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Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

  • Mustafa Mir
  • Armando Reimer
  • Michael Stadler
  • Astou Tangara
  • Anders S. Hansen
  • Dirk Hockemeyer
  • Michael B. Eisen
  • Hernan Garcia
  • Xavier DarzacqEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1814)

Abstract

In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.

Key words

Single molecule imaging Single molecule kinetics Lattice light-sheet microscopy Drosophila melanogaster Live embryo imaging Single molecule fluorescence Transcription factor dynamics Single particle tracking Selective plane illumination microscopy 

Notes

Acknowledgments

The authors thank the Betzig lab at HHMI Janelia Research Campus for designs and advice on setting up the LLSM. We thank all members of the Darzacq, Tjian, Garcia, and Eisen labs for reagents, suggestions, and useful discussions. This work was supported by the California Institute of Regenerative Medicine (CIRM) LA1-08013 and the National Institutes of Health (NIH) UO1-EB021236 & U54-DK107980 to X.D., by the Burroughs Wellcome Fund Career Award at the Scientific Interface, the Sloan Research Foundation, the Human Frontiers Science Program, the Searle Scholars Program, and the Shurl and Kay Curci Foundation to H.G., a Howard Hughes Medical Institute investigator award to M.E., NSF Graduate Research Fellowships A.R. D.H. is a Pew-Stewart Scholar for Cancer Research supported by the Pew Charitable Trusts, the Alexander and Margaret Stewart Trust, the Siebel Stem Cell Institute, and NIH R01-CA196884.

References

  1. 1.
    Izeddin I, Recamier V, Bosanac L, Cisse II, Boudarene L, Dugast-Darzacq C, Proux F, Benichou O, Voituriez R, Bensaude O, Dahan M, Darzacq X (2014) Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus. eLife 3:e02230. https://doi.org/10.7554/eLife.02230 CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Chen JJ, Zhang ZJ, Li L, Chen BC, Revyakin A, Hajj B, Legant W, Dahan M, Lionnet T, Betzig E, Tjian R, Liu Z (2014) Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156(6):1274–1285. https://doi.org/10.1016/j.cell.2014.01.062 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Morisaki T, Muller WG, Golob N, Mazza D, McNally JG (2014) Single-molecule analysis of transcription factor binding at transcription sites in live cells. Nat Commun 5:4456. https://doi.org/10.1038/Ncomms5456 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mazza D, Abernathy A, Golob N, Morisaki T, McNally JG (2012) A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res 40(15):e119. https://doi.org/10.1093/nar/gks701 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hansen AS, Pustova I, Cattoglio C, Tjian R, Darzacq X (2017) CTCF and cohesin regulate chromatin loop stability with distinct dynamics. eLife 6:e25776. https://doi.org/10.7554/eLife.25776 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cho WK, Jayanth N, English BP, Inoue T, Andrews JO, Conway W, Grimm JB, Spille JH, Lavis LD, Lionnet T, Cisse II (2016) RNA polymerase II cluster dynamics predict mRNA output in living cells. eLife 5:e13617. https://doi.org/10.7554/eLife.13617 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cisse II, Izeddin I, Causse SZ, Boudarene L, Senecal A, Muresan L, Dugast-Darzacq C, Hajj B, Dahan M, Darzacq X (2013) Real-time dynamics of RNA polymerase II clustering in live human cells. Science 341(6146):664–667. https://doi.org/10.1126/science.1239053 CrossRefPubMedGoogle Scholar
  8. 8.
    Chen BC, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XFS, Hammer JA, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins RD, Mitchell DM, Bembenek JN, Reymann AC, Bohme R, Grill SW, Wang JT, Seydoux G, Tulu US, Kiehart DP, Betzig E (2014) Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science 346(6208):439–439. https://doi.org/10.1126/science.1257998 CrossRefGoogle Scholar
  9. 9.
    Mir M, Reimer A, Haines JE, Li X-Y, Stadler M, Garcia H, Eisen MB, Darzacq X (2017) Dense Bicoid hubs accentuate binding along the morphogen gradient. Genes Dev 31(17):1784–1794CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Katz ZB, Wells AL, Park HY, Wu B, Shenoy SM, Singer RH (2012) Beta-actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration. Genes Dev 26(17):1885–1890. https://doi.org/10.1101/gad.190413.112 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Lucas JS, Zhang YJ, Dudko OK, Murre C (2014) 3D trajectories adopted by coding and regulatory DNA elements: first-passage times for genomic interactions. Cell 158(2):339–352. https://doi.org/10.1016/j.cell.2014.05.036 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chenouard N, Smal I, de Chaumont F, Maska M, Sbalzarini IF, Gong YH, Cardinale J, Carthel C, Coraluppi S, Winter M, Cohen AR, Godinez WJ, Rohr K, Kalaidzidis Y, Liang L, Duncan J, Shen HY, Xu YK, Magnusson KEG, Jalden J, Blau HM, Paul-Gilloteaux P, Roudot P, Kervrann C, Waharte F, Tinevez JY, Shorte SL, Willemse J, Celler K, van Wezel GP, Dan HW, Tsai YS, de Solorzano CO, Olivo-Marin JC, Meijering E (2014) Objective comparison of particle tracking methods. Nat Methods 11(3):281–U247. https://doi.org/10.1038/nmeth.2808 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hansen AS, Woringer M, Grimm JB, Lavis LD, Tjian R, Darzacq X (2017) Spot-On: robust model-based analysis of single-particle tracking experiments. bioRxiv. https://doi.org/10.1101/171983
  14. 14.
    Persson F, Linden M, Unoson C, Elf J (2013) Extracting intracellular diffusive states and transition rates from single-molecule tracking data. Nat Methods 10(3):265–269. https://doi.org/10.1038/Nmeth.2367 CrossRefPubMedGoogle Scholar
  15. 15.
    Serge A, Bertaux N, Rigneault H, Marguet D (2008) Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat Methods 5(8):687–694. https://doi.org/10.1038/nmeth.1233 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mustafa Mir
    • 1
  • Armando Reimer
    • 2
  • Michael Stadler
    • 1
  • Astou Tangara
    • 1
  • Anders S. Hansen
    • 1
  • Dirk Hockemeyer
    • 1
  • Michael B. Eisen
    • 1
    • 2
    • 3
    • 4
  • Hernan Garcia
    • 1
    • 2
    • 5
  • Xavier Darzacq
    • 1
    Email author
  1. 1.Department of Molecular and Cell BiologyUniversity of CaliforniaBerkeleyUSA
  2. 2.Biophysics Graduate GroupUniversity of CaliforniaBerkeleyUSA
  3. 3.Howard Hughes Medical InstituteUniversity of CaliforniaBerkeleyUSA
  4. 4.Department of Integrative BiologyUniversity of CaliforniaBerkeleyUSA
  5. 5.Department of PhysicsUniversity of CaliforniaBerkeleyUSA

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