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 twelve new bimolecular fluorescent complexes that correspond to seven different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency or subcellular sites of interactions between the bZIP domains of Fos and Jun. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation among alternative interaction partners.Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of developmental programs. Each protein typically has a large number of alternative interaction partners, and the selectivity of these interactions determines the developmental potential of the cell and its responses to extracellular stimuli. We recently described a new approach for the visualization of protein interactions in living cells designated bimolecular fluorescence complementation (BiFC) analysis 1 . The BiFC approach is based on the formation of a fluorescent complex by fragments of the yellow fluorescent protein (YFP) brought together by the association of two interaction partners fused to the fragments. This approach enables visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.Molecular engineering of the green fluorescent protein (GFP) has produced several variants with altered spectral characteristics 2 . These variants allow simultaneous visualization of the distributions of multiple proteins in living cells. Moreover, fluorescence resonance energy transfer (FRET) between different variants enables analysis of interactions between individual pairs of proteins in living cells 3, 4 . Thus far, it has not been possible to visualize multiple interactions in the same cell.Selected fragments of many proteins can associate to produce functional bimolecular complexes. Such bimolecular complementation provides a convenient approach for detection of protein interactions in cells if the protein fragments can associate only when they are brought together by interaction partners fused to the fragments 1, 5-9 . The unique characteristic of the BiFC approach is that the bright intrinsic fluorescence of the bimolecular complex allows direct * To whom correspondence should be addressed:Tel: (734) FAX: (734) RESULTSThe spectral characteristics of bimolecular fluorescent complexes formed by fragments of YFP were virtually identical t...

Intracellular protein interaction mapping with FRET hybrids

“…Consequently, they are not amenable to screening for reversible or dynamic phenomena. Given the limitations of two-hybrid assays, protein fragment complementation has been pursued as a means to directly report interactions without a requirement for secondary events (36,37) and may prove useful for large-scale interaction screening (36,38). In this report, FRET hybrids enabled direct quantitative and near-real-time detection of protein-ligand interactions in bacteria, yeast, and mammalian cells and a means to rapidly measure binding affinity.…”

Visualization of Ras-PI3K interaction in the endosome using BiFC

“…Hence, reconstitution of the fluorescent protein is enabled by the direct interaction between the two target proteins [16,17]. One of the advantages of BiFC is that a specific interaction produces a single color, making it amenable to combination with other fluorescent proteins and imaging techniques.…”

Lighting the Way to Protein-Protein Interactions: Recommendations on Best Practices for Bimolecular Fluorescence Complementation Analyses

“…Several distinct sites within the eYFP (or mVenus) protein have been found to allow for efficient reconstitution after splitting into separate fragments. Commonly, eYFP is split between Ala-154 and Asp-155 located between the seventh and the eighth b-sheet (Hu et al, 2002;Walter et al, 2004), between Glu-172 and Asp-173 within the linker separating the eighth and the ninth b-sheet (Hu and Kerppola, 2003;, and, more recently, after residue 210 within the loop separating the tenth and the eleventh b-sheet (Ohashi et al, 2012;Gookin and Assmann, 2014). Although fragmentation at position 172 appears to result in the strongest signal intensity of reconstituted YFP fluorescence, it also enhances unwanted background fluorescence.…”