Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis

Abstract

The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.

Figure 1

Visualization of bimolecular fluorescence complementation between fragments of different fluorescent proteins fused to bFos and bJun. (A) Diagram of amino acid substitutions among enhanced green fluorescent protein variants and the positions where they were fragmented (155 and 173). (B-K) Fluorescence images of COS-1 cells transfected with plasmids expressing the protein fragments indicated in each panel fused to the bZIP domains of Fos and Jun. The C-terminal fragments were fused to bFos and the N-terminal fragments were fused to bJun. Structural models of the bimolecular fluorescent complexes shown to the right of each image are based on the X-ray crystal structure of full-length GFP . The positions of fragmentation are indicated by arrows in the structures. The position and structure of the duplicated β-strand shown in panels H-J is unknown. The excitation/emission maxima of each complex are shown below each image. The bar represents 10 μm in all images.

Figure 2

Excitation and emission spectra of cells expressing fragments of different fluorescent proteins fused to bFos and bJun. Solid lines correspond to bimolecular fluorescent complexes and dashed lines to intact fluorescent proteins (A) The excitation spectra were collected by measuring emissions at 535 nm through a 530 nm long pass filter, and were normalized by the ratio between the emission maximum of each complex and the emission intensity at 535 nm. (B) The emission spectra of YN173-YC173 and YFP were collected through a 500 nm long-pass filter using excitation at 480 nm and the other emission spectra were collected through a 450 nm long-pass filter using excitation at 430 nm, and were normalized by the ratio between the excitation maximum of each complex and the excitation efficiencies at 480 nm and 430 nm respectively. The cells were transfected with 0.5 μg of the plasmids encoding the fluorescent protein fragments fused to bFos and bJun or with 0.05 μg of the plasmids encoding the full length proteins.

Figure 3

Effects of a deletion in the leucine zipper on the efficiencies of complementation between fragments of different fluorescent proteins fused to the bZIP domains of Fos and Jun. The efficiencies of fluorescence complementation were determined in individual cells by measuring the ratio between the fluorescence emissions of the bimolecular fluorescent complex and that of a co-expressed intact fluorescent protein. Plasmids encoding the proteins indicated in each panel were transfected into cells (0.25 μg of each fusion protein and 0.025 μg of the ECFP or EYFP internal control) and the ratio of fluorescence emissions produced by the bimolecular complex and the internal control (Y/C or C/Y) was measured. Note the logarithmic scaling of the categories in the histograms.

Figure 4

Comparison of the efficiencies of bimolecular complex formation by fragments of different fluorescent proteins fused to bFos and bJun. We examined the competition between different ratios of bJun fused to fragments of different fluorescent proteins for a limiting concentration of bFos fused to the complementary fragment. (A) The indicated ratios of GN173 and CN173 fused to bJun were mixed with a fixed concentration of YC173 fused to bFos. The excitation spectra were collected after 12 hours by measuring emissions at 530 nm. (B) The indicated ratios of YN173 and CN173 fused to bJun were mixed with a fixed concentration of YC173 fused to bFos. The emission spectra were collected after 12 hours during excitation at 450 nm.

Figure 5

Multicolor BiFC analysis of the subcellular sites of interactions between different proteins in the same cell. (A) Co-localization of bJunCN173-bFosYC173 and bJunYN173-bFosYC173 bimolecular fluorescent complexes. (B) Differential localization of bJunCN173-bFosYC155 and JunYN155-bFosYC55 complexes. Plasmids encoding the proteins indicated below the images were co-transfected into COS-1 cells. The images show the fluorescence emissions of the same cell using 436 nm and 470 nm (C) or 500 nm and 530 nm (Y) filters. The bar represents 10 μm in all images.

Figure 6

Multicolor BiFC analysis of the competition between alternative interaction partners in cells. Two combinations of interaction partners producing bimolecular complexes with different spectra were transfected into different cell populations (cyan and yellow bars) or co-transfected into the same cell population (green bars) as indicated above each graph. The ratio between the fluorescence emissions corresponding to each complex (Y/(C+Y)) was determined in individual cells in each population, and was plotted in the histograms. (A) Competition between bFosCN173 and bATF2YN155 for dimerization with bJunCC155. (B) Competition between bFosYN155 and bATF2CN173 for dimerization with bJunCC155. (C) Competition between bFosYN155 and bJunCN173 for dimerization with bJunCC155. Plasmids encoding the proteins indicated in each panel were co-transfected into cells. The plasmid encoding the shared interaction partner fused to CC155 was used at limiting concentration (0.1 μg), whereas the other plasmids were used at a higher concentration (0.5 μg). The fluorescence intensities of individual cells were quantified using 436 nm/470 nm (C) and 500 nm/535 nm (Y) filters and the Y/(Y+C) ratios were plotted in the histograms.