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Temperature-responsive optogenetic probes of cell signaling

Nature Chemical Biology volume 18, pages 152–160 (2022)Cite this article

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Abstract

We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

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Fig. 1: Single-component BcLOV4 fusions allow control of Ras and PI3K signaling.
Fig. 2: BcLOV-induced signaling dynamics depend on temperature and light exposure.
Fig. 3: BcLOV4 membrane translocation dynamics depend on temperature and light exposure.
Fig. 4: Modeling predicts BcLOV–SOScat-induced ppErk dynamics and reveals dynamic filtering properties of Ras/Erk signaling.
Fig. 5: BcLOV4 and BcLOV–SOScat in zebrafish embryos and Drosophila cells.
Fig. 6: BcLOV4 temperature sensitivity enables orthogonal multiplexing of multiple blue-light sensitive tools in single cells.

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Data availability

All raw data used to generate the figures can be found at the following link: https://drive.google.com/drive/folders/1h0eDceDplxYUguSpUNyg5CHA4uudZO39?usp=sharing

Code availability

MATLAB code used to fit data and model BcLOV4 activation dynamics can be found at the following link: https://drive.google.com/drive/folders/1h0eDceDplxYUguSpUNyg5CHA4uudZO39?usp=sharing

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Acknowledgements

We thank C. Seiler and the CHOP Aquatic Zebrafish Core for assistance in injecting and mounting zebrafish embryos, D. Wu and D. Chenoweth for assistance with TIRF microscopy and H. Johnson and J. Toettcher (Princeton) for providing Drosophila S2 cells. This work was supported by the National Institutes of Health (R35GM138211, R21GM132831 for L.J.B., R35GM133425 for H.D. and B.L., R01NS101106 for E.E.B and B.Y.C. and R01HL152086 to A.F.S.) and the National Science Foundation (Graduate Research Fellowship Program to W.B., CAREER MCB1652003 for E.E.B. and B.Y.C.).

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Authors and Affiliations

  1. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    William Benman, Erin E. Berlew, Ivan A. Kuznetsov, Brian Y. Chow & Lukasz J. Bugaj

  2. Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA

    Hao Deng & Bomyi Lim

  3. Department of Cell and Developmental Biology and Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA

    Caitlyn Parker & Arndt F. Siekmann

  4. Institute of Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Bomyi Lim, Arndt F. Siekmann & Lukasz J. Bugaj

  5. Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Lukasz J. Bugaj

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Contributions

W.B., B.Y.C. and L.J.B. conceived the study. W.B., E.E.B., H.D., I.A.K., A.F.S., C.P. and L.J.B. performed experiments and analyzed data. A.F.S., B.L., B.Y.C. and L.J.B. supervised the work. W.B. and L.J.B. wrote the manuscript and made figures, with editing from all authors.

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Correspondence to Lukasz J. Bugaj.

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Peer review information Nature Chemical Biology thanks Matias Zurbriggen, Maxwell Wilson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figures 1–16 and movie captions.

Reporting Summary

Supplementary Movie 1

Temperature-dependent decay of BcLOV-mCh membrane localization. HEK 293 T cells expressing BcLOV-mCh were stimulated with 488 nm light and imaged over 40 min. Membrane localization was sustained at lower temperatures (left), but decayed at higher temperatures (right). Time is mm:ss.

Supplementary Movie 2

BcLOV-mCh translocation in zebrafish embryos. 24 hpf zebrafish embryos expressing BcLOV-mCh were stimulated with 488 nm light and imaged with confocal microscopy. Time is mm:ss.

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Benman, W., Berlew, E.E., Deng, H. et al. Temperature-responsive optogenetic probes of cell signaling. Nat Chem Biol 18, 152–160 (2022). https://doi.org/10.1038/s41589-021-00917-0

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