Bimolecular Fluorescence Complementation (BiFC): A 5-year Update and Future Perspectives

Abstract

Over the past decade, bimolecular fluorescence complementation (BiFC) has emerged as a key technique to visualize protein-protein interactions in a variety of model organisms. The BiFC assay is based on reconstitution of an intact fluorescent protein when two complementary non-fluorescent fragments are brought together by a pair of interacting proteins. While the originally reported BiFC method has enabled the study of many protein-protein interactions, increasing demands to visualize protein-protein interactions under various physiological conditions have not only prompted a series of recent BiFC technology improvements, but also stimulated interest in developing completely new approaches. Here we review current BiFC technology, focusing on the development and improvement of BiFC systems, the understanding of split sites in fluorescent proteins, and enhancements in the signal-to-noise ratio. In addition, we provide perspectives on possible future improvements of the technique.

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

“…However, this should be the control of last resort: Since BiFC is a proximitybased assay, different protein structures can have a strong impact on protein reconstitution. Kodama and Hu (2012) have suggested an elegant competition-based assay that provides a more stringent alternative control that, however, has not yet been adapted for BiFC assays in plant cells. Inappropriate controls for BiFC experiments that, unfortunately, are frequently seen in the literature include, for example, combination of one of the interaction partners with an empty vector (expressing the unfused FP fragment) or the expression of only one of the fusion proteins (Horstman et al, 2014; Figures 1G to 1N).…”

Pumilio-Based RNA In Vivo Imaging

“…In BiFC, the fluorescent complex of N-and Cterminal split FP halves is extremely stable (31). As the BiFC constructs accumulate in a cell, the increasing frequency of random collisions will result in the formation of low levels of reassembled FP, independent of interactions of fused PUMHDs with their cognate RNA.…”

“…Several recently developed approaches that improve the signal-to-noise ratio of BiFC by introducing further modifications into the split FP fragments (31,32) are still unexplored for PUM-BiFC applications. Also, a tetramolecular fluorescence complementation system for Pumilio-based RNA detection was recently introduced (PUM-TetFC) (33) (Fig.…”

“…BiFC can be performed with various derivatives of both GFP and red FPs (31,(34)(35)(36)(37)(38)(39). This allows selection of an FP that will facilitate co-localising the RNA of interest with other fluorescent markers that may already be available.…”

“…Ideal BiFC controls are fusions with non-interacting variants of the proteins used in the actual experiments (31). In the case of PUM-BiFC this corresponds to either the absence of the target RNA as described here, or PUMHD fusions that do not bind the target RNA (24,33).…”