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A temperature-inducible protein module for control of mammalian cell fate

Nature Methods volume 22, pages 539–549 (2025)Cite this article

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Abstract

Inducible protein switches are currently limited for use in tissues and organisms because common inducers cannot be controlled with precision in space and time in optically dense settings. Here, we introduce a protein that can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization using temperature) oligomerizes and translocates to the plasma membrane when temperature is lowered. We generated a library of Melt variants with switching temperatures ranging from 30 °C to 40 °C, including two that operate at and above 37 °C. Melt was a highly modular actuator of cell function, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements and cell death. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer. Melt represents a versatile thermogenetic module for straightforward, non-invasive and spatiotemporally defined control of mammalian cells with broad potential for biotechnology and biomedicine.

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Fig. 1: Harnessing BcLOV4 thermosensitivity to generate a purely temperature-inducible protein.
Fig. 2: Characterization of Melt membrane association.
Fig. 3: Thermal control over diverse intracellular processes using Melt.
Fig. 4: Tuning of Melt membrane binding and thermal switch point enables application of Melt-based tools in mammalian temperature ranges.
Fig. 5: Thermal regulation of cell fate using Melt.
Fig. 6: Thermal control of Melt and cell fate in animal models.

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Acknowledgements

The authors thank E. Berlew and B. Chow for helpful discussions on BcLOV4 activity and for plasmids encoding BcLOV(Q355N) and BcLOV-ITSN1; and A. Hughes and M. Good for helpful comments on the manuscript. The authors also thank the Penn Cytomics and Cell Sorting Shared Resource Laboratory for assistance with cell sorting. This work was supported by funding from the National Institutes of Health (R35GM138211 for L.J.B.), the National Science Foundation (Graduate Research Fellowship Program to W.B., CAREER 2145699 to L.J.B.), and the Penn Center for Precision Engineering for Health (CPE4H). Cell sorting was performed on a BD FACSAria Fusion that was obtained through NIH S10 1S10OD026986.

Author information

Author notes

  1. These authors contributed equally: William Benman, Zikang Huang.

Authors and Affiliations

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

    William Benman, Zikang Huang, Delaney Wilde, Thomas R. Mumford & Lukasz J. Bugaj

  2. Department of Biophysics, University of Pennsylvania, Philadelphia, PA, USA

    Pavan Iyengar

  3. Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Lukasz J. Bugaj

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

    Lukasz J. Bugaj

Authors
  1. William Benman
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Contributions

W.B. and L.J.B. conceived the study. W.B. generated Melt and its integration into molecular circuits. Z.H. discovered and characterized thermostable Melt variants, which were then integrated into circuits by Z.H. and W.B. W.B. and P.I. developed and validated the thermoPlate. D.W. and T.R.M. validated cluster-induced cell killing. W.B., Z.H. and P.I. performed and analyzed all experiments. L.J.B. supervised the work. W.B., Z.H. and L.J.B. wrote the manuscript and produced the figures. All of the authors edited the manuscript.

Corresponding author

Correspondence to Lukasz J. Bugaj.

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Nature Methods thanks G. Woolley, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Supplementary information

Supplementary Information

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Supplementary Video 1

Reversible membrane binding of Melt using temperature.

Supplementary Video 2

Temperature-controlled nucleocytoplasmic shuttling of MeltNLS/NES.

Supplementary Video 3

Thermal control of Erk activity in mammalian temperature ranges using MeltEGFR-37.

Supplementary Video 4

Temperature-controlled nucleocytoplasmic shuttling of MeltNLS/NES-40 in mammalian temperature ranges.

Supplementary Video 5

Reversible changes in cell size through thermal control of MeltITSN1-37.

Supplementary Video 6

Temperature-inducible cell death using MeltCasp1-37.

Source data

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Benman, W., Huang, Z., Iyengar, P. et al. A temperature-inducible protein module for control of mammalian cell fate. Nat Methods 22, 539–549 (2025). https://doi.org/10.1038/s41592-024-02572-4

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