Improved validation of protein interactions using bicistronic BiFC (Bi2FC).

Refolding based Bimolecular Fluorescence Complementation (BiFC) has emerged as an important in vivo technique to identify protein interactions. Significant improvements have been made to enhance the detection capacities of BiFC, however less attention has been paid to the detection of expression levels of proteins. Here we demonstrate development and validation of an improved method to identify protein interactions that incorporates an expression control based on bicistronic expression of the protein of interest and a fluorescent protein separated by a self-cleaving peptide. This method gives robust identification of positive interactions and more reliably identifies absence of interactions. We also show an earlier identified non-interacting pair in yeast two-hybrid (Y2H) to be interacting in vivo. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01477-y.

Novel Antennapedia and Ultrabithorax trimeric complexes with TBP and Exd regulate transcription.

BACKGROUND: Hox proteins interact with DNA and many other proteins, co-factors, transcriptional factors, chromatin remodeling components, non-coding RNAs and even the extracellular matrix that assembles the Hox complexes. The number of interacting partners continues to grow with diverse components and more transcriptional factors than initially thought. Hox complexes present many activities, but their molecular mechanisms to modulate their target genes remain unsolved. RESULTS: In this paper we showed the protein-protein interaction of Antp with Ubx through the homeodomain using BiFC in Drosophila. Analysis of Antp-deletional mutants showed that AntpHD helixes 1 and 2 are required for the interaction with Ubx. Also, we found a novel interaction of Ubx with TBP, in which the PolyQ domain of TBP is required for the interaction. Moreover, we also detected the formation of two new trimeric complexes of Antp with Ubx, TBP and Exd using BiFC-FRET; these proteins, however, do not form a trimeric interaction with BIP2 or TFIIEß. The novel trimeric complexes reduced Antp transcriptional activity, indicating that they could confer specificity for repression. CONCLUSIONS: Our results increase the number of transcriptional factors in the Antp and Ubx interactomes that form two novel trimeric complexes with TBP and Exd. We also report a new Ubx interaction with TBP. These novel interactions provide important clues of the dynamics of Hox-interacting complexes involved in transcriptional regulation, contributing to better understand Hox function.

Comprehensive Review on Bimolecular Fluorescence Complementation and Its Application in Deciphering Protein-Protein Interactions in Cell Signaling Pathways.

Signaling pathways are responsible for transmitting information between cells and regulating cell growth, differentiation, and death. Proteins in cells form complexes by interacting with each other through specific structural domains, playing a crucial role in various biological functions and cell signaling pathways. Protein-protein interactions (PPIs) within cell signaling pathways are essential for signal transmission and regulation. The spatiotemporal features of PPIs in signaling pathways are crucial for comprehending the regulatory mechanisms of signal transduction. Bimolecular fluorescence complementation (BiFC) is one kind of imaging tool for the direct visualization of PPIs in living cells and has been widely utilized to uncover novel PPIs in various organisms. BiFC demonstrates significant potential for application in various areas of biological research, drug development, disease diagnosis and treatment, and other related fields. This review systematically summarizes and analyzes the technical advancement of BiFC and its utilization in elucidating PPIs within established cell signaling pathways, including TOR, PI3K/Akt, Wnt/ß-catenin, NF-κB, and MAPK. Additionally, it explores the application of this technology in revealing PPIs within the plant hormone signaling pathways of ethylene, auxin, Gibberellin, and abscisic acid. Using BiFC in conjunction with CRISPR-Cas9, live-cell imaging, and ultra-high-resolution microscopy will enhance our comprehension of PPIs in cell signaling pathways.

Novel nucleus-localized GRAM protein encoding OsGRAM57 gene enhances salt tolerance through ABA-dependent pathway and modulated carbohydrate metabolism.

