Multiplexing of Multicolor Bioluminescence Resonance Energy Transfer

Abstract

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.

The Parkinson's-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

“…For transient transfections, Flp-In-293 and SH-SY5Y cells (a kind gift of Mikko Hiltunen, University of Eastern Finland, Finland) were seeded onto 10-cm plates (3×10 6 cells/plate) and cultured for 24 h to 50-70% confluency. Cells were transfected with 5 µg of receptor constructs for 24 h using Lipofectamine 2000 according to the manufacturer's instructions, or alternatively, linear 25-kDa polyethyleneimine (PEI; Polysciences) as described previously (Breton et al, 2010). Both reagents were used at 1:3 DNA-to-reagent ratio.…”

Biased Signaling Favoring Gi over β-Arrestin Promoted by an Apelin Fragment Lacking the C-terminal Phenylalanine

“…The G␣ i -RlucII was previously described by Breton et al (37). G␤ 1 and G␥ 5 coding pcDNA3.1Ϯ vectors were obtained from Missouri University of Science and Technology.…”

“…A GFP10-G␥ 5 coding vector was subsequently produced by inserting the GFP10 sequence using the restriction sites NheI and Acc65I. The EPAC-based cAMP biosensor human ␤-arrestin1-RlucII and ␤-arrestin 2-RlucII plasmids were described by Breton et al (37) and Zimmerman et al (38).…”

“…cAMP production was monitored using the EPAC-BRET biosensor as previously described (37). Briefly, HEK293T cells were transfected with the EPAC-BRET construct along with the wild-type ApelinR coding plasmid.…”

A Perspective on Studying G-Protein–Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

“…For instance, the use of brighter versions of luminescent enzymes such as RLuc8 (Loening et al, 2006), RLuc8-S257G (Takai et al, 2015), and variants thereof such as RLuc2 (Leduc et al, 2009;Breton et al, 2010) in combination with luciferase substrates such as methoxy e-coelenterazine (Me-O-e-CTZ also known as purple coelenterazine), which is 13 times brighter than the coelenterazine-400a (deep-blue coelenterazine), will certainly contribute to shortening the image acquisition time and provide better spatial resolution.…”