Imaging Gene Expression pp 101-113

Part of the Methods in Molecular Biology book series (MIMB, volume 1042)

Imaging Nascent RNA Dynamics in Dictyostelium

  • Jonathan R. Chubb
  • Michelle Stevense
  • Danielle Cannon
  • Tetsuya Muramoto
  • Adam M. Corrigan
Protocol

Abstract

Dictyostelium cells have great utility for live imaging of single gene transcriptional dynamics. The cells allow efficient molecular genetics, for targeting of RNA reporters and fluorescent proteins to individual, defined loci. Dictyostelium cells share many signalling, chromatin and nuclear characteristics of larger eukaryotes, yet the cells have a relatively simple scattered differentiation programme, allowing imaging of transcriptional events in the context of stochastic developmental choices. This review will detail the methods and considerations for imaging nascent RNA dynamics at single genes in living Dictyostelium cells.

Key words

Chromatin Dictyostelium Morphogen Live-cell imaging Transcriptional bursting Pulsing Stochastic gene expression Transcriptional noise Transcriptional pausing 

References

  1. 1.
    Bertrand E et al (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445PubMedCrossRefGoogle Scholar
  2. 2.
    Chubb JR, Trcek T, Shenoy SM, Singer RH (2006) Transcriptional pulsing of a developmental gene. Curr Biol 16(10):1018–1025PubMedCrossRefGoogle Scholar
  3. 3.
    Golding I, Paulsson J, Zawilski SM, Cox EC (2005) Real-time kinetics of gene activity in individual bacteria. Cell 123(6):1025–1036PubMedCrossRefGoogle Scholar
  4. 4.
    Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4(10):e309PubMedCrossRefGoogle Scholar
  5. 5.
    Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135(2):216–226PubMedCrossRefGoogle Scholar
  6. 6.
    Chubb JR, Liverpool TB (2010) Bursts and pulses: insights from single cell studies into transcriptional mechanisms. Curr Opin Genet Dev 20(5):478–484PubMedCrossRefGoogle Scholar
  7. 7.
    Lionnet T, Singer RH (2012) Transcription goes digital. EMBO Rep 13(4):313–321PubMedCrossRefGoogle Scholar
  8. 8.
    Balazsi G, van Oudenaarden A, Collins JJ (2011) Cellular decision making and biological noise: from microbes to mammals. Cell 144(6):910–925PubMedCrossRefGoogle Scholar
  9. 9.
    Huh D, Paulsson J (2011) Non-genetic heterogeneity from stochastic partitioning at cell division. Nat Genet 43(2):95–100PubMedCrossRefGoogle Scholar
  10. 10.
    Stavreva DA et al (2009) Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription. Nat Cell Biol 11(9):1093–1102PubMedCrossRefGoogle Scholar
  11. 11.
    Harper CV et al (2011) Dynamic analysis of stochastic transcription cycles. PLoS Biol 9(4):e1000607PubMedCrossRefGoogle Scholar
  12. 12.
    Stevense M, Chubb JR, Muramoto T (2011) Nuclear organization and transcriptional dynamics in Dictyostelium. Dev Growth Differ 53(4):576–586PubMedCrossRefGoogle Scholar
  13. 13.
    Muramoto T, Chubb JR (2008) Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint. Development 135(9):1647–1657PubMedCrossRefGoogle Scholar
  14. 14.
    Stevense M, Muramoto T, Muller I, Chubb JR (2010) Digital nature of the immediate-early transcriptional response. Development 137(4):579–584PubMedCrossRefGoogle Scholar
  15. 15.
    Muramoto T, Muller I, Thomas G, Melvin A, Chubb JR (2010) Methylation of H3K4 Is required for inheritance of active transcriptional states. Curr Biol 20(5):397–406PubMedCrossRefGoogle Scholar
  16. 16.
    Muramoto T et al (2012) Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation. Proc Natl Acad Sci USA 109(19):7350–7355PubMedCrossRefGoogle Scholar
