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Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells

Nature Methods volume 10, pages 1232–1238 (2013)Cite this article

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Abstract

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.

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Figure 1: Design and biophysical characterization of tsGFP thermosensors.
Figure 2: Fluorescent properties of tsGFP1 in various conditions.
Figure 3: Temperature-dependent fluorescence characteristics of tsGFPs or GFPs targeted to subcellular organelles in HeLa cells.
Figure 4: tsGFP1-mito reveals heterogeneity in mitochondrial thermogenesis in HeLa cells.
Figure 5: Subcellular-targeted tsGFP1-mito visualizes endogenous thermogenesis in brown adipocytes.
Figure 6: Subcellular-targeted tsGFP1-ER visualizes endogenous thermogenesis in myotubes.

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Acknowledgements

We thank N. Okada (Kitasato University) for virulence plasmid from Salmonella enterica. Technical support was provided by S. Yano, Y. Suzuki and T. Sakoguchi (Kyoto University). This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan (to S.K. and Y.M.).

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Authors and Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

    Shigeki Kiyonaka, Taketoshi Kajimoto, Daisuke Shinmi, Itaru Hamachi & Yasuo Mori

  2. Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto, Japan

    Shigeki Kiyonaka & Yasuo Mori

  3. Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Tokyo, Japan

    Shigeki Kiyonaka & Yasuo Mori

  4. World Premier International Research Initiative-Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan

    Reiko Sakaguchi

  5. Department of Physiology, Shiga University of Medical Science, Shiga, Japan

    Mariko Omatsu-Kanbe & Hiroshi Matsuura

  6. The Hakubi Project, Kyoto University, Kyoto, Japan

    Hiromi Imamura

  7. Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan

    Takenao Yoshizaki

  8. Institute of Advanced Energy, Kyoto University, Uji, Japan

    Takashi Morii

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  1. Shigeki Kiyonaka
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Contributions

S.K., T.M. and Y.M. initiated and designed the project. S.K., T.K., R.S. and D.S. performed experiments and analyzed data. M.O.-K. and H.M. supervised brown adipocyte studies. H.I. supervised ATP sensor studies. T.Y., I.H. and T.M. contributed to analysis and interpretation of data. S.K., R.S. and Y.M. wrote the manuscript. Y.M. directed the research. All authors discussed and commented on the manuscript.

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Correspondence to Yasuo Mori.

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Kiyonaka, S., Kajimoto, T., Sakaguchi, R. et al. Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells. Nat Methods 10, 1232–1238 (2013). https://doi.org/10.1038/nmeth.2690

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