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.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

LEC–BiFC: a new method for rapid assay of protein interaction

Abstract

Protein–protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein–protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC–BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl–X_L and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC–BiFC can be applied easily in any lab equipped with a fluorescence microscope.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.     

In planta analysis of protein–protein interactions related to light signaling by bimolecular fluorescence complementation

The determination of protein-protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein-protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

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.     

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.     

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 (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.     

Significant reduction of BiFC non‐specific assembly facilitates in planta assessment of heterotrimeric G‐protein interactors

Protein networks and signaling cascades are key mechanisms for intra‐ and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein–protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal‐to‐noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined d ouble O RF e xpression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin‐like (CML) protein, and by showing that heterotrimeric G‐protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so‐called PLDα1 ‘DRY’ motif abolishes both of these interactions.     

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.     

A Computationally Guided Protein-Interaction Screen Uncovers Coiled-Coil Interactions Involved in Vesicular Trafficking

Mapping protein-protein interactions at a domain or motif level can provide structural annotation of the interactome. The α-helical coiled coil is among the most common protein-interaction motifs, and proteins predicted to contain coiled coils participate in diverse biological processes. Here, we introduce a combined computational/experimental screening strategy that we used to uncover coiled-coil interactions among proteins involved in vesicular trafficking in Saccharomyces cerevisiae. A number of coiled-coil complexes have already been identified and reported to play important roles in this important biological process. We identify additional examples of coiled coils that can form physical associations. The computational strategy used to prioritize coiled-coil candidates for testing dramatically improved the efficiency of discovery in a large experimental screen. As assessed by comprehensive yeast two-hybrid assays, computational prefiltering retained 90% of positive interacting pairs and eliminated > 60% of negatives from a set of interaction candidates. The coiled-coil-mediated interaction network elucidated using the combined computational/experimental approach comprises 80 coiled-coil associations between 58 protein pairs, among which 21 protein interactions have not been previously reported in interaction databases and 26 interactions were previously known at the protein level but have now been localized to the coiled-coil motif. The coiled-coil-mediated interactions were specific rather than promiscuous, and many interactions could be recapitulated in a green fluorescent protein complementation assay. Our method provides an efficient route to discovering new coiled-coil interactions and uncovers a number of associations that may have functional significance for vesicular trafficking.     

In vivo visualization of actin dynamics and actin interactions by BiFC

The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCdelta). Treatment with phorbol esters caused translocation of actin/PKCdelta complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function.     

New Gateway-compatible vectors for a high-throughput protein–protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis

Protein–protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC–CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.     

双分子荧光互补技术

双分子荧光互补（bimolecular fluorescence complementation, BiFC）是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术.该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新形成能具有活性的荧光蛋白.BiFC方法简单直观,既可以检测蛋白之间的相互作用,也可以定位相互作用蛋白质的位点.多色BiFC系统共用或与荧光共振能量转移（FRET）技术联用,还可以检测细胞内多个蛋白质的相互作用.     

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Genome-wide physical protein±protein interaction(PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeastenhanced green fluorescent protein-based bimolecular fluorescence complementation(y EGFPBiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied y EGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus.Two-thirds(9/14) of known interactions of EBOV were recap...     

双分子荧光互补技术在神经系统变性疾病蛋白质寡聚化研究中的应用

神经系统变性疾病(ND)的病理特征是蛋白质聚集在细胞内或细胞外形成异常的堆积体而导致神经毒性。双分子荧光互补技术(Bi FC)是研究ND相关蛋白质寡聚化分子机制的重要工具。与其他技术不同的是,Bi FC不但可以在生理环境中对蛋白质-蛋白质之间的相互作用进行可视化检测,而且能够提供详细的亚细胞定位信息。本文通过综述Bi FC技术的原理、优缺点及其在ND研究领域中的应用进展,探讨该技术对揭示寡聚体和包涵体形成原因方面的潜力,为神经系统疾病开发新的治疗策略提供重要参考。     

Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells

Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a bright-yellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.