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Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells

Abstract

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein–protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10–120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1–2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.

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Fig. 1: Overview of the setup for BRET microscopy.
Fig. 2: Comparison of spectral characteristics of BRET assay constructs.
Fig. 3: Comparison of RlucII luminescence with different substrates.
Fig. 4: Comparison of BRET dynamic range between BRET1, BRET2 and ebBRET images.
Fig. 5: Comparison of Nluc luminescence with different substrates.
Fig. 6: Comparison between cooled CCD and EMCCD camera.
Fig. 7: Examples of BRET imaging in GPCR signaling.
Fig. 8: Example of image quantification.

Data availability

The authors declare that all data supporting the findings in this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by a Foundation grant from the Canadian Institutes for Health Research (CIHR) (FDN148431) to M.B. L.-P.P. received scholarships from CIHR and the Fonds de la Recherche du Quebec–Santé (FRQ-S). A.-M.S. received a postdoctoral fellowship from FRQ-S. M.B. holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology. We are grateful to the Canadian Space Agency (CSA), which lent us the EMCCD camera for BRET imaging, and to NϋVϋ Cameras for technical assistance and development of the camera driver for the MetaMorph software. We are grateful to M. Lagacé for her critical reading of the manuscript.

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M.B. and H.K. conceptualized the method, designed the experiments and wrote the manuscript. H.K. assembled the imaging system, performed the imaging experiments and analyzed the images. A.-M.S. and L.-P.P. designed and generated constructs for BRET microscopy. L.-P.P. performed the comparison between the spectrometric characteristics of the different luciferase constructs and participated in the writing of the manuscript.

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Correspondence to Michel Bouvier.

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The authors declare no competing interests.

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Journal peer review information: Nature Protocols thanks Francisco Ciruela Alférez, Thomas Machleidt and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Namkung, Y. et al. Nat. Commun. 7, 12178 (2016): https://doi.org/10.1038/ncomms12178

Beautrait, A. et al. Nat. Commun. 8, 15054 (2017): https://doi.org/10.1038/ncomms15054

Integrated supplementary information

Supplementary Figure 1 Examples of image quantification.

a, Recruitment of β-arrestin to the plasma membrane. HEK293 cells were transfected with AT1R, βarrestin2-RlucII and rGFP-CAAX. Quantification was carried out using the same field of cells as Fig. 7b (top). 10 μM final concentration of Me-O-eCTZ was added, and ebBRET images were obtained before (control) and after treatment with 100 nM of angiotensin II for 15 min. Quantification was carried out using the method described in Fig. 8. P value was calculated by paired t-test (two tails), n=6. b, GPCR endocytosis. HEK293 cells were transfected with AT1R-RlucII and rGFP-CAAX. Quantification was carried out using the same field of cells as Fig. 7b (middle). 10 μM final concentration of Me-O-eCTZ was added, and ebBRET images were obtained before (control) and after treatment with 100 nM of angiotensin II for 60 min. Quantification was carried out using the method described in Fig. 8. P value was calculated by unpaired t-test (two tails), n=4.

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Supplementary Figure 1

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Supplementary Data 1

Raw image data, the cell masks and the MATLAB script

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Kobayashi, H., Picard, LP., Schönegge, AM. et al. Bioluminescence resonance energy transfer–based imaging of protein–protein interactions in living cells. Nat Protoc 14, 1084–1107 (2019). https://doi.org/10.1038/s41596-019-0129-7

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