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. 2007 Aug 1;67(15):7175-83.
doi: 10.1158/0008-5472.CAN-06-4623.

An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects

Abhijit De  1 , Andreas Markus Loening, Sanjiv Sam Gambhir
Affiliations

An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects

Abhijit De et al. Cancer Res. .

Abstract

Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP(2) acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals.

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Figures

Figure 1
Figure 1
Mammalian expression of the new BRET vectors using Renilla luciferase mutants as donor. A, fluorescent photomicrographs of transiently transfected 293T cells expressing either GFP2-RLUC or GFP2-RLUC8 fusion 24 h after transfection. B, semiquantitative Western blot analysis showing donor (RLUC) and acceptor (GFP2) protein expression in 293T cells transiently transfected with donor alone and fusion plasmids as marked. GFP2-Rluc-C, GFP2-Rluc-M, and GFP2-Rluc8 indicate BRET fusions using the single-mutation C124A RLUC, double-mutation C124A/M185V RLUC, and eight-mutation RLUC8 donors, respectively. α-Tubulin was used as loading control. C, the same cells as mentioned in (B) were plated (10,000 per well in a 48-well plate) and imaged with a CCD camera after adding equal amount of Clz400 substrate in each well. Mean photon values were determined by drawing regions of interest over triplicate samples. The chart represents the normalized mean BRET ratio (columns) and RLUC emission light outputs (line). Bars, SEM. D, semiquantitative assessment of BRET donor and acceptor proteins by Western blotting in selected clonal populations of HT1080 cells expressing the fusion constructs. α-Tubulin was used as a loading control. After checking the fusion protein expression in clonal populations, a fixed number of each cell type was plated and, within 4 h, CCD camera imaging was done by adding equal amount of Clz400 in well plates. Region of interest values from corresponding wells were plotted as obtained from image data using either a donor or acceptor filter. Bars, SEM.
Figure 2
Figure 2
BRET signal can be spectrally resolved from mammalian cells expressing the BRET fusion vector. A, CCD camera image of a few HT1080 cells stably transfected with Rluc8 and GFF2-Rluc8 plasmid vector from individual wells of a 96-well plate. Cell imaging was done by adding Clz400 substrate (0.5 µg/well) 4 h after plating. Spectral separation of emission light from individual clonal cells transfected with native Rluc or GFP2-Rluc plasmid was not possible. Pseudocolor scale bar represents luminescence photon output averaged for the three filters. B, to confirm the true nature of signals from individual cells, parallel wells containing cells were imaged at 3 and 22 h after plating. As the cells divide over time, acceptor and donor signal intensities are doubled. Individual cells of the marked region of interest (ROI) locations were photographed with a light microscope after CCD imaging.
Figure 3
Figure 3
Localization of BRET signal from subcutaneous and deep tissue structures of a nude mouse implanted with cells constitutively overexpressing GFP2-RLUC8. A, CCD camera image of a representative mouse implanted with 5 × 105 GFP2-Rluc cells on the left shoulder (L) and the same number of GFP2-Rluc8 cells on the right flank (R). The mice were injected with 25-µg Clz400 substrate via tail vein and imaged using a 2-min image acquisition time. B, CCD camera image of a representative mouse injected with 2 × 106 GFP2-Rluc8 cells by tail-vein injection. Thirty minutes later, the mouse was injected with 75-µg Clz400 and imaged immediately using a 3-min acquisition time. Unlike cells that stably express GFP2-RLUC, both donor and acceptor signals from GFP2-Rluc8 expression can be measured from the lungs. For both (A) and (B), images were first captured using the GFP filter followed by the DBC filter after a single injection of Clz400.
Figure 4
Figure 4
Characterization of a BRET sensor for testing a small-molecule dimerizer drug in mammalian cells. A, diagram showing the BRET vector construct, where two individual mTOR pathway protein sequences (FRB and FKBP12) were cloned between the donor and acceptor molecules using the specified amino acid linkers. FKBP12 and FRB domains dimerize only in the presence of the small-molecule dimerizer rapamycin, bringing the acceptor and donor in close proximity. B, HT1080 cells constitutively overexpressing the sensor vector were exposed to measured quantities of rapamycin for 20 h and then the BRET signal was quantitated by imaging with the Clz400 substrate. C, the same cells were exposed to 40 nmol/L rapamycin and the BRET signal was measured at various time points after addition of drug. D, a few cells were plated in a 96-well black plate and the BRET signal (line) was determined from individual cells or cells dividing over time, showing that although the acceptor and donor signals (columns) increase, the BRET ratio remains constant (at a specific drug concentration).
Figure 5
Figure 5
CCD camera images of individual HT1080 cells constitutively overexpressing the GFP2-FRB-FKBP12-Rluc8 fusion in the presence or absence of rapamycin. A, HT1080 cells expressing the GFP2-FRB-FKBP12-RLUC8 fusion were also used to determine the reversible nature of the BRET signal. Positive control (dark dotted line) cells were constantly incubated in medium containing 40 nmol/L rapamycin and negative control (light dotted line) cells were incubated in normal medium. The experimental cells (solid line) were first incubated in rapamycin (40 nmol/L)–containing medium for 4 h, imaged, and then maintained in rapamycin-free medium until the signal dropped significantly (120-h scan time point). After imaging at this time point, the cells were reexposed to rapamycin (40 nmol/L) for 5 h and imaged again showing increased BRET signal. B, a 96-well plate containing a few stably selected HT1080 cells expressing the GFP2-FRB-FKBP12-RLUC8 fusion were subjected to different doses of rapamycin as marked and imaged using a CCD camera 4 h after plating. Individual cells were below detectable threshold with the substrate concentration (0.5 µg/well) and the CCD integration time (1 min) used. With increasing drug concentration, as the interacting partners dimerize, the BRET partners come in closer proximity, leading to a higher BRET signal and thus enabling detection of BRET-specific GFP signal from individual cells. Pseudocolor scale bar represents the average luminescence photon output.
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