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Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging

Abstract

Taking advantage of the phenomenon of bioluminescence resonance energy transfer (BRET), we developed a bioluminescent probe composed of EYFP and Renilla reniformis luciferase (RLuc)—BRET-based autoilluminated fluorescent protein on EYFP (BAF-Y)—for near-real-time single-cell imaging. We show that BAF-Y exhibits enhanced RLuc luminescence intensity and appropriate subcellular distribution when it was fused to targeting-signal peptides or histone H2AX, thus allowing high spatial and temporal resolution microscopy of living cells.

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Figure 1: Characterization of BAF-Y.
Figure 2: Improvement of BAF-Y using enhanced RLuc mutant.
Figure 3: Single-cell imaging with BAF-Y derivatives.

References

  1. Welsh, D.K., Yoo, S.H., Liu, A.C., Takahashi, J.S. & Kay, S.A. Curr. Biol. 14, 2289–2295 (2004).

    Article  CAS  Google Scholar 

  2. Welsh, D.K., Imaizumi, T. & Kay, S.A. Methods Enzymol. 393, 269–288 (2005).

    Article  CAS  Google Scholar 

  3. Bertrand, L. et al. J. Recept. Signal Transduct. Res. 22, 533–541 (2002).

    Article  CAS  Google Scholar 

  4. Jensen, A.A., Hansen, J.L., Sheikh, S.P. & Brauner-Osborne, H. Eur. J. Biochem. 269, 5076–5087 (2002).

    Article  CAS  Google Scholar 

  5. Nakamura, H., Wu, C., Murai, A., Inouye, S. & Shimomura, O. Tetrahedr. Lett. 38, 6405–6406 (1997).

    Article  CAS  Google Scholar 

  6. Gales, C. et al. Nat. Methods 2, 177–184 (2005).

    Article  CAS  Google Scholar 

  7. Perroy, J., Pontier, S., Charest, P.G., Aubry, M. & Bouvier, M. Nat. Methods 1, 203–208 (2004).

    Article  CAS  Google Scholar 

  8. De, A. & Gambhir, S.S. FASEB J. 19, 2017–2019 (2005).

    Article  CAS  Google Scholar 

  9. Ward, W.W. & Cormier, M.J. J. Biol. Chem. 254, 781–788 (1979).

    CAS  PubMed  Google Scholar 

  10. Lorenz, W.W., McCann, R.O., Longiaru, M. & Cormier, M.J. Proc. Natl. Acad. Sci. USA 88, 4438–4442 (1991).

    Article  CAS  Google Scholar 

  11. Xu, Y., Piston, D.W. & Johnson, C.H. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).

    Article  CAS  Google Scholar 

  12. Qing, G. et al. Nat. Biotechnol. 22, 877–882 (2004).

    Article  CAS  Google Scholar 

  13. Loening, A.M., Fenn, T.D., Wu, A.M. & Gambhir, S.S. Protein Eng. Des. Sel. 19, 391–400 (2006).

    Article  CAS  Google Scholar 

  14. Siino, J.S. et al. Biochem. Biophys. Res. Commun. 297, 1318–1323 (2002).

    Article  CAS  Google Scholar 

  15. Hoffman, R.M. Nat. Rev. Cancer 5, 796–806 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. Ogoh, K. Niwa and C. Wu (AIST) for discussion, S. Ohgiya (AIST) and K. Igarashi (Tohoku University) for discussion and valuable advice, T. Ishihara, T. Enomoto and H. Kubota (ATTO Corp.), for technical support with single-cell imaging, and T. Ikura (Tohoku University) for histone H2AX cDNA and S. Tashiro (Hiroshima University) for experimental advice using H2AX. This study was supported in part by a NEDO grant (Dynamic Biology Project; to Y.O.) from the Ministry of Economy, Trade and Industry of Japan.

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Authors and Affiliations

Authors

Contributions

H.H. developed BAF probes, designed and performed all the experiments and prepared the manuscript. Y.N. and Y.O. contributed to development and optimization of the Cellgraph systems. Y.O. directed the bioluminescence imaging project.

Corresponding author

Correspondence to Hideto Hoshino.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Methods. (PDF 582 kb)

Supplementary Movie 1

Time-lapse bioluminescence imaging of H2AX-eBAF-Y. Time-lapse images were acquired with 10 sec exposure at 1 min intervals using a Nikon S Fluor 40 × objective (N.A. 0.90). Sequential images were converted into a movie with MetaMorph software (Molecular Devices). Number in the movie represents the time point (min) when each image was obtained. Note that the bioluminescence images obtained using eBAF-Y gave high signal-to-noise ratio and high temporal resolution. (MOV 2496 kb)

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Hoshino, H., Nakajima, Y. & Ohmiya, Y. Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging. Nat Methods 4, 637–639 (2007). https://doi.org/10.1038/nmeth1069

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