Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues

Abstract

FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.

Fig. 1.

BRET macroimaging of tobacco seedlings using the light-tight box apparatus (see SI Text). (A–L) Tobacco seedlings ≈7 days after germination. Shown are seedlings transformed with (i) P_35S::Rluc (A–D), (ii) P_35S::Rluc·EYFP (E–H), and (iii) P_35S::Rluc·COP1 + P_35S::EYFP·COP1 (I–L). (A, E, and I) Bright-field images. (B, F, and J) Short-pass luminescence images (blue). (C, G, and K) Long-pass luminescence images (yellow). (D, H, and L) BRET ratios (Y÷B) over the entire image (pseudocolor scale is shown above D). Quantification of the average BRET ratios (Y÷B ± SD) over the entire images for these samples are as follows: for RLUC, Y÷B = 0.85 ± 0.08 SD (n = 4, including D); for RLUC·EYFP, Y÷B = 1.31 ± 0.15 SD (n = 3, including H); and for RLUC·COP1/EYFP·COP1, Y÷B = 1.26 ± 0.12 SD (n = 4, including L). A ×4 noninfinity-corrected microscopic objective was coupled directly to the Dual-View, which was connected in turn to the EB-CCD camera. Exposure time averaged 7.5 min. Coelenterazine concentration was 10 μM at 22°C.

Fig. 2.

BRET microimaging of Arabidopsis seedlings using an inverted fluorescence microscope apparatus. (A) Spectra of RLUC and RLUC·YFP emission from whole Arabidopsis seedlings. Luminescence spectra were normalized to the emission at 480 nm. Shown are seedlings transformed with (i) P_35S::Rluc (B–D) (no bright-field image is available for this sample, and because RLUC is not fluorescent, there is no fluorescence image), and (ii) P_35S::Rluc·EYFP (E–I) (optics arrangement 1 with ×2 objective, 5-min exposure time). (J–N) A single isolated Arabidopsis cell expressing RLUC·EYFP from a suspension culture [optics arrangement 2 with ×40 objective, N.A. 1.30 (oil immersion), 7.5-min exposure time]. (E and J) Bright-field images. (F and K) RLUC·EYFP fusion protein's fluorescence images. (B, G, and L) Short-pass luminescence (blue) images. (C, H, and M) Long-pass luminescence images (yellow). (D, I, and N) BRET ratios (Y÷B) over the entire image (pseudocolor scale shown above D). Quantification of the average BRET ratios (Y÷B ± SD) over the entire images for these samples are as follows: for RLUC, Y÷B = 0.58 ± 0.02 SD (n = 6, including D); and for RLUC·EYFP, Y÷B = 1.18 ± 0.05 SD (n = 6, including I). Coelenterazine concentration was 10 μM at 22°C.

Fig. 3.

Correction of BRET images from plants for differential absorption of luminescence. (A) RLUC·EYFP emission spectra for light-grown (green) and dark-grown (etiolated) tobacco seedlings, normalized to the emission at 480 nm. (B) RLUC·EYFP emission spectra for green and etiolated tobacco seedlings, normalized to the emission at 530 nm. (C) Absorption spectra of an ethanol extraction of pigments from green and etiolated tobacco seedlings. (D and E) D shows the BRET ratio image from Fig. 1D, shown with a red box encasing the pigmented (cotyledon) portion that is corrected in E. (F and G) F shows the BRET ratio image from Fig. 1H, shown with a red box encasing the cotyledon portion that is corrected in G. Correction factor for boxed regions of E and G was 1.27 (see SI Text).

Fig. 4.

Subcellular imaging of BRET in single mammalian cells. Cells were imaged with an Olympus IX71 microscope by using optics arrangement 3 with a ×60 oil immersion objective (NA 1.45). The luminescence images are integrations of 20 sequential 100-msec exposures. Cells were treated with 10 μM ViviRen in DMEM with 10% FBS (36°C). (A–D) HEK293 cells expressing hRLUC; Y÷B = 0.32 ± 0.05 SD (n = 10) over the luminescent portion of the cell. (E) Spectra of hRLUC versus hRLUC·Venus emission from HEK293 cells. (F–J) HEK293 cells expressing hRLUC·Venus; Y÷B = 0.82 ± 0.07 SD (n = 8) over the luminescent portion of the cell. (K–O) HEK293 cells expressing unfused hRLUC and Venus; Y÷B = 0.36 over the luminescent portion of this cell. (P–S) Mouse GHFT1 cells expressing hRLUC·C/EBPα; Y÷B = 0.24 ± 0.06 SD (n = 11) over the luminescent portion of the cell. (T) Spectra of hRLUC·C/EBPα versus hRLUC·C/EBPα + Venus·C/EBPα emission from mouse GHFT1 cells. (U–Y) Mouse GHFT1 cells expressing hRLUC·C/EBPα + Venus·C/EBPα; Y÷B = 0.39 ± 0.04 SD (n = 9) over the luminescent portion of the cell. (Z–D′) Mouse GHFT1 cells expressing hRLUC + Venus·C/EBPα; Y÷B = 0.30 over the luminescent portion of this cell. A, F, K, P, U, and Z are bright-field images; B, G, L, Q, V, and A′ are blue luminescence images; C, H, M, R, W, and B′ are yellow luminescence images; D, I, N, S, X, and C′ are BRET ratios (Y÷B) over the entire image (pseudocolor scale shown above D); and J, O, Y, and D′ are fluorescence images from Venus in fusion proteins.