GRAM (Glucosyltransferases-like GTPase activators and Myotubularin) domain-encoding proteins play pivotal roles in plant growth and responses to biotic stresses. Yet, their influence on abiotic stress responses has remained enigmatic. This study unveils a novel nucleus-localized OsGRAM57, a GRAM protein-encoding gene and its profound regulatory functions in enhancing salt stress tolerance using Arabidopsis thaliana as a model plant. OsGRAM57-OEX (OsGRAM57-OEX) lines displayed significant enhancement in salt tolerance, modulated physiological, biochemical, K+/Na+ ratios, and enzymatic indices as compared to their wild-type (WT). Furthermore, OsGRAM57-OEX seedlings demonstrate increased levels of endogenous abscisic acid (ABA) and other phytohormones, while metabolic profiling revealed enhanced carbohydrate metabolism. Delving into the ABA signaling pathway, OsGRAM57 emerged as a central regulator, orchestrating the expression of genes crucial for salt stress responses, carbohydrate metabolism, and ABA signaling. The observed interactions with target genes and transactivation assays provided additional support for OsGRAM57's pivotal role. These findings underscore OsGRAM57's positive influence on the ABA pathway and affirm its capacity to enhance salt tolerance through an ABA-dependent pathway and fine-tuned carbohydrate metabolism. In summary, this new study reveals the previously undiscovered regulatory roles of OsGRAM57 in Arabidopsis abiotic stress responses, offering promising ways for strengthening plant resilience in the face of adverse environmental conditions.

Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva.

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

Dual-transgenic BiFC vector systems for protein-protein interaction analysis in plants.

Protein-protein interaction (PPI) play a pivotal role in cellular signal transduction. The bimolecular fluorescence complementation (BiFC) assay offers a rapid and intuitive means to ascertain the localization and interactions of target proteins within living cells. BiFC is based on fluorescence complementation by reconstitution of a functional fluorescent protein by co-expression of N- and C-terminal fragments of this protein. When fusion proteins interact, the N- and C-terminal fragments come into close proximity, leading to the reconstitution of the fluorescent protein. In the conventional approach, the N-terminal and C-terminal fragments of the fluorescent protein are typically expressed using two separate vectors, which largely relies on the efficiency of the transformation of the two vectors in the same cells. Furthermore, issues of vector incompatibility can often result in loss of one plasmid. To address these challenges, we have developed novel dual-transgenic BiFC vectors, designed as pDTQs, derived from the previously published pDT1 vector. This set of BiFC vectors offers the following advantages: 1) Both fluorescent fusion proteins are expressed sequentially within a single vector, enhancing expression efficiency; 2) Independent promoters and terminators regulate the expression of the two proteins potentially mitigating vector compatibility issues; 3) A long linker is inserted between the fluorescent protein fragment and the gene of interest, facilitating the recombination of the fused fluorescent protein into an active form; 4) Four distinct types of fluorescent proteins, namely, EYFP, mVenus, mRFP1Q66T and mCherry are available for BiFC analysis. We assessed the efficiency of the pDTQs system by investigating the oligomerization of Arabidopsis CRY2 and CRY2-BIC2 interactions in N. benthamiana. Notably, the pDTQs were found to be applicable in rice, underscoring their potential utility across various plant species.

An optimum study on the laser scanning confocal microscopy techniques for BiFC assay using plant protoplast.

BACKGROUND: The bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein-protein interactions. While several BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in BiFC assays. RESULTS: When the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing. CONCLUSION: When the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.

A Ubiquitin-Based Module Directing Protein-Protein Interactions in Chloroplasts.

A promising approach for the genetic engineering of multiprotein complexes in living cells involves designing and reconstructing the interaction between two proteins that lack native affinity. Thylakoid-embedded multiprotein complexes execute the light reaction of plant photosynthesis, but their engineering remains challenging, likely due to difficulties in accurately targeting heterologous membrane-bound proteins to various sub-compartments of thylakoids. In this study, we developed a ubiquitin-based module (Nub-Cub) capable of directing interactions in vivo between two chloroplast proteins lacking native affinities. We applied this module to genetically modify thylakoid multiprotein complexes. We demonstrated the functionality of the Nub-Cub module in the model organism Arabidopsis thaliana. Employing this system, we successfully modified the Photosystem II (PSII) complex by ectopically attaching an extrinsic subunit of PSII, PsbTn1, to CP26-a component of the antenna system of PSII. Surprisingly, this mandatory interaction between CP26 and PsbTn1 in plants impairs the efficiency of electron transport in PSII and unexpectedly results in noticeable defects in leaf development. Our study not only offers a general strategy to modify multiprotein complexes embedded in thylakoid membranes but it also sheds light on the possible interplay between two proteins without native interaction.

Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein-Protein Interactions and Cellular Protein Complex Localizations.

Investigations of protein-protein interactions (PPIs) are of paramount importance for comprehending cellular processes within biological systems. The bimolecular fluorescence complementation (BiFC) assay presents a convenient methodology for visualizing PPIs within live cells. While a range of fluorescent proteins have been introduced into the BiFC system, there is a growing demand for new fluorescent proteins to accommodate the expanding requirements of researchers. This study describes the introduction of Tagged blue fluorescent protein 2 (TagBFP2) into the BiFC assay to verify the interaction between two proteins, with Enhanced yellow fluorescent protein (EYFP) employed as a positive control. Both fluorescent proteins demonstrated optimal performance in this study. Compared to EYFP, the BiFC system utilizing TagBFP2 yielded a higher signal-to-noise ratio, which facilitated differentiation of the signal of PPIs from noise and enabled employment of other fluorescent proteins within the BiFC assay. Notably, the utilization of a fluorescent secondary antibody in immunofluorescence applications or the tagging of an interest protein with a fluorescent protein occupied the green or yellow channel. Overall, the present article introduces a BiFC assay that is highly straightforward, reliable, and replicable, with the ability to be completed within 1 week. This method requires neither expensive instrumentation nor technical skills of a high order.

Highly Sensitive and Specific Detection of Influenza A Viruses Using Bimolecular Fluorescence Complementation (BiFC) Reporter System.

In this study, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV)-sensing system by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) made up of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, which was derived from 6'-silalyllactose and used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range extending from 1 × 100 to 1 × 107 TCID50/mL, and it had a detection limit of 1.1 × 100 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while it displayed weak fluorescence signals when exposed to NA-deficient viruses, such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Overall, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC that is capable of detecting ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.

Bimolecular Fluorescence Complementation Assay to Evaluate HSP90-Client Protein Interactions in Cells.

Protein-protein interactions (PPI) in cells play a pivotal role in cellular function and dynamics. Cellular proteostasis is maintained by PPI networks between molecular chaperones, co-chaperones, and client proteins. Consequently, strategies to visualize and analyze PPI in cells are useful in understanding protein homeostasis regulation. The Bimolecular Fluorescence Complementation (BiFC) assay has emerged as a useful tool for studying PPI between proteins in live or fixed cells. BiFC is based on the detection of fluorescence generated when interacting protein pairs, produced as fusion proteins with either the N- or C-terminal fragment of a fluorescent protein, are in sufficient proximity to permit reconstitution of the split fluorophore. Here, we describe the application of the BiFC assay to a model of chaperone-client interactions using Hsp90 and the validated client protein CDK4. This assay allows for the distribution and spatiotemporal analysis of HSP90-CDK4 complexes in live or fixed cells and is amenable to studying the effects of inhibitors and mutations on chaperone-client protein networks.

Bimolecular Fluorescence Complementation (BiFC) Assay to Visualize Protein-Protein Interactions in Living Cells.

Bimolecular fluorescence complementation (BiFC) assay is a method to visualize the protein-protein interaction in living cells. This technique is based on ability of the non-fluorescent fragment of fluorescent protein to form fluorescent complex when they are fused to two interacting proteins. In this chapter, we describe the widely used split yellow fluorescent protein (YFP) system to visualize the protein-protein interaction in plant cells.

Detecting Protein-Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC) and Luciferase Complementation Assays (LCA).

