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Bimolecular Fluorescence Complementation: Visualization of Molecular Interactions in Living Cells
Abstract
A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally diverse interaction partners in many different cell types. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.
... Plasmid construction α-Synuclein fusion protein constructs, i.e., α-synuclein + N-terminal luciferase protein (SLN) and α-synuclein + C-terminal luciferase protein (SLC), were generated previously [17,18]. SLN and SLC were subcloned into pcDNA3.1 (+) at the Hind III and Kpn І restriction sites by GENEWIS (South Plainfield, NJ, USA). ...
... The small molecule 03A10 was identified as a candidate inhibitor of α-synuclein aggregation The bimolecular PCA is becoming a general strategy for studying the dynamics of protein-protein interactions in vivo using humanized Gaussia luciferase [17] or fluorescent-tagged protein(-Venus YFP) [18,20]. We fused α-synuclein to N-terminal Gaussia luciferase (SLN) or C-terminal Gaussia luciferase (SLC) and coexpressed these two plasmids in SH-SY5Y cells for highthroughput screening (Fig. 1a). ...
Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson’s disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 μM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3 mg· kg−1 ·d−1, 1 mg ·kg−1 ·d−1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg−1 ·d−1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice.
... However, visualization of cell-cell interactions using this technology is inhibited by some limitations. For example, the emergence of fluorescence from the reconstituted split components by cell-cell interactions requires a time delay derived from the maturation of chromophores, and the irreversibility of the process makes it impossible to monitor the dissociation of cell-cell interactions 25,28,30 . ...
... mGRASP showed green fluorescence at the cell adhesion sites similar to INCIDERs and its signal was more than twice as high as that of INCIDERs (Fig. 5d, e). Split-GFP-based indicators for cell-cell interactions are potentially unable to monitor the dissociation of cell-cell interactions 25,28,30 . To evaluate the relative dissociation property, we examined the effect of EGTA treatment on pre-formed cell-cell interactions. ...
N-cadherin (NCad) is a classical cadherin that mediates cell–cell interactions in a Ca ²⁺ -dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.
... To minimize the size of fluorescent protein tagging, the split fluorescent protein technique or BiFC is introduced ( Table 1) where a fluorescent protein is split into two non-fluorescent fragments to reduce the size of fusion as well as background fluorescence. 77 Two types of BiFC based biosensors are available: ...
... [113][114][115] However, they differ in terms of the type of interactions they can detect, sensitivity and signal-to-noise ratio, the possibility of false-positive or false-negative signals, capability of spatiotemporal monitoring of interactions, and the instrumentation needed. 77,116,117 We have summarized the advantages and limitations of each technique ( Table 2). In general, expressing these tau biosensors in living cells have the key advantage of preserving the native surroundings, such as the presence of molecular chaperones or other molecular constituents, in which the interaction takes place and is monitored. ...
Tau is a microtubule binding protein which plays an important role in physiological functions but it is also involved in the pathogenesis of Alzheimer's disease and related tauopathies. While insoluble and β‐sheet containing tau neurofibrillary tangles have been the histopathological hallmark of these diseases, recent studies suggest that soluble tau oligomers, which are formed prior to fibrils, are the primary toxic species. Substantial efforts have been made to generate tau oligomers using purified recombinant protein strategies to study oligomer conformations as well as their toxicity. However, no specific toxic tau species has been identified to date, potentially due to the lack of cellular environment. Hence, there is a need for cell‐based models for direct monitoring of tau oligomerization and aggregation. This review will summarize the recent advances in the cellular biosensor technology, with a focus on fluorescence resonance energy transfer, bimolecular fluorescence complementation, and split luciferase complementation approaches, to monitor formation of tau oligomers and aggregates in living cells. We will discuss the applications of the cellular biosensors in examining the heterogeneous tau conformational ensembles and factors affecting tau self‐assembly, as well as detecting cell‐to‐cell propagation of tau pathology. We will also compare the advantages and limitations of each type of tau biosensors, and highlight their translational applications in biomarker development and therapeutic discovery.
... These disadvantages have been partially overcome by applying different microscopy techniques directly in living cells, e.g. FRET or BiFC (reviewed in Kerppola (2006) and De Los Santos et al. (2015)). In both approaches, proteins predicted to interact are genetically fused to fluorescent proteins. ...
