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PLoS One
2009 Jun 12;46:e5868. doi: 10.1371/journal.pone.0005868.
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High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases.
Hida N
,
Awais M
,
Takeuchi M
,
Ueno N
,
Tashiro M
,
Takagi C
,
Singh T
,
Hayashi M
,
Ohmiya Y
,
Ozawa T
.
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Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1-Smad4 and Smad2-Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.
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19536355
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Figure 1. Schematic illustration showing structures of luciferases composed of different luciferase fragments' complementation.Structural models of each luciferase (upper part of each image) are based on the X-ray crystal structure of full-length Photinus pyralis (Firefly) luciferase. The N-terminal and C-terminal domains of respective luciferases are shown in different colors. Bioluminescence images of COS-7 cells transfected with plasmids expressing the protein fragments fused to FKBP and FRB are shown below the luciferase structures. The cells were cultured on the 24-well plate and incubated in the presence (+Rap) and absence (âRap) of rapamycin.
Figure 2. Quantitative and time-course evaluation of rapamycin-induced luciferase complementation.(AâC; upper) Absolute photon counts for COS-7 cells cotransfected with the plasmids of a pair of luciferase fragments fused to FKBP and FRB in the absence (white bars) and presence (black bars) of rapamycin. (AâC; lower) Normalized photon counts for COS-7 cells transiently cotransfected with plasmids expressing the luciferase fragments fused to FKBP and FRB, and Renilla luciferase. The Renilla luciferase was used to normalize the transfection efficiency. Results are expressed as the relative luminescence unit (RLU) ratio; values of which the luminescence intensity were normalized to the intensity of Renilla Luciferase for rapamycin-treated cells were divided by those for rapamycin-untreated cells (black bars). Differences of heights between white and black bars indicate rapamycin-induced luminescence. (D) Reversibility of the complementation between McLuc1 and N-terminal FLuc. The lysates extracted from COS-7 cells expressing FKBP and FRB fused to the luciferase fragments were treated with rapamycin for 1â10 min (50 nM, upper data). The cells were treated successively with different concentrations of FK506 (1 µM, 10 µM, 100 µM) for 0â25 min in the presence of 50 nM rapamycin (middle data). Photon counts were taken every 60 s. The luminescence values were normalized against the maximum luminescence values. Western blotting analysis of the cells including LucN and McLuc1 in the presence or absence of rapamycin and FK506 with cycloheximide (lower). Error bars represent s.d. calculated for three independent samples. (**P<0.01, ***P<0.001)
Figure 3. Bioluminescence analysis of Smad1âSmad4 and Smad2âSmad4 interactions.(A) Characterization of the probes. a and b: COS-7 cells were transfected with the plasmids, Smad4-McLuc1 plus FLucN-Smad1 or Smad4-McLuc1 plus CBRN-Smad1, in the absence (Mock) and presence of either ALK3CA or ALK3DN receptor. Smad1(AXA) and Smad2(AXA) indicate double mutants of Smad1(S462A,S464A) and Smad2(S465A,S467A), respectively. The cells were incubated for 12â16 h and the luciferase activities were measured. c: COS-7 cells were transfected with the plasmids, ELucN-Smad2 plus Smad4-McLuc1 or ELucN-Smad2(AXA) plus Smad4-McLuc1 in the absence (Mock) and presence of either ALK5CA or ALK5DN receptor. d: COS-7 cells transfected with the plasmids FLucN-Smad1 and Smad4-McLuc1 were stimulated with BMP-2 (50 nM) for 2 h and luciferase activities were measured. Error bars represent s.d. calculated for three independent samples. e: Western blotting analysis of the expression of Smad1âSmad4 or Smad2âSmad4 protein in the presence of ALK3CA, ALK3DN, ALK5CA or ALK5DN. (*P<0.05, **P<0.01, ***P<0.001) (B)â(D) Real-time bioluminescence images of Smad1âSmad4 and Smad2âSmad4 interactions using CBRN-Smad1 and Smad4-McLuc1 (B), CBRN-Smad2 and Smad4-McLuc1 (C), and CBRN-Smad1, ELucN-Smad2 and Smad4-McLuc1 (D), in a Xenopus embryo. RNAs encoding the Smad probes were injected into the animal pole of a 2-cell stage Xenopus embryo. The embryo was incubated for 24 h at 13°C and thereafter, digital images of Venus (gray) and bioluminescence (pseudocolor) were acquired using a microscopic system equipped with an EM-CCD camera. Bar, 1 mm.
Figure 4. Dual-color assay for Smad1âSmad5 and Smad2âSmad3 interactions using different fragments of luciferases.(A) Ligand-induced Smad1âSmad5 and Smad2âSmad3 interactions. COS-7 cells were cotransfected with CBRN-Smad1, Smad5-McLuc1, ELucN-Smad2 and Smad3-McLuc1 and their bioluminescence intensities were obtained with a luminometer. (*P<0.05) (B) Analysis of bioluminescence with different combination of Smads. COS-7 cells were cotransfected with either CBRN-Smad1 and Smad3-McLuc1 or ELucN-Smad2 and Smad5-McLuc1. Representative data were shown.
Figure 5. Analysis of IRS1-p85β interaction based on luminescence intensities of McLuc1 and FLucN complementation.The CHO-IR cells were transiently transfected with IRS1-McLuc1 and FLucN-p85β; luciferase activities were measured at each time point. Error bars represent s.d. calculated for three independent samples.
Figure 6. Analysis of Bad phosphorylation based on luminescence intensities of the McLuc1 and N-terminal ELuc complementation.(A) Results of analyses of interactions between 14-3-3 and Bad mutants. The COS-7 cells were transiently transfected with BadâMcLuc1 and ELucNâ14-3-3; luciferase activities were tested. Error bars represent s.d. calculated for three independent samples. (B) Results of Western blotting analysis of Bad phosphorylation. Expression levels of Bad and its mutants were determined using immunoblotting with the anti-V5 antibody (left). Mutations of Bad at S112A, S136A, and S155A were confirmed by immunoblotting with the respective antibodies (right). (C) An inhibitory effect of antimycin or HA14-1 on the bioluminescence was developed by the Bad-Bcl-2 interaction. Antimycin (10 µM) or HA14-1 (10 µM) was added to the cells after transfection and incubated for 20 h. The cells were harvested and the photon counts were analyzed. Error bars represent s.d. calculated for three independent samples. (D) Dual color imaging of mice with a single substrate. The images shown are superimposed on the optical CCD bioluminescence image without a filter (Open) or with a band-pass filter of BP(ELuc) (525±25 nm) or BP(SLRLuc) (630±37.5 nm). A nude mouse was imaged after implantation of COS-7 cells that had been transiently transfected with plasmids SLRLuc (site 1), BadâMcLuc1 and ELucNâ14-3-3 (site 2), SLRLuc plus BadâMcLuc1 and ELucNâ14-3-3 (site 3), and SLRLuc plus Bad(S112A, A136A, A155A) âMcLuc1 and ELucNâ14-3-3 (site 4). To obtain photon flux information from mice, the bioluminescence intensity was shown as pseudocolors. Photon counts with BP(ELuc) divided by those with BP(SLRLuc) are shown at the right side of the image.
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