Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects

Abstract

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular intermolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.

Fig. 1.

Schematic representation of the BRET fusion constructs used in this study. The bioluminescence spectra illustrate the emission spectra of the RLuc mutants and the red fluorescent acceptor proteins. All constructs contained an 18-aa linker inserted between the donor and acceptor proteins. Luciferase substrates used in each case were either CLZ or CLZ-v as indicated. Spectral resolution of each system is marked as a bidirectional arrow.

Fig. 2.

Characterization of the designed BRET systems. (A) Western blot analysis of protein expression for all BRET fusions and donor-only proteins contained in lysates of HT1080 cells stably expressing the reporter proteins. (B) Spectral imaging of HT1080 cells expressing either RLuc8.6 or BRET6 using 20-nm filers in the 460–720 nm range. EnduRen luciferase substrate was used for this experiment. Error bars = SD. (C) BRET ratios of the newly designed systems measured in cell culture. HT1080 cells (6.4 × 10⁴), stably expressing each of the BRET fusion and donor-only proteins (RLuc8 or RLuc8.6), were plated in black 96-well plates and imaged on the addition of CLZ or CLZ-v. The graph shows the average BRET ratios calculated as the A/D of the BRET system minus the A/D of donor-only cells. (D) BRET ratios calculated for the BRET6 system using various number of HT1080 cells. Experiments were performed as described in C. The dotted line represents the linear fitting for the data points. Error bars = SEM.

Fig. 3.

Performance of the BRET systems for deep-tissue imaging in mice. (A and B) Representative bioluminescence images of HT1080 cells stably expressing BRET fusion proteins accumulated in the lungs of nude mice. Cells (3 × 10⁶ in 150 μL PBS) were injected through the tail vein, resulting in significant trapping in the lungs. Mice were injected through the tail vein with luciferase substrate at 1.5 h after cell injection and imaged using sequentially open/donor/acceptor filters. Mice from (A) BRET3.1 (n = 10) and (B) BRET6.1 (n = 10) groups are shown. Average radiance from the thorax region was measured and used for calculating the BRET ratios. Substrate-only control mice (n = 5) were used for background subtraction. CLZ-v substrate was used. (C) Average A/Ds for BRET6, BRET6.1, RLuc8.6 (CLZ), and RLuc8.6 (CLZ-v) calculated from mice imaging experiments. *P = 3.1 × 10⁻⁹; **P = 3.5 × 10⁻¹⁰. (D) Average BRET ratios obtained from imaging of mice injected i.v. with cells expressing various BRET fusion proteins. A/D from mice injected with donor-only cells (RLuc8 or RLuc8.6) was subtracted. *P = 1.1 × 10⁻⁸; **P = 7.7 × 10⁻⁹. (E and F) Average A/Ds calculated from bioluminescence imaging of mice injected with increasing number of cells expressing either RLuc8.6 (n = 4 for each cell number) or BRET6.1 cells (n = 4 for each cell number). CLZ-v was used as substrate. The dotted line represents the linear fit for the data points. Error bars = SEM.

Fig. 4.

Characterization of the genetically encoded FRB-FKBP12 BRET6 sensor. (A) Schematic illustration of the BRET6 sensor for monitoring the rapamycin-induced FRB-FKBP12 association. (B) Rapamycin dose–response curve for BRET6 FRB-FKBP12 sensor in HT1080 cells. HT1080 cells (1 × 10⁵) expressing both FRB-RLuc8.6 and FKBP12-TurboFP635 sensor components were plated in black 24-well plates and incubated with increasing concentrations of rapamycin. After 6 h, the plates were imaged using donor- and acceptor-specific filters on an IVIS-200 imager. A/Ds were calculated and plotted against rapamycin concentration. The data were fitted to a sigmoidal curve fitting (EC₅₀ = 0.7 ± 0.2 nM); error bars = SD. (C) Inhibition of rapamycin-induced FRB-FKBP12 association by FK506. HT1080 cells (1 × 10⁵) were incubated for 6 h with rapamycin (0, 0.5, and 1 nM) and with and without FK506 (10 μM), and they were imaged as described above. A/Ds were calculated for each condition; error bars = SD. *P = 2.2 × 10⁻³; **P = 4.1 × 10⁻³. (D) Representative bioluminescence images of HT1080 cells stably expressing FRB-FKBP12 BRET6 sensor accumulated in the lungs of nude mice. Cells (3 × 10⁶ in 150 μL PBS) were injected through the tail vein, resulting in significant trapping in the lungs. One group of mice (n = 8) was injected 2 h before cell injection with 40 μg rapamycin dissolved in 20 μL DMSO and further diluted in 130 μL PBS administered through the tail vein. A second group of mice (n = 8) was injected with DMSO (20 μL in 130 μL PBS). Two hours after cells injection, the mice were injected i.v. with CLZ luciferase substrate and sequentially imaged using open/donor/acceptor filters. Substrate-only control mice (n = 4) were used for background subtraction. (E) Average A/D values for BRET6 FRB-FKBP12 sensor (rapamycin and DMSO-treated groups) and donor-only FRB-RLuc8.6 calculated from mice lung-trapping model imaging experiments; error bars = SEM. *P = 1.7 × 10⁻⁴; **P = 2.7 × 10⁻⁴.