Split YFP Research Articles

DNA-Regulated Multi-Protein Complement Control.

In nature, the interactions between proteins and their complements/substrates can dictate complex functions. Herein, we explore how DNA on nucleic acid modified proteins can be used as scaffolds to deliberately control interactions with a peptide complement (by adjusting length, sequence, and rigidity). As model systems, split GFPs were covalently connected through DNA scaffolds (36-58 bp). Increasing the length or decreasing the rigidity of the DNA scaffold (through removal of the duplex) increases the extent of intramolecular protein binding (up to 7.5-fold) between these GFP fragments. Independent and dynamic control over functional outputs can also be regulated by DNA hybridization; a multi-protein (split CFP and YFP) architecture was synthesized and characterized by fluorescence. This ternary construct shows that DNA displacement strands in different stoichiometric ratios can be used deliberately to regulate competitive binding between two unique sets of proteins. These studies establish a foundation for creating new classes of biological machinery based upon the concept of DNA-regulated multi-protein complement control.

Probing protein ubiquitination in live cells.

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1β as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1β is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.

Late Embryogenesis Abundant (LEA)5 Regulates Translation in Mitochondria and Chloroplasts to Enhance Growth and Stress Tolerance.

The late embryogenesis abundant (LEA)5 protein is predominantly expressed in Arabidopsis leaves in the dark, the levels of LEA5 transcripts decreasing rapidly upon illumination. LEA5 is important in plant responses to environmental stresses but the mechanisms involved have not been elucidated. We therefore explored LEA5 functions in Arabidopsis mutants (lea5) and transgenic Arabidopsis plants constitutively expressing LEA5 (OEX 2-5), as well as in transgenic barley lines expressing the Arabidopsis LEA5 gene. The OEX 2-5 plants grew better than controls and lea5 mutants in the presence of the prooxidants methyl viologen and menadione. Confocal microscopy of Arabidopsis mesophyll protoplasts expressing a LEA5-YFP fusion protein demonstrated that LEA5 could be localized to chloroplasts as well as mitochondria in Arabidopsis protoplasts. Tandem affinity purification (TAP) analysis revealed LEA5 interacts with the chloroplast DEAD-box ATP-dependent RNA helicase 22 (RH22) in Arabidopsis cells. Split YFP analysis confirmed the interaction between RH22 and LEA5 in chloroplasts. The abundance of translated protein products in chloroplasts was decreased in transgenic Arabidopsis plants and increased in lea5 knockout mutants. Conversely, the abundance of translated mitochondrial protein products was increased in OEX 2-5 plants and decreased in lea5 mutants. Mitochondrial electron transport rates were higher in the OEX 2-5 plants than the wild type. The transformed barley lines expressing the Arabidopsis LEA5 had increased seed yields, but they showed a greater drought-induced inhibition of photosynthesis than controls. Taken together, these data demonstrate that LEA5 regulates organellar translation, in order to enhance respiration relative to photosynthesis in response to stress.

Engineering an efficient and bright split Corynactis californica green fluorescent protein

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein–protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

Simple Fluorogenic Cellular Assay for Histone Deacetylase Inhibitors Based on Split-Yellow Fluorescent Protein and Intrabodies.

Histone deacetylase (HDAC) inhibitors that regulate the posttranslational modifications of histone tails are therapeutic drugs for many diseases such as cancers, neurodegenerative diseases, and asthma; however, convenient and sensitive methods to measure the effect of HDAC inhibitors in cultured mammalian cells remain limited. In this study, a fluorogenic assay was developed to detect the acetylation of lysine 9 on histone H3 (H3K9ac), which is involved in several cancers, Alzheimer’s disease, and autism spectrum disorder. To monitor the changes in H3K9ac levels, an H3K9ac-specific intrabody fused with a small fragment FP11 of the split-yellow fluorescent protein (YFP) (scFv-FP11) was expressed in mammalian cells, together with a larger YFP fragment FP1-10 fused with a nuclear localization signal. When the intranuclear level of H3K9ac is increased, the scFv-FP11 is more enriched in the nucleus via passive diffusion through the nuclear pores from the cytoplasm, which increases the chance of forming a fluorescent complex with the nuclear YFP1-10. The results showed that the YFP fluorescence increased when the cells were treated with HDAC inhibitors. Moreover, the sensitivity of the split YFP reporter system to three HDAC inhibitors was higher than that of a conventional cell viability test. The assay system will be a simple and sensitive detection method to evaluate HDAC inhibitor activities at the levels of both single cells and cell populations.

