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.     

MCM-GINS and MCM-MCM interactions in vivo visualised by bimolecular fluorescence complementation in fission yeast

Background  

Each of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM.     

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.     

Monitoring the interference of protein-protein interactions in vivo by bimolecular fluorescence complementation: the DnaK case

Many cellular processes depend on protein-protein interactions. The identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Nevertheless, finding antagonists of protein interactions that work efficiently within the cell is a challenging task. Here, we describe the novel use of bimolecular fluorescence complementation (BIFC) to detect compounds that block the interaction of target proteins in vivo. In the BIFC method, each interaction partner is fused to a complementary fragment of a fluorescent protein and interactions are detected by fluorescence restoration after reporter reassembly. Here, we demonstrate that the inhibition of specific intracellular protein interactions results in a concomitant decrease in fluorescence emission. We also show that integration of BIFC with flow cytometry might provide an effective means to detect interaction modulators by directly reading out changes in the reporter signal. The in vivo application of this approach is illustrated through monitoring the inhibition of the interaction between the Escherichia coli Hsp70 chaperone and a short peptidic substrate by pyrrhocoricin-derived antibacterial peptides.     

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.     

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.     

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.     

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.     

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.     

Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case

Protein-protein interactions are essential in most biological processes. Many proteomic approaches have succeeded in the identification of strong and obligatory interactions but the study of weak and transient protein-protein interactions is still a challenge. The aim of the present study was to test the ability of bimolecular fluorescence complementation to detect and discriminate in vivo weak intracellular protein interactions. As a test case, the interaction of the SH3 domain from the c-Abl tyrosine kinase with both natural and designed targets has been chosen. The reassociation of functional yellow fluorescent protein (YFP) from its fragments requires previous binding between the SH3 domain and its partners; but once this occurs, the complex is trapped, turning transient SH3 interactions into stable, easily detectable ones. The method is very sensitive and can be implemented for proteomic analysis of weak protein interactions using flow cytometry. The fluorescence emission is dependent on the strength of the interaction, in such a way that it can be used, at least qualitatively, to screen for best binding candidates among similar proline-rich peptides. In addition, it is illustrated how this method can be used to gain structural insights into particular c-Abl SH3 interactions.     

Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high‐resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0‐CaM complex on the AQP0 C‐terminus is also demonstrated since the C‐terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X‐ray crystallography or single particle electron microscopy.     

Dimerization of ABCG2 analysed by bimolecular fluorescence complementation

ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis.     

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.     

Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

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.     

An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein

Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein, which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further, since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.