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.     

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.     

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.     

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.     

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.     

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.     

Bimolecular fluorescence complementation: illuminating cellular protein interactions

Many cellular processes depend on the establishment of selective stable or transient interactions between proteins. Therefore, the ability to identify and characterize these contacts in physiologically relevant environments is crucial to understanding the networks of contacts that allow the transmission and integration of biological information in living cells. Protein-fragment complementation assays (PCA) have emerged as approaches that report on the proximity of two given proteins in the cell at a given location and time. In particular, bimolecular fluorescence complementation (BIFC) allows noninvasive imaging of protein binding in living cells at high spatial resolution and without the requirement for exogenous substrates. In the present review, we discuss PCA and BIFC fundamentals, the implementation of BIFC assays and selected applications of BIFC in drug discovery, developmental studies or neurological disorders.     

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.     

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.     

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.     

Gap-junctional coupling measured by flow cytometry

A method is described which reliably quantifies the degree of intercellular communication via gap junctions by combining a dye-loading technique with fluorescence-activated flow cytometry. Our experiments expand former measurements of other groups by analyzing the time- and density-dependent onset of coupling with a fixed ratio of donor to recipient cells. The high sensitivity of this technique provides a better resolution than the microelectrode technique and allows the detection of small changes in gap-junctional coupling by examining a large number of cells in a single experiment. Suspended cells were loaded with the membrane-permeable dye calcein AM, which is intracellularly hydrolyzed by nonspecific esterases, and the resulting polyanionic calcein is thus trapped inside these donor cells. Gap junctions, however, are permeable for this fluorescent dye, as can be observed when suspended donor cells are added to recipient cells (i.e., monolayer cultures) in which case cell-cell contact is established within less than 60 min. In addition, one of these two cell populations can also be stained with a membrane-resident dye (e.g., DiI), which facilitates the identification of different cell populations (donors, recipients, and noncoupled cells) not only by epifluorescence microscopy but also by flow cytometry. Our analyses reveal that junctional coupling depends not only on the connexin type (homo- or heterotypic junction) but also on the origin (species) of the contacting cells (homo- or heterospecific contact). We confirm earlier reports in which homotypic-homospecific coupling was demonstrated with different techniques in connexin-transfected HeLa and RIN cells as well as in BICR/M1R(k) and 3T3/SV40 cells. In contrast to other publications, we show that a significant heterotypic-homospecific coupling between Cx40- and Cx43-HeLa transfectants can be resolved, whereas no coupling was detected for heterotypic-heterospecific contacts between Cx40-HeLa transfectants and the Cx43-expressing cell lines BICR/M1R(k), 3T3/SV40, and RIN.     

Detection of protein interactions based on GFP fragment complementation by fluorescence microscopy and spectrofluorometry

We have developed a set of simple modifications of the green fluorescent protein (GFP)fragment reassembly assay in bacteria that permits: (i)fluorescent microscopy visualization of GFP reassembly only 1-2 h after induction of protein expression, thus approximating the detection of GFP reassembly to the real-time dynamics of protein complex formation in living cells; (ii) spectrofluorometric detection of reassembled GFP fluorescent signals directly in lysates from cell suspension thereby avoiding, in many cases, the need for tag-affinity isolation of protein complexes; and (iii) comparative quantification of signal intensity in numerous cell-sample lysates using a Bio-Rad IQ5 spectrofluorometric detection system (Bio-Rad Laboratories, Madrid, Spain). Collectively, the results demonstrate that the combination of microscopic and spectrofluorometric detection provides a time-saving and sensitive alternative to existing methods of fluorescence complementation analysis.     

Mucin-lectin interactions assessed by flow cytometry

The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.