  17. 17.
    Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332(6028):475–478PubMedCrossRefGoogle Scholar
  18. 18.
    Lionnet T et al (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8(2):165–170PubMedCrossRefGoogle Scholar
  19. 19.
    Sussman R, Sussman M (1967) Cultivation of Dictyostelium discoideum in axenic medium. Biochem Biophys Res Commun 29(1):53–55PubMedCrossRefGoogle Scholar
  20. 20.
    Thomson I, Gilchrist S, Bickmore WA, Chubb JR (2004) The radial positioning of chromatin is not inherited through mitosis but is established de novo in early G1. Curr Biol 14(2):166–172PubMedCrossRefGoogle Scholar
  21. 21.
    Muller I, Boyle S, Singer RH, Bickmore WA, Chubb JR (2010) Stable morphology, but dynamic internal reorganisation, of interphase human chromosomes in living cells. PLoS One 5(7):e11560PubMedCrossRefGoogle Scholar
  22. 22.
    Faix J, Kreppel L, Shaulsky G, Schleicher M, Kimmel AR (2004) A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system. Nucleic Acids Res 32(19):e143PubMedCrossRefGoogle Scholar
  23. 23.
    Spencer SL, Gaudet S, Albeck JG, Burke JM, Sorger PK (2009) Non-genetic origins of cell-to-cell variability in TRAIL-induced apoptosis. Nature 459(7245):428–432PubMedCrossRefGoogle Scholar
  24. 24.
    Sigal A et al (2006) Variability and memory of protein levels in human cells. Nature 444(7119):643–646PubMedCrossRefGoogle Scholar
  25. 25.
    Williams JG (2006) Transcriptional regulation of Dictyostelium pattern formation. EMBO Rep 7(7):694–698PubMedCrossRefGoogle Scholar
  26. 26.
    Verkerke-van Wijk I, Brandt R, Bosman L, Schaap P (1998) Two distinct signaling pathways mediate DIF induction of prestalk gene expression in Dictyostelium. Exp Cell Res 245(1):179–185PubMedCrossRefGoogle Scholar
  27. 27.
    Alcantara F, Monk M (1974) Signal propagation during aggregation in the slime mould Dictyostelium discoideum. J Gen Microbiol 85(2):321–334PubMedCrossRefGoogle Scholar
  28. 28.
    Maiuri P et al (2011) Fast transcription rates of RNA polymerase II in human cells. EMBO Rep 12(12):1280–1285PubMedCrossRefGoogle Scholar
  29. 29.
    Darzacq X et al (2007) In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol 14(9):796–806PubMedCrossRefGoogle Scholar
  30. 30.
    Yunger S, Rosenfeld L, Garini Y, Shav-Tal Y (2010) Single-allele analysis of transcription kinetics in living mammalian cells. Nat Methods 7(8):631–633PubMedCrossRefGoogle Scholar
  31. 31.
    Boireau S et al (2007) The transcriptional cycle of HIV-1 in real-time and live cells. J Cell Biol 179(2):291–304PubMedCrossRefGoogle Scholar
  32. 32.
    Phair RD, Misteli T (2000) High mobility of proteins in the mammalian cell nucleus. Nature 404(6778):604–609PubMedCrossRefGoogle Scholar
  33. 33.
    Daigle N, Ellenberg J (2007) LambdaN-GFP: an RNA reporter system for live-cell imaging. Nat Methods 4(8):633–636PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Jonathan R. Chubb
    • 1
  • Michelle Stevense
    • 2
  • Danielle Cannon
    • 3
  • Tetsuya Muramoto
    • 4
  • Adam M. Corrigan
    • 1
  1. 1.MRC Laboratory for Molecular Cell Biology, Department of Cell and Developmental BiologyUniversity College LondonLondonUK
  2. 2.Institut für Physiologische Chemie, Medizinische FakultätTU DresdenDresdenGermany
  3. 3.Department of Cell and Developmental Biology and MRC Laboratory for Molecular Cell BiologyUniversity College LondonLondonUK
  4. 4.Laboratory for Cell Signaling Dynamics, Quantitative Biology CenterRIKENOsakaJapan

Personalised recommendations