscopic observation of specific genomic domains and the nucleolus, followed by image analysis to allow quantification. The fluorescent repressor operator system (FROS) is an invaluable method to fluorescently tag DNA sequences and investigate chromosome position and dynamics in living cells. This chapter describes a combination of methods to define motion and region of confinement of a locus relative to the nucleolus in cell’s nucleus, from fluorescence acquisition to automated image analysis using two dedicated pipelines.

Key words

Nucleolus FROS Nuclear organization Live-cell imaging Fluorescence microscopy Chromosome domain dynamics Yeast Saccharomyces cerevisiae 



This work was supported by ATS-Nudgene and Emergence-CLEMgene of the Toulouse-IDEX. O.G. and C.N. are supported by Agence Nationale de la Recherche (ANDY). We thank I. Léger-Silvestre for thoughtful discussions and technical advices. Julien Mathon wrote the MTT-based executable files.


  1. 1.
    van Steensel B, Dekker J (2010) Genomics tools for unraveling chromosome architecture. Nat Biotechnol 28(10):1089–1095CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ay F, Noble WS (2015) Analysis methods for studying the 3D architecture of the genome. Genome Biol 16:183CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Williamson I, Berlivet S, Eskeland R, Boyle S, Illingworth RS, Paquette D, Dostie J, Bickmore WA (2014) Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization. Genes Dev 28(24):2778–2791CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Dion V, Gasser SM (2013) Chromatin movement in the maintenance of genome stability. Cell 152(6):1355–1364CrossRefPubMedGoogle Scholar
  5. 5.
    Huet S, Lavelle C, Ranchon H, Carrivain P, Victor JM, Bancaud A (2014) Relevance and limitations of crowding, fractal, and polymer models to describe nuclear architecture. Int Rev Cell Mol Biol 307:443–479CrossRefPubMedGoogle Scholar
  6. 6.
    Hajjoul H, Mathon J, Ranchon H, Goiffon I, Mozziconacci J, Albert B, Carrivain P, Victor JM, Gadal O, Bystricky K, Bancaud A (2013) High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome. Genome Res 23(11):1829–1838CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wang R, Mozziconacci J, Bancaud A, Gadal O (2015) Principles of chromatin organization in yeast: relevance of polymer models to describe nuclear organization and dynamics. Curr Opin Cell Biol 34:54–60CrossRefPubMedGoogle Scholar
  8. 8.
    Albert B, Mathon J, Shukla A, Saad H, Normand C, Leger-Silvestre I, Villa D, Kamgoue A, Mozziconacci J, Wong H, Zimmer C, Bhargava P, Bancaud A, Gadal O (2013) Systematic characterization of the conformation and dynamics of budding yeast chromosome XII. J Cell Biol 202(2):201–210CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Meister P, Gehlen LR, Varela E, Kalck V, Gasser SM (2010) Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol 470:535–567CrossRefPubMedGoogle Scholar
  10. 10.
    Berger AB, Cabal GG, Fabre E, Duong T, Buc H, Nehrbass U, Olivo-Marin JC, Gadal O, Zimmer C (2008) High-resolution statistical mapping reveals gene territories in live yeast. Nat Methods 5(12):1031–1037CrossRefPubMedGoogle Scholar
  11. 11.
    Serge A, Bertaux N, Rigneault H, Marguet D (2008) Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5(8):687–694CrossRefPubMedGoogle Scholar
  12. 12.
    Hajjoul HM, Mathon J, Viero Y, Bancaud A (2011) Optimized micromirrors for three-dimensional single-particle tracking in living cells. Appl Phys Lett 98:243701CrossRefGoogle Scholar
  13. 13.
    Therizols P, Duong T, Dujon B, Zimmer C, Fabre E (2010) Chromosome arm length and nuclear constraints determine the dynamic relationship of yeast subtelomeres. Proc Natl Acad Sci U S A 107(5):2025–2030CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Weber SC, Thompson MA, Moerner WE, Spakowitz AJ, Theriot JA (2012) Analytical tools to distinguish the effects of localization error, confinement, and medium elasticity on the velocity autocorrelation function. Biophys J 102(11):2443–2450CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Backlund MP, Joyner R, Weis K, Moerner WE (2014) Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope. Mol Biol Cell 25(22):3619–3629CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Michaelis C, Ciosk R, Nasmyth K (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91(1):35–45CrossRefPubMedGoogle Scholar
  17. 17.
    Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135(6 Pt 2):1685–1700CrossRefPubMedGoogle Scholar
  18. 18.
    Lassadi I, Bystricky K (2011) Tracking of single and multiple genomic loci in living yeast cells. Methods Mol Biol 745:499–522CrossRefPubMedGoogle Scholar
  19. 19.
    Loiodice I, Dubarry M, Taddei A (2014) Scoring and manipulating gene position and dynamics using FROS in budding yeast. Curr Protoc Cell Biol 62: Unit 22 17 21–14Google Scholar
  20. 20.
    Dubarry M, Loiodice I, Chen CL, Thermes C, Taddei A (2011) Tight protein-DNA interactions favor gene silencing. Genes Dev 25(13):1365–1370CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Saad H, Gallardo F, Dalvai M, Tanguy-le-Gac N, Lane D, Bystricky K (2014) DNA dynamics during early double-strand break processing revealed by non-intrusive imaging of living cells. PLoS Genet 10(3):e1004187CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shaham S (2006) WormBook: methods in cell biology. The C. elegans Research Community (ed), WormBookGoogle Scholar
  23. 23.
    Winey M, Yarar D, Giddings TH Jr, Mastronarde DN (1997) Nuclear pore complex number and distribution throughout the Saccharomyces cerevisiae cell cycle by three-dimensional reconstruction from electron micrographs of nuclear envelopes. Mol Biol Cell 8(11):2119–2132CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhang B, Zerubia J, Olivo-Marin J (2007) Gaussian approximations of fluorescence microscope point-spread function models. Appl Opt 46(10):1819CrossRefPubMedGoogle Scholar
  25. 25.
    Backlund MP, Joyner R, Moerner WE (2015) Chromosomal locus tracking with proper accounting of static and dynamic errors. Phys Rev E Stat Nonlin Soft Matter Phys 91(6):062716CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS (2006) Long-range directional movement of an interphase chromosome site. Curr Biol 16(8):825–831CrossRefPubMedGoogle Scholar
  27. 27.
    Saner N, Karschau J, Natsume T, Gierlinski M, Retkute R, Hawkins M, Nieduszynski CA, Blow JJ, de Moura AP, Tanaka TU (2013) Stochastic association of neighboring replicons creates replication factories in budding yeast. J Cell Biol 202(7):1001–1012CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Renjie Wang
    • 1
  • Christophe Normand
    • 1
  • Olivier Gadal
    • 1
    Email author
  1. 1.Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI)Université de Toulouse, CNRS, UPSToulouseFrance

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