Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans

Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two non-fluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here, we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis.     

Visualizing protein interactions by bimolecular fluorescence complementation in Xenopus

Bimolecular fluorescence complementation (BiFC) provides a simple and direct way to visualise protein–protein interactions in vivo and in real-time. In this article, we describe methods by which one can implement this approach in embryos of the South African claw-toed frog Xenopus laevis. We have made use of Venus, an improved version of yellow fluorescent protein (YFP), so as to achieve rapid detection of protein interactions. To suppress spontaneous interactions between the N- and C-terminal fragments of Venus, a point mutation (T153M) was introduced into the N-terminal fragment. We have used this reagent to monitor signalling by members of the transforming growth factor type β family in cells of the Xenopus embryo.     

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.     

Visualization of APP dimerization and APP-Notch2 heterodimerization in living cells using bimolecular fluorescence complementation

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.     

Detection of protein-protein interactions in plants using bimolecular fluorescence complementation

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.     

Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis

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 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.     

Localizing protein-protein interactions by bimolecular fluorescence complementation in planta

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein–protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein–protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.     

Olig1 and ID4 interactions in living cells visualized by bimolecular fluorescence complementation technique

Olig1, a member of class B basic-helix-loop-helix (bHLH), plays key roles in early oligodendrocyte specification. Inhibitors of DNA binding (Id) is another sub-class of HLH proteins, act as dominant-negative regulators of bHLH proteins, which can form heterodimers with class A or B bHLH proteins, but lack the critical basic DNA binding domain. Id4 was recently found to interact with olig1 and inhibit oligodendrocyte differentiation. However, there still no direct evidence to reveal the spatial and temporal interaction of olig1 and ID4 in living cells. In this study, we performed bimolecular fluorescence complementation (BiFC) analysis to further characterize the distinct subcellular localization of olig1, ID4 and their dimer in living SW1116 cells. To examine the subcellular localization of olig1 and ID4 by themselves, the olig1-EGFP or ID4-DsRed2 fusion proteins were also expressed in SW1116 cells, respectively. As predicted, the olig1-EGFP fusion proteins were located in the nucleus, and ID4-DsRed2 fusion proteins were located in the cytoplasm. When olig1-EGFP and ID4-DsRed2 fusion proteins were co-expressed, the green and red signals were co-located in the cytoplasm. Using BiFC, the strong BiFC signals could be detected in pBiFC-olig1VN173 and pBiFC-ID4VC155 co-transfected cells and the fluorescence signal was located in the cytoplasm. These results collectively confirmed that olig1 and ID4 could interact and form dimer in living cells, and ID4 could block the transport of olig1 from cytoplasm to nucleus.     

Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments

The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.     

Visualization of molecular interactions by fluorescence complementation

The visualization of protein complexes in living cells enables the examination of protein interactions in their normal environment and the determination of their subcellular localization. The bimolecular fluorescence complementation assay has been used to visualize interactions among multiple proteins in many cell types and organisms. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin-family peptides.     

Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.     

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.     

Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro

Here, we present a protocol for isolating the large N-terminal fragment of enhanced green fluorescent protein (EGFP) with a preformed chromophore. By itself, the chromophore-containing EGFP fragment exhibits very weak fluorescence, but it rapidly becomes brightly fluorescent upon complementation with the corresponding small, C-terminal EGFP fragment. Each EGFP fragment is cloned and overexpressed in E. coli as a fusion with self-splitting intein. After solubilizing and refolding these fusions from inclusion bodies, both EGFP fragments are cleaved from intein and purified using chitin columns. When these EGFP fragments are linked with the two complementary oligonucleotides and combined in equimolar amounts, fluorescence develops within a few minutes. The isolation of profluorescent protein fragments from recombinant E. coli cells requires approximately 3 d, and their conjugation to oligonucleotides requires 1-4 h.     

Tat-mediated protein delivery in living Caenorhabditis elegans

The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in various biological models, but never in the nematode worm Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.     

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.     

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.