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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The authors declare that all data supporting the findings in this study are available from the corresponding author upon request.
Förster, T. Zwischenmolekulare energiewanderung und fluoreszenz. Ann. Phys. 437, 55–75 (1948).
Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).
Berney, C. & Danuser, G. FRET or no FRET: a quantitative comparison. Biophys. J. 84, 3992–4010 (2003).
Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).
Xu, Y., Piston, D. W. & Johnson, C. H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).
Ayoub, M. A. et al. Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 21522–21528 (2002).
Mercier, J.-F., Salahpour, A., Angers, S., Breit, A. & Bouvier, M. Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J. Biol. Chem. 277, 44925–44931 (2002).
Galés, C. et al. Real-time monitoring of receptor and G-protein interactions in living cells. Nat. Methods 2, 177–184 (2005).
Galés, C. et al. Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat. Struct. Mol. Biol. 13, 778–786 (2006).
Kobayashi, H., Ogawa, K., Yao, R., Lichtarge, O. & Bouvier, M. Functional rescue of beta-adrenoceptor dimerization and trafficking by pharmacological chaperones. Traffic 10, 1019–1033 (2009).
Hamdan, F. F., Audet, M., Garneau, P., Pelletier, J. & Bouvier, M. High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J. Biomol. Screen. 10, 463–475 (2005).
Charest, P. G. & Bouvier, M. Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation. J. Biol. Chem. 278, 41541–41551 (2003).
Terrillon, S., Barberis, C. & Bouvier, M. Heterodimerization of V1a and V2 vasopressin receptors determines the interaction with beta-arrestin and their trafficking patterns. Proc. Natl. Acad. Sci. USA 101, 1548–1553 (2004).
Bertrand, L. et al. The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J. Recept. Signal Transduct. Res. 22, 533–541 (2002).
Azzi, M. et al. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA 100, 11406–11411 (2003).
Héroux, M., Breton, B., Hogue, M. & Bouvier, M. Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET. Biochemistry 46, 7022–7033 (2007).
Perroy, J., Pontier, S., Charest, P. G., Aubry, M. & Bouvier, M. Real-time monitoring of ubiquitination in living cells by BRET. Nat. Methods 1, 203–208 (2004).
Stoddart, L. A. et al. Application of BRET to monitor ligand binding to GPCRs. Nat. Methods 12, 661–663 (2015).
Stoddart, L. A., Kilpatrick, L. E. & Hill, S. J. NanoBRET approaches to study ligand binding to GPCRs and RTKs. Trends Pharmacol. Sci. 39, 136–147 (2018).
Loening, A. M., Fenn, T. D., Wu, A. M. & Gambhir, S. S. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng. Des. Sel. 19, 391–400 (2006).
Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).
Xu, X. et al. Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc. Natl. Acad. Sci. USA 104, 10264–10269 (2007).
Coulon, V. et al. Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. Biophys. J. 94, 1001–1009 (2008).
Kim, J. & Grailhe, R. Nanoluciferase signal brightness using furimazine substrates opens bioluminescence resonance energy transfer to widefield microscopy. Cytometry A 89, 742–746 (2016).
Goyet, E., Bouquier, N., Ollendorff, V. & Perroy, J. Fast and high resolution single-cell BRET imaging. Sci. Rep. 6, 28231 (2016).
Namkung, Y. et al. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nat. Commun. 7, 12178 (2016).
Beautrait, A. et al. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling. Nat. Commun. 8, 15054 (2017).
De, A., Ray, P., Loening, A. M. & Gambhir, S. S. BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals. FASEB J. 23, 2702–2709 (2009).
Yeh, H.-W. et al. Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging. Nat. Methods 14, 971–974 (2017).
Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol. 20, 473–477 (2002).
Kerppola, T. K. Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell Biol. 7, 449–456 (2006).
Pelletier, J. N., Campbell-Valois, F. X. & Michnick, S. W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).
Ozawa, T., Kaihara, A., Sato, M., Tachihara, K. & Umezawa, Y. Split luciferase as an optical probe for detecting protein–protein interactions in mammalian cells based on protein splicing. Anal. Chem. 73, 2516–2521 (2001).