In multicellular organisms, establishing the full body plane involves cell-cell signaling where protein associations are important for the diverse cellular functions within the cells. For the study of protein-protein interactions (PPI), bimolecular fluorescence complementation (BiFC) and luciferase complementation assays (LCA) have proven to be reliable tools that can be used to confirm the physical association of two proteins in a semi-in vivo environment. This chapter provides a detailed description of these two techniques using Nicotiana benthamiana as a semi-in vivo transient expression system. As an example, we will use the interaction of the two well-described transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR), which are known as regulators of asymmetric cell division and stem cell specification in the root meristem of the model plant Arabidopsis thaliana. While the BiFC assay provides subcellular information by displaying a fluorescence signal, nuclear in this case, resulting from the reconstituted fluorophore, the LCA generates a quantitative readout of the SCR-SHR interaction. The combination of both assays provides information on the localization and strength of the PPI.

Determination of Complex Formation between Drosophila Nrf2 and GATA4 Factors at Selective Chromatin Loci Demonstrates Transcription Coactivation.

Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila. Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.

The SAH7 Homologue of the Allergen Ole e 1 Interacts with the Putative Stress Sensor SBP1 (Selenium-Binding Protein 1) in Arabidopsis thaliana.

In this study, we focused on a member of the Ole e 1 domain-containing family, AtSAH7, in Arabidopsis thaliana. Our lab reports for the first time on this protein, AtSAH7, that was found to interact with Selenium-binding protein 1 (AtSBP1). We studied by GUS assisted promoter deletion analysis the expression pattern of AtSAH7 and determined that the sequence 1420 bp upstream of the transcription start can act as a minimal promoter inducing expression in vasculature tissues. Moreover, mRNA levels of AtSAH7 were acutely increased under selenite treatment in response to oxidative stress. We confirmed the aforementioned interaction in vivo, in silico and in planta. Following a bimolecular fluorescent complementation approach, we determined that the subcellular localization of the AtSAH7 and the AtSAH7/AtSBP1 interaction occur in the ER. Our results indicate the participation of AtSAH7 in a biochemical network regulated by selenite, possibly associated with responses to ROS production.

Studies on alpha-synuclein and islet amyloid polypeptide interaction.

Introduction: Parkinson's disease and type 2 diabetes have both elements of local amyloid depositions in their pathogenesis. In Parkinson's disease, alpha-synuclein (aSyn) forms insoluble Lewy bodies and Lewy neurites in brain neurons, and in type 2 diabetes, islet amyloid polypeptide (IAPP) comprises the amyloid in the islets of Langerhans. In this study, we assessed the interaction between aSyn and IAPP in human pancreatic tissues, both ex vivo and in vitro. Material and Methods: The antibody-based detection techniques, proximity ligation assay (PLA), and immuno-TEM were used for co-localization studies. Bifluorescence complementation (BiFC) was used for interaction studies between IAPP and aSyn in HEK 293 cells. The Thioflavin T assay was used for studies of cross-seeding between IAPP and aSyn. ASyn was downregulated with siRNA, and insulin secretion was monitored using TIRF microscopy. Results: We demonstrate intracellular co-localization of aSyn with IAPP, while aSyn is absent in the extracellular amyloid deposits. ASyn reactivity is present in the secretory granules of ß-cells and some α-cells in human islets. The BiFC-expression of aSyn/aSyn and IAPP/IAPP in HEK293 cells resulted in 29.3% and 19.7% fluorescent cells, respectively, while aSyn/IAPP co-expression resulted in ∼10% fluorescent cells. Preformed aSyn fibrils seeded IAPP fibril formation in vitro, but adding preformed IAPP seeds to aSyn did not change aSyn fibrillation. In addition, mixing monomeric aSyn with monomeric IAPP did not affect IAPP fibril formation. Finally, the knockdown of endogenous aSyn did not affect ß cell function or viability, nor did overexpression of aSyn affect ß cell viability. Discussion: Despite the proximity of aSyn and IAPP in ß-cells and the detected capacity of preformed aSyn fibrils to seed IAPP in vitro, it is still an open question if an interaction between the two molecules is of pathogenic significance for type 2 diabetes.

Methods to detect AUTOphagy-Targeting Chimera (AUTOTAC)-mediated Targeted Protein Degradation in Tauopathies.