SpMnn9p and SpAnp1p form a protein complex involved in mannan synthesis in the fission yeast Schizosaccharomyces pombe

The cell walls of yeast cells possess a large mannan structure mainly comprising of a linear α1,6-linked mannose oligomer on the N-linked glycans. The biosynthesis of the mannan is initiated by ScOch1p α1,6-mannosyltransfease, and elongated by the mannan polymerase complexes M-Pol I and II in the Golgi of Saccharomyces cerevisiae. Here, we functionally characterized SpMnn9 and SpAnp1 proteins in the fission yeast Schizosaccharomyces pombe; these proteins are homologs of S.cerevisiae M-Pol II complex proteins ScMnn9p and ScAnp1p. Cells harboring disruptions in Spmnn9+ and Spanp1+ genes showed slower growth at 37°C and an increased sensitivity to hygromycin B, characteristic of a glycosylation defect. Results obtained from the acid phosphatase assay and high-performance liquid chromatography analysis of N-linked glycans in Spmnn9Δ and Spanp1Δ mutants suggested that the mannan structure in S.pombe is synthesized sequentially by the α-mannosyltransferases in the order of SpOch1p, SpMnn9p and SpAnp1p. Immunoprecipitation and split YFP analyses demonstrated that SpMnn9p and SpAnp1p form the M-Pol-II like complex. Together, these results provided an improved understanding of the mechanism of mannan synthesis by SpMnn9p and SpAnp1p in S.pombe.

A cell-based high-content screen identifies isocotoin as a small molecule inhibitor of the meiosis-specific MEIOB-SPATA22 complex†.

MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The MeiobD383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins.

Genetic encoding of DNA nanostructures and their self-assembly in living bacteria.

The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32–205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3′-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 μm2) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells.

Abstract 5112: In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation

Abstract Hypoxia is a common feature of many solid tumors that promotes tumor progression and leads to radiation and chemotherapy resistance. Hypoxia-inducible factor 1 (HIF-1), as one of the major mediators of the hypoxic response, has been shown to activate hypoxia-responsive genes, which are involved in multiple aspects of tumorigenesis and cancer progression. Previously, we have described an interaction between septin 9 isoform 1 (SEPT9_i1) protein and HIF-1α, the oxygen regulated subunit of HIF-1. SEPT9_i1 is a member of the conserved family of septins, which are GTP-binding proteins that form cytoskeleton-like filaments essential for many functions in eukaryotic organisms including neoplasia. The interaction of SEPT9_i1 with HIF-1α increases protein stability and HIF-1 transcriptional activity in vitro and promotes proliferation, tumor growth and angiogenesis in vivo. Recent data indicated that SEPT9_i1 was also involved in the trafficking of cytoplasmic HIF-1α into the nucleus by direct interaction to the nuclear transporter importin-α. Herein, we utilized split YFP bimolecular fluorescence complementation (BiFC) methodology in order to monitor HIF-1α/SEPT9_i1 interactions in vivo. First, we generated split YFP protein chimeras fused to HIF-1α and to SEPT9_i1 on both their amino and carboxyl termini. Different pairs of HIF-1α and SEPT9_i1 chimeras were tested to functional complementation of the split YFP fragments using transient transfection in HEK293 cells. The complemented proteins capable of fluorescence were identified as SEPT9_i1-YN (the amino terminus of YFP fused to the carboxyl terminal of SEPT9_i1) and YC-HIF-1α (the carboxyl terminus of YFP fused to the N-terminal of HIF-1α). This pair was selected for further studies in PC-3 human prostate cancer cells. YFP complementation fluorescence derived from these chimeras was increased in the presence of hypoxia mimicking agents, such as CoCl2 and the iron chelators deferoxamine (DFO) and dibenzoylmethane (DBM). The chimera, YCΔHLH lacking the HLH domain that is essential for the interaction with SEPT9_i1 gave a significant reduction in YFP complementation fluorescence. Furthermore, expression of SEPT9_i1 252-379aa fragment, the domain essential for the interaction with HIF-1α, abolished almost completely the YFP complementation fluorescence. These results reconfirm our previous studies on HIF-1α/SEPT9_i1 interactions and make this system attractive for identifying new compounds capable of disrupting this complex in order to inhibit tumor growth and angiogenesis. Citation Format: Maya Golan, Nicola J. Mabjeesh. In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2015-5112

Interaction specificity and coexpression of rice NPR1 homologs 1 and 3 (NH1 and NH3), TGA transcription factors and Negative Regulator of Resistance (NRR) proteins

