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Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics

Nature Chemistry volume 9, pages 279–286 (2017)Cite this article

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Abstract

Alterations in glutathione (GSH) homeostasis are associated with a variety of diseases and cellular functions, and therefore, real-time live-cell imaging and quantification of GSH dynamics are important for understanding pathophysiological processes. However, existing fluorescent probes are unsuitable for these purposes due to their irreversible fluorogenic mechanisms or slow reaction rates. In this work, we have successfully overcome these problems by establishing a design strategy inspired by Mayr's work on nucleophilic reaction kinetics. The synthesized probes exhibit concentration-dependent, reversible and rapid absorption/fluorescence changes (t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values (1–10 mM: within the range of intracellular GSH concentrations). We also developed FRET-based ratiometric probes, and demonstrated that they are useful for quantifying GSH concentration in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds.

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Figure 1: Si-rhodamine as a candidate for rapid and reversible detection of GSH.
Figure 2: Properties of the QuicGSH probes (buffer: 0.2 M sodium phosphate buffer, pH 7.4, containing 5% DMSO).
Figure 3: Quantification of GSH in several cell lines.
Figure 4: Real-time GSH imaging with QG3.0 (1 μM) in A549 cells upon H2O2 treatment with a perfusion system.
Figure 5: Real-time GSH imaging with QG3.0 (1 μM) in A549 cells under glucose-deprivation with a perfusion system.

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Change history

  • 13 December 2016

    In the version of this Article originally published, the y-axis of the middle panel in Fig. 1d was mislabelled. Molecular structures were also not present above Table 1. Both errors have been fixed in all versions of the Article.

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Acknowledgements

This research was supported in part by AMED-CREST, by JST, PRESTO, by MEXT/JSPS KAKENHI grant numbers JP16H02606 and JP26111012 (to Y.U.), JP15H05951 ‘Resonance Bio’ (to M.K.), by JSPS Core-to-Core Program, by a grant from Hoansha Foundation (to Y.U.). The authors thank H. Takahashi for advices on intracellular imaging and statistical analysis, A. Morozumi for providing 2′Me SiR620, and Y. Kagami for advice on synthesis of Si-rhodamines.

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Author notes

  1. Keitaro Umezawa and Masafumi Yoshida: These authors contributed equally to this work

Authors and Affiliations

  1. Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113 0033, Japan

    Keitaro Umezawa, Masafumi Yoshida, Mako Kamiya & Yasuteru Urano

  2. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113 0033, Japan

    Keitaro Umezawa & Yasuteru Urano

  3. Department of Otolaryngology - Head and Neck Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113 0033, Japan

    Masafumi Yoshida & Tatsuya Yamasoba

  4. PRESTO, Japan Science and Technology (JST) Agency, Honcho, Kawaguchi-shi, Saitama, 332 0012, Japan

    Mako Kamiya

  5. CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100 0004, Japan

    Yasuteru Urano

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Contributions

K.U., M.Y., M.K. and Y.U. designed the research. K.U. and M.Y. performed experiments and analysed the data. M.K., T.Y. and Y.U. supervised the project. All authors discussed the results and co-wrote the manuscript.

Corresponding authors

Correspondence to Mako Kamiya or Yasuteru Urano.

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Umezawa, K., Yoshida, M., Kamiya, M. et al. Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics. Nature Chem 9, 279–286 (2017). https://doi.org/10.1038/nchem.2648

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