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

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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.

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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

  1. Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Osaka, 563-8577, Japan

    Hideto Hoshino, Yoshihiro Nakajima & Yoshihiro Ohmiya

  2. Department of Photobiology, Hokkaido University, Graduate School of Medicine, Sapporo, 060-8638, Japan

    Yoshihiro Ohmiya

Authors
  1. Hideto Hoshino
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  2. Yoshihiro Nakajima
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  3. Yoshihiro Ohmiya
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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.

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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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  • DOI: https://doi.org/10.1038/nmeth1069

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