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Optogenetic Downregulation of Protein Levels to Control Programmed Cell Death in Mammalian Cells with a Dual Blue-Light Switch

Part of the Methods in Molecular Biology book series (MIMB,volume 2173)

Abstract

Optogenetic approaches facilitate the study of signaling and metabolic pathways in animal cell systems. In the past 10 years, a plethora of light-regulated switches for the targeted control over the induction of gene expression, subcellular localization of proteins, membrane receptor activity, and other cellular processes have been developed and successfully implemented. However, only a few tools have been engineered toward the quantitative and spatiotemporally resolved downregulation of proteins. Here we present a protocol for reversible and rapid blue light-induced reduction of protein levels in mammalian cells. By implementing a dual-regulated optogenetic switch (Blue-OFF), both repression of gene expression and degradation of the target protein are triggered simultaneously. We apply this system for the blue light-mediated control of programmed cell death. HEK293T cells are transfected with the proapoptotic proteins PUMA and BID integrated into the Blue-OFF system. Overexpression of these proteins leads to programmed cell death, which can be prevented by irradiation with blue light. This experimental approach is very straightforward, requires just simple hardware, and therefore can be easily implemented in state-of-the-art equipped mammalian cell culture labs. The system can be used for targeted cell signaling studies and biotechnological applications.

Key words

  • Optogenetics
  • Protein downregulation
  • Blue-light degron
  • Blue-light gene repression
  • Blue-OFF
  • Dual optogenetic switch
  • Optogenetic apoptosis control

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  • DOI: 10.1007/978-1-0716-0755-8_11
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Acknowledgments

We thank J. Andres, L. Koch, and T. Blomeier for experimental support and fruitful discussions, and R. Wurm, M. Gerads, and J. Müller for valuable experimental support. This work was supported by the German Research Foundation (DFG) (grant ZU259/2-1 to M.D.Z. and under Germany’s Excellence Strategy CEPLAS—EXC-2048/1—Project ID 390686111 to M.D.Z., BIOSS—EXC-294 and CIBSS—EXC-2189—Project ID 390939984 to W.W.) and the European Commission—Research Executive Agency (H2020 Future and Emerging Technologies (FET-Open) Project ID 801041 CyGenTiG to J.A.D. and M.D.Z.).

Author contributions: P.F. designed the system and performed the experiments, analyzed the data, and wrote the protocol. P.G. and J.B. designed the system. J.D. designed experiments and analyzed the data. W.W. designed the system and experiments, and analyzed the data. M.D.Z. designed the system and experiments, analyzed the data, and wrote the protocol.

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Correspondence to Matias D. Zurbriggen .

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Fischbach, P., Gonschorek, P., Baaske, J., Davies, J.A., Weber, W., Zurbriggen, M.D. (2020). Optogenetic Downregulation of Protein Levels to Control Programmed Cell Death in Mammalian Cells with a Dual Blue-Light Switch. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-0755-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0754-1

  • Online ISBN: 978-1-0716-0755-8

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