Targeted protein degradation (TPD) facilitates the selective elimination of unwanted and pathological cellular cargoes via the proteasome or the lysosome, ranging from proteins to organelles and pathogens, both within and outside the cell. Currently, there are several in vitro and in vivo protocols that assess the degradative potency of a given degrader towards a myriad of targets, most notably soluble, monomeric oncoproteins. However, there is a clear deficiency of methodologies to assess the degradative potency of heterobifunctional chimeric degraders, especially those in the autophagy space, against pathological, mutant tau species, such as detergent-insoluble oligomers and high-molecular aggregates. The protocol below describes both in vitro and in vivo biochemical assays to induce tau aggregation, as well as to qualitatively and quantitatively measure the degradative potency of a given degrader towards said aggregates, with specific applications of the AUTOTAC (AUTOphagy-TArgeting Chimera) platform provided as an example. A well-defined set of methodologies to assess TPD-mediated degradation of pathological tau species will help expand the scope of the TPD technology to neurodegeneration and other proteinopathies, in both the lab and the clinic. Graphical abstract Overview of assays observing elimination of tauP301L aggregates with AUTOTAC. (A) Description of the biological working mechanism of heterobifunctional chimeric AUTOTAC degraders. (B) Schematic illustration of assays described in this paper.

Overexpression of PvSVP1, an SVP-like gene of bamboo, causes early flowering and abnormal floral organs in Arabidopsis and rice.

Bamboo is a nontimber woody plant featuring a long vegetative stage and uncertain flowering time. Therefore, the genes belonging to flowering repressors might be essential in regulating the transition from the vegetative to reproductive stage in bamboo. The Short Vegetative Phase ( SVP) gene plays a pivotal role in floral transition and development. However, little is known about the bamboo SVP homologues. In this study, Phyllostachys violascens PvSVP1 is isolated by analysis of the P. edulis transcriptome database. Phylogenetic analysis shows that PvSVP1 is closely related to OsMADS55 (rice SVP homolog). PvSVP1 is ubiquitously expressed in various tissues, predominantly in vegetative tissues. To investigate the function of PvSVP1, PvSVP1 is overexpressed in Arabidopsis and rice under the influence of the 35S promoter. Overexpression of PvSVP1 in Arabidopsis causes early flowering and produces abnormal petals and sepals. Quantitative real-time PCR reveals that overexpression in Arabidopsis produces an early flowering phenotype by downregulating FLC and upregulating FT and produces abnormal floral organs by upregulating AP1, AP3 and PI expressions. Simultaneously, overexpression of PvSVP1 in rice alters the expressions of flowering-related genes such as Hd3a, RFT1, OsMADS56 and Ghd7 and promotes flowering under field conditions. In addition, PvSVP1 may be a nuclear protein which interacts with PvVRN1 and PvMADS56 on the yeast two-hybrid and BiFC systems. Our study suggests that PvSVP1 may play a vital role in flowering time and development by interacting with PvVRN1 and PvMADS56 in the nucleus. Furthermore, this study paves the way toward understanding the complex flowering process of bamboo.

A Live Cell Protein Complementation Assay for ORFeome-Wide Probing of Human HOX Interactomes.

Biological pathways rely on the formation of intricate protein interaction networks called interactomes. Getting a comprehensive map of interactomes implies the development of tools that allow one to capture transient and low-affinity protein-protein interactions (PPIs) in live conditions. Here we presented an experimental strategy: the Cell-PCA (cell-based protein complementation assay), which was based on bimolecular fluorescence complementation (BiFC) for ORFeome-wide screening of proteins that interact with different bait proteins in the same live cell context, by combining high-throughput sequencing method. The specificity and sensitivity of the Cell-PCA was established by using a wild-type and a single-amino-acid-mutated HOXA9 protein, and the approach was subsequently applied to seven additional human HOX proteins. These proof-of-concept experiments revealed novel molecular properties of HOX interactomes and led to the identification of a novel cofactor of HOXB13 that promoted its proliferative activity in a cancer cell context. Taken together, our work demonstrated that the Cell-PCA was pertinent for revealing and, importantly, comparing the interactomes of different or highly related bait proteins in the same cell context.