Paulmurugant, R. & Gambhir, S. S. Monitoring protein–protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal. Chem. 75, 1584–1589 (2003).
Dixon, A. S. et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 400–408 (2016).
Kerppola, T. K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Protoc. 1, 1278–1286 (2006).
Rebois, R. V. et al. Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells. Methods 45, 214–218 (2008).
Héroux, M., Hogue, M., Lemieux, S. & Bouvier, M. Functional calcitonin gene-related peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like receptor homo-oligomer and a monomer of receptor activity-modifying protein-1. J. Biol. Chem. 282, 31610–31620 (2007).
Armando, S. et al. The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and βarrestin. FASEB J. 28, 4509–4523 (2014).
Fichter, K. M., Flajolet, M., Greengard, P. & Vu, T. Q. Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots. Proc. Natl. Acad. Sci. USA 107, 18658–18663 (2010).
Ahn, S., Shenoy, S. K., Wei, H. & Lefkowitz, R. J. Differential kinetic and spatial patterns of β-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J. Biol. Chem. 279, 35518–35525 (2004).
Lohse, M. J., Maiellaro, I. & Calebiro, D. Kinetics and mechanism of G protein-coupled receptor activation. Curr. Opin. Cell Biol. 27, 87–93 (2014).
Breton, B. et al. Multiplexing of multicolor bioluminescence resonance energy transfer. Biophys. J. 99, 4037–4046 (2010).
Leduc, M. et al. Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands. J. Pharmacol. Exp. Ther. 331, 297–307 (2009).
Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).
Inouye, S. & Shimomura, O. The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem. Biophys. Res. Commun. 233, 349–353 (1997).
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
Pfleger, K. D. G. & Eidne, K. A. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat. Methods 3, 165–174 (2006).
Molinari, P., Casella, I. & Costa, T. Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells. Biochem. J. 409, 251–261 (2008).
Wampler, J. E., Hori, K., Lee, J. W. & Cormier, M. J. Structured bioluminescence. Two emitters during both the in vitro and the in vivo bioluminescence of the sea pansy, Renilla. Biochemistry 10, 2903–2909 (1971).
Yamakawa, Y., Ueda, H., Kitayama, A. & Nagamune, T. Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. J. Biosci. Bioeng. 93, 537–542 (2002).
Li, F. et al. Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease. Anal. Chim. Acta 724, 104–110 (2012).
Inouye, S., Sato, J., Sahara-Miura, Y., Yoshida, S. & Hosoya, T. Luminescence enhancement of the catalytic 19kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions. Biochem. Biophys. Res. Commun. 445, 157–162 (2014).
Inouye, S., Watanabe, K., Nakamura, H. & Shimomura, O. Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase. FEBS Lett. 481, 19–25 (2000).
Czupryna, J. & Tsourkas, A. Firefly luciferase and Rluc8 exhibit differential sensitivity to oxidative stress in apoptotic cells. PLoS ONE 6, e20073 (2011).
Shigehisa, M. et al. Stabilization of luciferase from Renilla reniformis using random mutations. Protein Eng. Des. Sel. 30, 7–13 (2017).
Hu, M.-J. et al. Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation. Amino Acids 50, 1111–1119 (2018).
Stoddart, L. A. et al. Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells. Sci. Rep. 8, 1572 (2018).
Chu, J. et al. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat. Biotechnol. 34, 760–767 (2016).
Laviv, T. et al. Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat. Methods 13, 989–992 (2016).
Machleidt, T. et al. NanoBRET—a novel BRET platform for the analysis of protein–protein interactions. ACS Chem. Biol. 10, 1797–1804 (2015).
Robbins, M. S. & Hadwen, B. J. The noise performance of electron multiplying charge-coupled devices. IEEE Trans. Electron Devices 50, 1227–1232 (2003).
Basden, A. G., Haniff, C. A. & Mackay, C. D. Photon counting strategies with low-light-level CCDs. Mon. Not. R. Astron. Soc. 345, 985–991 (2003).
Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).
Schink, K. O., Raiborg, C. & Stenmark, H. Phosphatidylinositol 3-phosphate, a lipid that regulates membrane dynamics, protein sorting and cell signalling. Bioessays 35, 900–912 (2013).
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.
The authors declare no competing interests.
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
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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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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