... Only when both proteins interact, the fragments will be brought into close proximity, allowing the fluorescent protein to reconstitute into its native structure, thus emitting a fluorescent signal. However, this reconstitution can take up to 50 min, thereby strongly reducing temporal resolution (reviewed in Kerppola (2006)). Although both methods overcome spatial and partially temporal resolution limits, both methods are highly distance dependent. ...
Im Unterschied zu anderen RNA Viren vervielfältigen Influenzaviren ihr Genom im Zellkern infizierter Zellen. Für die erfolgreiche Vermehrung müssen neu gebildete Genomsegmente (virale Ribonukleoproteine, vRNPs) wieder aus dem Zellkern exportiert werden. Dafür nutzt Influenza einen Exportkomplex, der sich aus dem viralen Matrixprotein 1 (M1) und Nukleusexportprotein (NEP) zusammensetzt und vRNPs unter Verwendung des zellulären Exportproteins CRM1 aus dem Zellkern transportiert. Zahlreiche Fragen im Zusammenhang mit dem Exportkomplex sind noch unbeantwortet: Wie viele Exportkomplexe werden pro vRNP gebunden? Wie interagieren die Proteine innerhalb des Komplexes mit vRNPs? Wie wird die zeitliche und räumliche Präsenz der beteiligten Proteine im Verlauf der Infektion reguliert? Um zu einem besseren Verständnis beizutragen, wurden in der vorliegenden Arbeit Fluoreszenzfluktuationsspektroskopie und molecular brightness-Analysen genutzt, um die Oligomerisierung der beteiligten Exportkomplexproteine zu quantifizieren. Werden Fluoreszenzproteine für solche Untersuchungen verwendet, treten häufig nicht-fluoreszente Zustände auf, die die Bestimmung des Oligomerzustandes beeinflussen. Daher wurde in dieser Arbeit ein einfaches Korrekturmodel vorgestellt, das die Population an nicht-fluoreszenten Zuständen berücksichtigt, und somit die genaue Bestimmung des Oligomerzustandes erlaubt. Dadurch konnte zum ersten Mal gezeigt werden, dass NEP Homodimere im Zytoplasma ausbildet, wohingegen eine um das 2,5-fach geringere Homodimerpopulation im Zellkern vorhanden war. Durch die Integration von Informationen über den Lokalisationsphänotyp und den Oligomerzustand von NEP sowie mehrerer Mutanten, konnte ein Modell abgeleitet werden, dass den Regulationsmechanismus beschreibt: Durch vorrübergehendes Maskieren und Demaskieren der beiden Nukleusexportsignale wird der Transport von NEP reguliert. Die Dimerisierung im Zytoplasma und Monomerisierung im Zellkern unterstützen diesen Mechanismus.
... To determine the most efficient split site of superfolder GFP (sfGFP) (Cabantous et al., 2005;Pedelacq et al., 2006), we tested the reconstitution activity of two probe pairs containing sfGFP fragments cut at 1-7/8-11 and 1-10/11 within its 11 b-sheets ( Figure 1B). The 1-7/8-11 split site is frequently used in the BiFC (bimolecular fluorescence complementation) method (Kerppola, 2008;Shyu and Hu, 2008), whereas the 1-10/11 split site is used for all previous intercellular probes (Feinberg et al., 2008;Kim et al., 2012;Tsetsenis et al., 2014). In this system, we found that the 1-7/8-11 combination possessed higher reconstitution activity than the 1-10/11 combination ( Figure S1). ...
... Our present study revealed that the 1-7/8-11 split pair in the mGRASP system (mGRASP(7/8)) showed higher reconstitution activity than 1-10/11 split pair. In the detection of intracellular molecular interactions, 1-7/8-11 is the most popular split site owing to its higher reconstitution activity, although it may increase non-specific molecular interactions (Kerppola, 2008;Shyu and Hu, 2008). To detect intercellular contacts, however, GFP is theoretically not reconstituted unless the cell-cell distance becomes close enough for intercellular molecular interaction. ...
