Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells

Abstract

A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally divers interaction partners in many different cell types and. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation (UbFC) assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes

“…BiFC assays were performed as described (56). An improved BiFC assay with a high signal-to-noise ratio was selected to avoid background interference (57,58).…”

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

“…FSP27 was highly expressed in the fat cake (lane 1) and coimmunopre cipi tated with NFAT5 (lanes 3 and 5) but not when non immune IgG (lanes 2 and 4) was used for the immunoprecipitation, thus substantiating the physical with the N-terminal (VN173) or C-terminal (VC155) of Venus for the BiFC assay. If FSP27 and NFAT5 interact with each other, the interaction should bring VN173 and VC155 into close proximity and reconstitute an intact Venus molecule, which can be detected by direct visualization with fl uorescence imaging ( 21 ). To confi rm the interaction between the fusion proteins, we transiently expressed the proteins in HEK293 cells that had been loaded with fatty acids (FAs) to promote LD formation.…”

Establishment of Bimolecular Complementation System for the Interaction Detection between Epstein-barr Virus Nuclear Antigen 1 and Host Proteins

“…The result also showed that the interaction took place in the nuclear (Figure 4). is that it enables characterization of protein-protein interactions in the natural cellular environment [5][6][7].…”

“…Bimolecular complementation (BiMC) assay is a useful approach method for the detection, visualization and map of protein-protein interactions in live mammalian cells [5]. In this system, the enhanced yellow fluorescent protein (EYFP) is split into N-and Cterminal fragments, and reconstitution of these two fragments takes place by the mediation of a proteinprotein interaction, thus resulting in irreversible fluorescent signal [6]. In order to improve our understanding of the interaction patterns of EBNA1 and host proteins, in this study, we established a BiMC assay system expressing a fusion protein of EBNA1 and the C-termini of EYFP, and this system was employed in the assay for a interaction detection of EBNA1 with one host protein in living 293T cells.…”