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.

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Figure 1: Visualization of complementation between fragments of different fluorescent proteins fused to bFos and bJun.
Figure 2: Excitation and emission spectra of cells expressing fragments of different fluorescent proteins fused to bFos and bJun.
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.
Figure 4: Comparison of the efficiencies of bimolecular complex formation by fragments of different fluorescent proteins fused to bFos and bJun.
Figure 5: Multicolor BiFC analysis of the subcellular sites of interactions between different proteins in the same cell.
Figure 6: Multicolor BiFC analysis of the competition between alternative interaction partners in cells.

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Acknowledgements

We thank members of the Kerppola laboratory for helpful discussions.

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Correspondence to Tom K. Kerppola.

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The authors have applied for a patent (application no. 60/375,949) on the molecular fluorescence complementation assay described in this paper.

Supplementary information

Supplementary Fig. 1. Comparison of expression of bFos, bJun and bATF2 fused to fragments of different fluorescent proteins. (A) Plasmids encoding the fragments indicated fused to bJun and bFos respectively were co-transfected into cells. The YN155 used in this experiment (but not in others presented in this manuscript) was fused to Jun225-334, resulting in a larger fusion protein (lane 1). (B) Plasmids encoding the proteins indicated above each lane were co-transfected into cells. Cell lysates were analyzed by Western blotting using antibodies directed against the FLAG and HA epitopes. (JPG 82 kb)

Supplementary Fig. 2. Selectivity of bimolecular fluorescence complementation between fragments of fluorescent proteins fused to bFos and bJun. The excitation/emission maxima of all complexes that exhibited detectable fluorescence complementation are shown. None of the fragments exhibited detectable fluorescence alone. Cells with no detectable complementation (indicated by -) produced less than 1% of the fluorescence emissions of cells expressing YN155 and YC155 or CN155 and CC155 at every wavelength tested. (GIF 25 kb)

Supplementary Table 1. Effects of a deletion in the leucine zipper of Fos on complementation between fragments of different fluorescent proteins fused to bFos and bJun. The relative fluorescence intensities of cells transfected with the expression vectors indicated were evaluated by fluorescence microscopy. For a calibration of the qualitative scale, please compare with the quantitative data in Fig. 3. (GIF 38 kb)

Supplementary Table 2. Selectivity of dimerization among the bZIP domains of bFos, bJun and bATF2. The fluorescence intensities of cells transfected with plasmids expressing the proteins indicated were measured using 436/470 nm (C) or 500/530 nm (Y) filters. The average fluorescence intensities and the average of the ratios for all cells examined are shown. (GIF 44 kb)

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Hu, C., Kerppola, T. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21, 539–545 (2003). https://doi.org/10.1038/nbt816

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