Intercellular contacts are essential for precise organ morphogenesis, function, and maintenance; however, spatiotemporal information of cell-cell contacts or adhesions remains elusive in many systems. We developed a genetically encoded fluorescent indicator for intercellular contacts with optimized intercellular GFP reconstitution using glycosylphosphatidylinositol (GPI) anchor, GRAPHIC (GPI anchored reconstitution-activated proteins highlight intercellular connections), which can be used for an expanded number of cell types. We observed a robust GFP signal specifically at the interface between cultured cells, without disrupting natural cell contact. Application of GRAPHIC to the fish retina specifically delineated cone-bipolar connection sites. Moreover, we showed that GRAPHIC can be used in the mouse central nervous system to delineate synaptic sites in the thalamocortical circuit. Finally, we generated GRAPHIC color variants, enabling detection of multiple convergent contacts simultaneously in cell culture system. We demonstrated that GRAPHIC has high sensitivity and versatility, which will facilitate the analysis of the complex multicellular connections without previous limitations.
... [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] Bimolecular fluorescence complementation (BiFC) (Figure 3) uses a split fluorescent protein that upon reconstitution regains its fluorescence. [44][45][46][47] The Split TEV assay (Figure 3) uses the split NIa protease from Tobacco Etch Virus (TEV). 48 Reconstitution of this protein restores its proteolytic activity for previously silent fluorescent or luminescent reporter proteins, and compatible proteolytically cleavable inactive transcription factors also exist that can drive the expression of reporter proteins in a comparable way as in the original Y2H. ...
... BiFC is for example a PCA that is notorious for quasi-irreversible protein reconstitution. 46,50 The membrane-based yeast two-hybrid (MYTH) ( Figure 3) is a typical example of a Split ubiquitin assay, although also others exist. [51][52][53] The MYTH-acronym already indicates that this technology is performed in yeast and is focused on the detection of membrane interactions, while membrane interactions are notoriously difficult to detect with most PPI technologies. ...
The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption often leads to diseases. Hence, many technologies have been developed to study protein‐protein interactions (PPIs) in a cellular context. The expansion of the PPI technology toolbox however complicates the selection of optimal approaches for diverse biological questions. This review gives an overview of the binary and co‐complex technologies, with the former evaluating the interaction of two co‐expressed genetically tagged proteins, and the latter only needing the expression of a single tagged protein or no tagged proteins at all. Mass spectrometry is crucial for some binary and all co‐complex technologies. After the detailed description of the different technologies, the review compares their unique specifications, advantages, disadvantages, and applicability, while highlighting opportunities for further advancements.
... As predicted, impaired lysosomal acidification was observed (using LysoTracker) after HBSS (Fig. 7a) or rapamycin treatment (Supplement 7a) of VRK2 shRNA-transduced cells; this impairment coincided with inhibition of conversion of LC3I to LC3II (Fig. 6a). 3 Kinase activities of both Akt and VRK2 are important for lysosomal accumulation of VRK2-Akt complexes. a By BiFC assays [36,101], intracellular spatial localization of Akt and VRK2A with lysosomes was examined. Schematic view of BiFC is shown. ...
... BiFC analysis was essentially described previously [36,101] using pCMV-VN-Empty, pCMV-VC-Empty, pCMV-VN-VRK2A, pCMV-VC-Akt2, pCMV-VN-VRK2A-Kinase Dead, or pCMV-VC-Akt2-T308A/S473A. Transfected cells were immunostained with the indicated antibodies combined with DAPI staining, mounted on Fluoromount/Plus (VWR, 95041-480), or stained with Lysotracker Red DND-99 (Thermo Fisher) and analyzed using confocal laser microscope [98]. ...
Serine–threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt–Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight–mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine–threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2–Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2–Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2–Akt complexes in the lysosomes via modulation of autophagy.
... This study introduces a high-throughput screening breeding system incorporating a bimolecular fluorescent protein that is tailored to detect pullulan. Bimolecular fluorescence complementation (BIFC) refers to using two proteins with interaction affinity to bring the fluorescent protein fragments connected to them closer and assemble them into a complete fluorescent protein, thereby characterizing the occurrence and spatial location of protein interactions [11]. When combined with high-throughput screening, this system enabled the selection of high-yielding strains following UV mutagenesis ultimately enhancing the ability of A. pullulans to produce pullulan. ...
