Deciphering protein–protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) is one of extensively used PCAs over the last decade in many different native contexts, including human live cells. It is based on the reconstitution of a monomeric fluorescent protein (FP) such as cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) from two complementary nonfluorescent sub-fragments (N- and C-terminal sub-fragments) upon spatial proximity. Interaction between a bait protein and a prey protein fused to such complementary sub-fragments is sufficient to lead to the reconstitution of the FP, resulting in an emission signal upon excitation (Figure 1). In this comprehensive review, we commence by tracing the development of fluorescent proteins (FPs) tailored for BiFC, followed by the highlighting of BiFC advantages over other PPI detection methods. Concurrently, the essential factors and nuanced considerations when employing BiFC are thoroughly explored. To vividly showcase the versatility of BiFC, the implementations at different throughputs are also presented, encompassing both single PPI experiments and large-scale studies, along with noteworthy variants of BiFC.

For a comprehensive exploration of the Bimolecular Fluorescence Complementation (BiFC) approach, we critically assess whether protein pairs establish direct or indirect interactions within this context. Emphasizing the multifaceted nature of protein interactions, this review underscores the imperative of delineating macromolecular complex characteristics across five distinct levels: composition, stoichiometry, copy number, topology, and dynamics. A pivotal discussion revolves around the challenges introduced by the overexpression of target proteins and the potential artificial ramifications of fusion proteins. The significance of stoichiometry in protein interactions is illuminated, with the HOXA9/PBX1 interaction serving as a pertinent case study. The research identifies and elaborates on the inherent limitations of prevailing large-scale BiFC screenings, advocating for the incorporation of rigorous negative controls. A novel proposition is introduced in the form of a high-throughput multicolor BiFC strategy, exemplified using the HOXA9 and PBX1 proteins. The paper culminates with an insightful discourse on the implications of tags within the BiFC system, presenting the TriFC (Tripartite Fluorescence Complementation) assay as a promising alternative.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied for the analysis of different types of PPIs. In particular, BiFC has been applied for analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that could be coupled with BiFC. Here, we present a novel experimental strategy that we called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables to precisely quantify the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more largely in other tissues during Drosophila development. Our work set up the experimental basis for these future applications.

Herein, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV) sensing system created by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, derived from 6’-silalyllactose used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range from 1 x 100 TCID50/mL to 1 x 107 TCID50/mL, with a detection limit of 4 x 101 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while displaying weak fluorescence signals when exposed to NA-deficient viruses such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Taken together, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC capable of detecting the ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.

The PII protein is an evolutionary highly conserved regulatory protein from bacteria to higher plants. In bacteria it modulates the activity of several enzymes, transporters and regulatory factors by interacting with them and thereby regulating important metabolic hubs like carbon/nitrogen homeostasis. More than two decades ago the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behaviour of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies we found that AtPII interacts in chloroplasts with itself as well as with known interactors like NAGK in dot-like aggregates, which we named PII foci. In these novel protein aggregates AtPII interacts also with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal part of AtPII is crucial for the formation of PII foci. Altogether, the presented results indicate a novel mode of interaction for PII proteins with other proteins in plants, which may be a new starting point for the elucidation of physiological functions of PII proteins in plants.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman’s disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the characterization of the protein-protein interactions of the vPIC factors has not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral factors has not been sufficiently revealed. Here, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Gln36, His41 and Trp170) on ORF18 that were responsible for its interaction with ORF30. The artificial intelligence (AI) system AlphaFold2 predicted that identified four residues are exposed to the surface of ORF18 and located in proximity to each other in the surface of ORF18. Thus, AI-predicted model supports the importance of four residues for binding of ORF18 to ORF30. Our results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.