Bimolecular Fluorescence complementation (BiFC) and beyond

Abstract

Protein interactions are key determinants of signal transduction in cells, and identification of interacting proteins is critical for deciphering molecular mechanisms by which proteins function in cells. Since the first demonstration of the reassembly of split green fluorescent protein (GFP) in vitro (1) and the development of a yellow fluorescent protein (YFP)-based bimolecular fluorescence complementation (BiFC) assay for visualization of protein interactions in living cells (2), many fluorescent protein fragments have been identified to support fluorescence complementation (for principle, see Figure 1) (3-5). The availability of these fluorescent protein fragments increases the spectra of BiFC analysis. Consequently, many BiFC-based novel applications have been explored, further extending the applications of the BiFC assay to various aspects of proteins, RNA-protein interactions (6), and DNA hybridization (7). These developments have increased our capability to study various aspects of large molecules, and met the needs of researchers working on different model systems. One example is the demonstration that the fluorescent protein Venus, an improved YFP variant (8), can support BiFC under physiological conditions (4). The use of Venus for BiFC assay eliminates the need of a preincubation of cells at 30C or lower temperatures, which had hindered the use of YFP-based BiFC for many signaling molecules. Further, it can also be paired with cyan fluorescent protein (CFP) and Cerulean, an improved CFP variant for FRET analysis (9), for multicolor BiFC analysis. Recently, we have combined Venus-based BiFC assay with Cerulean, and developed a BiFC-based FRET (BiFC-FRET) assay for identification and visualization of ternary complexes in living cells (for principle, see Figure 2). Using the BiFC-FRET, we have demonstrated that activator protein (AP-1), consisting of Fos-Jun heterodimers, interacts with NFκB subunit p65 and forms a ternary complex. This finding reveals a novel crosstalk between AP-1 and NF-κB. Given that many signaling molecules form multiple protein complexes including ternary complexes, we anticipate that the BiFC-FRET will facilitate future identification and visualization of ternary complexes in living cells. The BiFC-FRET along with other new BiFC-based applications will be discussed.