This study addresses the production enhancement of pullulan, an extracellular polysaccharide with various applications. Pullulan is primarily produced by Aureobasidium pullulans (A. pullulans), and genetic modification is commonly used to increase its yield. However, there is a need for a more efficient and safer method. To achieve this, we designed a high-throughput screening system utilizing a unique fluorescent protein specific to pullulan. Ultraviolet (UV) mutagenesis was applied to create a pool of mutant strains, and flow cytometry allowed for single-cell screening. Our approach yielded strain M1-B3, which exhibited a substantial increase in pullulan production from 26.5 g/L to 76.88 g/L. Additionally, the molecular weight of the produced pullulan significantly increased, expanding its potential commercial application. This study demonstrates an efficient and safe method to enhance pullulan production in A. pullulans. The UV mutagenesis and flow cytometry based on screening not only increased yield but also improved pullulan’s molecular weight. The adaptability of this method to other polysaccharides and its potential for genomic analysis and broader applications make it a valuable tool in bioproduction.
... Entdeckungen von Muniz und seinen Kollegen (2006Kollegen ( , 2010, welche eine spezifische Expression der Response Regulatoren ZmTCRR-1 und ZmTCRR-2 in basalen endospermalen (Fashena et al., 2000;Uetz et al., 2000;Ito et al., 2001) und die Fluoreszence resonance energy transfer (FRET)-Methode (Gordon et al., 1998;Immink et al., 2002;Kerppola, 2006b ist ausreichend, wenn ein kleiner Teil der Fluorophorfragmente zusammengeführt wird, (Kerppola, 2006b(Kerppola, , 2013, so dass sich BiFC durch eine hohe Sensitivität auszeichnet. Auch können vielfarbige BiFC mit mehreren Farbstoffen und verschiedenen Proteinen durchgeführt werden (Kerppola, 2006a(Kerppola, , 2008Gehl et al., 2009). ...
In the last two decades it was shown that two component systems (TCS), which originally were discovered as an essential regulatory mechanism in bacteria, also play an integral role in differentiation processes of fungi and plants. TCS were identified as an essential signal transduction pathway in endospermal transfer cells (ETCs) of barley by a tissue-specific transcriptome sequencing analysis. Tissue specific expression studies point out a coordinated function of these signaling components in defined phosphorelays which possibly regulate cellularisation, cell identity and other differentiation processes of ETC. In this thesis protein- protein interactions were investigated using Bimolecular fluorescence complementation (BiFC). For the first time the studies show interactions of barley TCS-elements in possible phosphorelays. Two detected interactions of a putative ethylen receptor protein (HvETR2) with a phosphor transfer protein (HvHP1) as soon as the formed complex of HvHP1 and the type-C Response Regulator 15 (HvRR15) together show a multi-step phosphorelay which might be linked to the signaling transduction of the phytohormone ethylene. This correlates with the co-expression of the transcripts of these elements in ETC after cellularisation. Two other TCS elements (the transfer protein HvHP2 and the type-A response regulator HvRR1), which were co-expressed before cellularisation starts, also show a positive reaction in BiFC assay and refer to a coordinated function in another phosphorelay. Interestingly a not supposed interaction between HvHP2 and HvRR15 could be detected which suggest possible links between different phosphorelays.
... Transformation cassette of HK1::GFP construct for localization of promoter activity of HvHK1 Fig. S7: Scheme of BiFC principle, modified fromKerppola, 2006 Pre-experiment for optimization of protein expression from TCS elements For getting maximum yield of expressed protein in transient transformed tobacco leaves the direction of expression cassettes in binary vector pCB301 and the optimal incubation time of transient transformed tobacco leaves were analyzed; (A) Schematic illustration of expression cassette directions in pCB301; (B) Results of western blots with detection of His-tags linked to transfer proteins HP1 and HP2; (C) Results of western blots with detection of cmyc-tags linked to response regulators RR1 and RR15 and histidine kinase receiver domain, marker: PageRuler TM Prestained Protein Ladder 10-180kDa, NK-negative control (tobacco raw extract), PK-positive controls with His-or cmyc-tagged proteins, fwd/ revconforward/reverse direction of expression construct; (D+E) Schematic models of expression cassette from different TCS element expression constructs, p35S/ t35S -35S-promoter/ terminator of cauliflower mosaic virus, LeB4SPsignal peptide for ER localization, KDEL -ERtarbget amino-acid-sequence, His/cmyctags for specific antibody detection Coomassie staining and Western blots of purified proteins left: Coomassie stainings of different fractions during purification procedure; right: Western blot of different fractions from purification procedure with anti-His antibody; RE-raw extract, M-marker, F-flow through, W1-W4-washing fractions with increasing imidazole content (5, 10, 20, 50mM), E1-E5-eluate fractions, where protein-concentration could be detected, nc-negative control tobacco leaf, pc-positive control: His-tagged protein; marker: PageRuler TM Prestained Protein Ladder 10-180kDaFig. S10: Quantification of HP2-amount in eluatesWestern blot with HP2 eluates and protein-standard containing His-tag for estimation of protein amount; E1-2: eluate fractions; marker: ...