BackgroundThe nonexpressor of pathogenesis-related genes 1, NPR1 (also known as NIM1 and SAI1), is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis. In rice, the NPR1 homolog 1 (NH1) interacts with TGA transcriptional regulators and the Negative Regulator of Resistance (NRR) protein to modulate the SAR response. Though five NPR1 homologs (NHs) have been identified in rice, only NH1 and NH3 enhance immunity when overexpressed. To understand why NH1 and NH3, but not NH2, NH4, or NH5, contribute to the rice immune response, we screened TGA transcription factors and NRR-like proteins for interactions specific to NH1 and NH3. We also examined their co-expression patterns using publicly available microarray data.ResultsWe tested five NHs, four NRR homologs (RHs), and 13 rice TGA proteins for pair-wise protein interactions using yeast two-hybrid (Y2H) and split YFP assays. A survey of 331 inter-family interactions revealed a broad, complex protein interaction network. To investigate preferred interaction partners when all three families of proteins were present, we performed a bridged split YFP assay employing YFPN-fused TGA, YFPC-fused RH, and NH proteins without YFP fusions. We found 64 tertiary interactions mediated by NH family members among the 120 sets we examined. In the yeast two-hybrid assay, each NH protein was capable of interacting with most TGA and RH proteins. In the split YFP assay, NH1 was the most prevalent interactor of TGA and RH proteins, NH3 ranked the second, and NH4 ranked the third. Based on their interaction with TGA proteins, NH proteins can be divided into two subfamilies: NH1, NH2, and NH3 in one family and NH4 and NH5 in the other.In addition to evidence of overlap in interaction partners, co-expression analyses of microarray data suggest a correlation between NH1 and NH3 expression patterns, supporting their common role in rice immunity. However, NH3 is very tightly co-expressed with RH1 and RH2, while NH1 is strongly, inversely co-expressed with RH proteins, representing a difference between NH1 and NH3 expression patterns.ConclusionsOur genome-wide surveys reveal that each rice NH protein can partner with many rice TGA and RH proteins and that each NH protein prefers specific interaction partners. NH1 and NH3 are capable of interacting strongly with most rice TGA and RH proteins, whereas NH2, NH4, and NH5 have weaker, limited interaction with TGA and RH proteins in rice cells. We have identified rTGA2.1, rTGA2.2, rTGA2.3, rLG2, TGAL2 and TGAL4 proteins as the preferred partners of NH1 and NH3, but not NH2, NH4, or NH5. These TGA proteins may play an important role in NH1- and NH3-mediated immune responses. In contrast, NH4 and NH5 preferentially interact with TGAL5, TGAL7, TGAL8 and TGAL9, which are predicted to be involved in plant development.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-461) contains supplementary material, which is available to authorized users.

Mapping regions of Epstein–Barr virus (EBV) glycoprotein B (gB) important for fusion function with gH/gL

Glycoproteins gB and gH/gL are required for entry of Epstein–Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB–gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.

Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO(2)-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO(2)-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO(2)-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO(2)-related transport processes, a structure built up by the tetramer is the basis of this function.

Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco

Tobacco is a commonly used heterologous system for studying combinatorial regulation of the flavonoid biosynthetic pathway by the bHLH-MYB transcription factor (TF) complex in plants. However, little is known about the endogenous tobacco bHLH and MYB TFs involved in the pathway. Ectopic expression in tobacco of heterologous bHLH TF genes, such as maize Lc, leads to increased anthocyanin production in the reproductive tissues, suggesting the presence of a reproductive tissue-specific MYB TF that interacts with the Lc-like bHLH TFs. We isolated a gene (NtAn2) encoding a R2R3 MYB TF from developing tobacco flowers. NtAn2 shares high sequence homology with other known flavonoid-related MYB TFs and is mostly expressed in developing flowers. Constitutive ectopic expression of NtAn2 induces whole-plant anthocyanin production in tobacco and Arabidopsis. In transgenic tobacco and Arabidopsis expressing NtAn2, both subsets of early and late flavonoid pathway genes are up-regulated. Suppression of NtAn2 by RNAi in tobacco resulted in a white-flowered phenotype and the inhibition of the late pathway genes. Yeast two-hybrid assays demonstrated that NtAn2 can interact with five heterologous bHLH TFs known to induce anthocyanin synthesis in other species including maize, perilla, snapdragon and Arabidopsis. Bimolecular fluorescent complementation using split YFP demonstrated that NtAn2 interacts with Lc in tobacco cells and that the complex is localized to nuclei. Transient co-expression of NtAn2 and Lc or Arabidopsis TT8 in tobacco protoplasts activated the promoters of two key flavonoid pathway genes, chalcone synthase and dihydroflavonol reductase. These results suggest that NtAn2 is a key gene controlling anthocyanin production in reproductive tissues of tobacco.

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

BackgroundThe investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.ResultsWe have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin α-1 in the cytoplasm, whereas interaction of VirE2 with a different importin α isoform, importin α-4, occurs predominantly in the nucleus.ConclusionMulti-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods.

Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques.