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. 2010 Aug;8(4):437–458. doi: 10.1089/adt.2010.0281

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Copyright 2010, Mary Ann Liebert, Inc.

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Fig. 1. — p53-hDM2 protein–protein interaction biosensor components, principle, and characterization. (A) Cartoon schematic of the protein–protein interaction partner components and the 2 interaction states of the p53-hDM2 positional biosensor. The p53-hDM2 protein–protein interaction biosensor design incorporates 2 recombinant adenovirus constructs driving expression of the N-terminal domains of the p53 and hDM2 interacting partners fused to fluorescent proteins (green fluorescent protein [GFP] or red fluorescent protein [RFP]) at their carboxy-terminus. The N-terminal residues 1–131 of p53 encompass the p53 transactivating domain that contains the binding site for hDM2 and is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by the inclusion of a nuclear localization sequence (NLS). The N-terminal residues 1–118 of hDM2 encompass the binding site for the N-terminal transactivating domain of p53 and is expressed as an RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells that are coinfected with both adenovirus constructs the binding interactions between the hDM2 and p53 components of the biosensor resulted in both proteins becoming localized to the nucleolus, producing a yellow signal in composite images. Upon disruption of the p53-hDM2 protein–protein interaction with a compound such as Nutlin-3, the p53-GFP interaction partner remained nucleolar, while the shuttling hDM2-RFP interaction partner redistributed into the cytoplasm, and in the composite images of these cells, the nucleolus was predominantly light green/blue and the cytoplasm was predominantly red. (B–D) Individual gray-scale and 3-color composite images of U-2 OS cells from 3 fluorescent channels (Hoechst Ch1, GFP Ch2, and RFP Ch3) were sequentially acquired on the ArrayScan V^TI platform using a 20 × 0.4 NA objective with the XF93 excitation and emission filter set (Hoechst, blue; FITC, green; and TRITC, red). U-2 OS cells were infected with (B) only the p53-GFP biosensor adenovirus, (C) only the hDM2-RFP biosensor adenovirus, or (D) both the p53-GFP and hDM2-RFP biosensor adenoviruses. Adenovirus-infected cells were seeded at 2,500 cells per well in 384-well Greiner collagen-coated assay plates, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and were then treated for 90 min with 0.5% dimethyl sulfoxide (DMSO) or 10 μM Nutlin-3 in 0.5% DMSO prior to fixation with 3.7% formaldehyde containing 2 μg/mL Hoechst 33342. Images from a single representative experiment of numerous experiments are presented.

Fig. 1. — p53-hDM2 protein–protein interaction biosensor components, principle, and characterization. (A) Cartoon schematic of the protein–protein interaction partner components and the 2 interaction states of the p53-hDM2 positional biosensor. The p53-hDM2 protein–protein interaction biosensor design incorporates 2 recombinant adenovirus constructs driving expression of the N-terminal domains of the p53 and hDM2 interacting partners fused to fluorescent proteins (green fluorescent protein [GFP] or red fluorescent protein [RFP]) at their carboxy-terminus. The N-terminal residues 1–131 of p53 encompass the p53 transactivating domain that contains the binding site for hDM2 and is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by the inclusion of a nuclear localization sequence (NLS). The N-terminal residues 1–118 of hDM2 encompass the binding site for the N-terminal transactivating domain of p53 and is expressed as an RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells that are coinfected with both adenovirus constructs the binding interactions between the hDM2 and p53 components of the biosensor resulted in both proteins becoming localized to the nucleolus, producing a yellow signal in composite images. Upon disruption of the p53-hDM2 protein–protein interaction with a compound such as Nutlin-3, the p53-GFP interaction partner remained nucleolar, while the shuttling hDM2-RFP interaction partner redistributed into the cytoplasm, and in the composite images of these cells, the nucleolus was predominantly light green/blue and the cytoplasm was predominantly red. (B–D) Individual gray-scale and 3-color composite images of U-2 OS cells from 3 fluorescent channels (Hoechst Ch1, GFP Ch2, and RFP Ch3) were sequentially acquired on the ArrayScan V^TI platform using a 20 × 0.4 NA objective with the XF93 excitation and emission filter set (Hoechst, blue; FITC, green; and TRITC, red). U-2 OS cells were infected with (B) only the p53-GFP biosensor adenovirus, (C) only the hDM2-RFP biosensor adenovirus, or (D) both the p53-GFP and hDM2-RFP biosensor adenoviruses. Adenovirus-infected cells were seeded at 2,500 cells per well in 384-well Greiner collagen-coated assay plates, cultured overnight at 37°C, 5% CO₂, and 95% humidity, and were then treated for 90 min with 0.5% dimethyl sulfoxide (DMSO) or 10 μM Nutlin-3 in 0.5% DMSO prior to fixation with 3.7% formaldehyde containing 2 μg/mL Hoechst 33342. Images from a single representative experiment of numerous experiments are presented.