Two-component signaling (TCS) systems are important regulatory mechanisms of signal transduction. First, they were described in bacteria where they are involved in stress regulation and response to environmental stimuli. Since the last two decades further research uncovered the importance of these signaling systems in fungi and plants. In barley, several TCS elements were found which are transcriptionally activated in endosperm transfer cells (ETCs). These cells are localized at the maternal-filial boundary of barley grains and have an outstanding importance for assimilate supply to the endosperm. Co-expression studies pointed to a coordinated function of TCS elements in defined multistep phosphorelays, which possibly regulate cellularization and differentiation of ETCs. The histidine kinase HvHK1 showed a very specific expression during early cellularization of ETCs. Grains of transgenic lines, where HvHK1-expression was reduced by RNAi, were analyzed by a histological approach. The reduction of HvHK1-expression revealed distinct alterations in grain development especially in the ETC layer, the endosperm tissue and in differentiation of the aleurone layer.
Combinations of TCS elements, which are co-expressed in ETCs, were investigated by bimolecular fluorescence complementation (BiFC) and enzyme-linked immunosorbent assay (ELISA). One phosphorelay shows complex formation between the receiver domain of histidine kinase HvHK1 and phosphotransfer protein HvHP2 which itself forms a complex with type-A response regulator 1 (HvRR1). This phosphorelay might be associated with abscisic acid (ABA) signaling because of known cis-binding sites in the promoter regions of these elements and a detected transcriptional response to external ABA. In a second phosphorelay, the putative ethylene receptor HvETR2 forms a complex with co-expressed phosphotransfer protein HvHP1 which itself interacts with the type-C response regulator HvRR15. These elements are expressed in the differentiation phase of ETCs. This second phosphorelay might be involved in ethylene signaling. One additional interaction between HvHP2 and HvRR15 gives further possibilities of signal pathways in ETC differentiation.
Expression studies revealed further candidates with importance for TCS in other tissues. Co-expressed ethylene receptors, transfer protein HvHP3 and type-A response regulator HvRR3 were detected in vascular tissue, spike meristems and root tips, where they might act in multistep phosphorelays. Additionally, the putative cytokinin receptor HvHK4 is co-expressed with HvHP3 and HvRR3 in the spike meristem, suggesting a linkage between cytokinin and ethylene signaling.
... To further determine the interactions in live cells and the subcellular localisation of these complexes, we performed bimolecular fluorescence complementation assays with selected GPCRs and MRAPs, as described previously. 32 Basically, the C-terminus of these GPCRs were fused to a fragment of YFP which called F1, while the C-end of MRAP1 or MRAP2 were fused to the complementary fragment of YFP (F2). With this approach, YFP fluorescence can only be detected if the fused GPCR and MRAP1 or MRAP2 are co-expressed and come close to allow the YFP to complement (Figures 5 and S6). ...
Background:
The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown.
Methods:
Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs.
Results:
36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner.
Conclusions:
Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.
... In recent years, important subunits of these translocators have been identified and used to clarify the exact location of proteins (Gould et al., 2015;Lau et al., 2016;. To test protein-protein interactions, the split GFP assay, or bimolecular fluorescence complementation (BiFC) assay are often applied, which allows detection of protein-protein interactions in vivo as the two GFP fragments can only re-assemble if they are in close proximity to each other (Kerppola, 2006) and not if they are located in the same compartment only without being fused to interacting proteins (Hempel et al., 2009). ...
Diatoms succeed to survive the low iron conditions of the contemporary ocean. Despite their relatively large cell sizes, diatoms tolerate iron limitation and frequently dominate iron-stimulated phytoplankton blooms, both natural and artificial. However, our understanding of diatom iron physiology remains limited and is the subject of ongoing research. The objective of the present work was to investigate iron metabolism in diatoms, first, by reviewing the main iron use strategies of diatoms, relying on published literature and complementing published data with a search of 82 diatom transcriptomes. Then, through transgenic fluorescent lines, the subcellular localizations of ferritin (FTN) and iron starvation induced protein 3 (ISIP3) were visualized for the first time. It was found that FTN targete to the chloroplast, whereas ISIP3 localizes to the vicinity of the chloroplast and aggregates close by exhibiting similar localization to the iron starvation induced proteins ISIP1 and ISIP3. Furthermore, phenotypes of FTN and ISIP3 knockout (KO) mutants grown in different conditions of iron availability and intracellular iron status were compared. The FTN-KO mutants showed decreased growth in all conditions, indicating a potential function in iron storage. The ISIP3-KO mutants showed decreased growth when iron was available and no difference when grown in iron-free medium compared with control cells, suggesting that ISIP3 was required for iron acquisition. When copper was removed from the medium, these phenotypes disappeared, confirming the copper-dependency of iron uptake. Decreased growth of iron-repleted ISIP3-KO mutants supplied with siderophore (FOB) further suggested an involvement in siderophore uptake. Overall, the results presented in this thesis have added novel insights to the study of iron metabolism in diatoms.
... It also underlines the relevance of adenosine, dopamine and cannabinoid signalling and their pivotal contribution to various signalling mechanisms. The A 2A R-CB 1 R-DRD 2 heterooligomer was identified for the first time in 2008 [857] using a method combining BiFC and BRET techniques [858][859][860][861][862][863][864]. In 2009, the A 2A R-DRD 2 -mGlu 5 R was discovered in HEK293 cells, using BiFC and BRET approaches [865]. ...
Neurodegenerative diseases affect over 30 million people worldwide with an ascending trend. Most individuals suffering from these irreversible brain damages belong to the elderly population, with onset between 50 and 60 years. Although the pathophysiology of such diseases is partially known, it remains unclear upon which point a disease turns degenerative. Moreover, current therapeutics can treat some of the symptoms but often have severe side effects and become less effective in long-term treatment. For many neurodegenerative diseases the involvement of G protein-coupled receptors (GPCRs), which are key players of neuronal transmission and plasticity, has become clearer and holds great promise in elucidating their biological mechanism. With this review, we introduce and summarize class A and class C GPCRs, known to form heterodimers or oligomers to increase their signalling repertoire. Additionally, the examples discussed here were shown to display relevant alterations in brain signalling and have already been associated with the pathophysiology of certain neurodegenerative diseases. Lastly, we classified the heterodimers into two categories of crosstalk, positive or negative, for which there is known evidence.
... Like BiFC, Förster resonance energy transfer (FRET) is one of the most commonly employed PPI detection methods to visualize PPIs in a variety of model organisms (Hu and Kerppola, 2003;Kerppola, 2006a). It allows real-time quantitative analysis in living cells with the best spatial and temporal resolutions to study PPIs (Pollok and Heim, 1999). ...
Proteins are crucial for most cellular functions and typically participate in biological processes in concert with other proteins. Hence, identification of key protein players and characterization of protein-protein interactions (PPIs) are highly important. Owing to substantial advances in current biotechnology, a wide range of methods has been developed to dissect the PPI landscape. Given their popularity and power, the bimolecular fluorescence complementation (BiFC) assay, based on the reconstitution of a fluorescent protein in vivo, has emerged as the most popular protein-fragment complementation method in cellular biology over the past years. My team has previously established BiFC for probing different binary protein interactions in live Drosophila. During my PhD work, I expanded the utility of the BiFC in mammalian live cells. In particular, I standardized the protocol of the BiFC analysis to investigate protein binding affinities, in an applicable and simple manner. This quantitative BiFC approach was used in a systematic analysis of HOX/PBX/MEIS interaction properties in live cells and revealed novel interaction interfaces in several human HOX proteins. Furthermore, I applied the BiFC from low to high throughput PPI detection. Pairing sequence-verified human ORF collections with next generation sequencing, I participated in the conception of a powerful tool for performing a large-scale BiFC interaction screen in live cells. Benefited chiefly from this approach, a synoptic view of comprehensive HOX interactomes was substantially contributed to the current limited knowledge on human HOX protein partners and provided a novel tool in the cell biology arsenal. Along with the contemporary development of proximity labeling methods, in my side project, I depicted and tested a new cell-based PPI detection approach, which combines BiFC and BioID (proximity-dependent biotinylation identification) technologies, and allows deciphering the endogenous interactome of a protein complex. In summary, my PhD work demonstrates that the BiFC is a versatile and powerful approach to study PPIs in the live cellular context, on either small or large scale. In addition, my work further enlarged the potential of BiFC applications by combining it with other tools.
... Indeed, co-expression of H4R-V1 and V2-TSPAN4 in HEK293T resulted in BiFC ( Figure 4C), which matches the localization of mVenus-TSPAN4 ( Figure 4D). BiFC detection of protein-protein interactions by mVenus reconstitution is known to be virtually irreversible and as anticipated no decrease in fluorescence was observed upon histamine stimulation ( Figure 4E,F) [45,46]. In addition, histamine seemed not to affect the localization of the H4R-TSPAN4 complexes as compared to vehicle-stimulated cells. ...
The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits -arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.
... Like BiFC, Förster resonance energy transfer (FRET) is one of the most commonly employed PPI detection methods to visualize PPIs in a variety of model organisms (Hu and Kerppola, 2003;Kerppola, 2006a). It allows real-time quantitative analysis in living cells with the best spatial and temporal resolutions to study PPIs (Pollok and Heim, 1999). ...
Proteins are crucial for most cellular functions and typically participate in biological processes in concert with other proteins. Hence, identification of key protein players and characterization of protein-protein interactions (PPIs) are highly important. Owing to substantial advances in current biotechnology, a wide range of methods has been developed to dissect the PPI landscape. Given their popularity and power, the bimolecular fluorescence complementation (BiFC) assay, based on the reconstitution of a fluorescent protein in vivo, has emerged as the most popular protein-fragment complementation method in cellular biology over the past years.
My team has previously established BiFC for probing different binary protein interactions in live Drosophila. During my PhD work, I expanded the utility of the BiFC in mammalian live cells. In particular, I standardized the protocol of the BiFC analysis to investigate protein binding affinities, in an applicable and simple manner. This quantitative BiFC approach was used in a systematic analysis of HOX/PBX/MEIS interaction properties in live cells and revealed novel interaction interfaces in several human HOX proteins.
Furthermore, I applied the BiFC from low to high throughput PPI detection. Pairing sequence-verified human ORF collections with next generation sequencing, I participated in the conception of a powerful tool for performing a large-scale BiFC interaction screen in live cells. Benefited chiefly from this approach, a synoptic view of comprehensive HOX interactomes was substantially contributed to the current limited knowledge on human HOX protein partners and provided a novel tool in the cell biology arsenal.
Along with the contemporary development of proximity labeling methods, in my side project, I depicted and tested a new cell-based PPI detection approach, which combines BiFC and BioID (proximity-dependent biotinylation identification) technologies, and allows deciphering the endogenous interactome of a protein complex.
In summary, my PhD work demonstrates that the BiFC is a versatile and powerful approach to study PPIs in the live cellular context, on either small or large scale. In addition, my work further enlarged the potential of BiFC applications by combining it with other tools.
... BiFC is used to investigate interactions between proteins in a cellular milieu as well as subcellular localization. 16,25 We used BiFC to verify interactions between CPPED1 and PAK4 and PIK3R2 and to determine the subcellular localization of the pro- When single plasmids, either VN or VC variants, were transfected into HEK293T cells, we did not observe a fluorescence signal (Fig. S6A,B). After cotransfection with JUN-VN and FOS-VC, there was a strong fluorescence signal in the nucleus, which confirmed the validity of the method for the identification of protein interactions and subcellular localization of protein complexes (Fig. S6C). ...
Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K-AKT signalling pathway. We previously showed that CPPED1 levels are down-regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K-AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K-AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K-AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.
... The ORF sequence of TaAP2-15 was cloned into a pUC-PSYNE vector and fused with the N-terminal fragment of the yellow fluorescent protein (YFP) to generate TaAP2-15-nYFP construct. The full-length coding sequence of TaRZFP34 was subcloned into a pUC-pSPYCE vector as a fusion with the C-terminal fragment of YFP to generate TaRZFP34-cYFP [73]. To verify if there is change in expression with exchange of the constructs in the vectors, we cloned TaAP2-15 into pUC-PSYCE to produce TaAP2-15-cYFP and TaRZFP34 into pUC-PSYNE to form TaRZFP34-nYFP. All the generated constructs TaAP2-15-nYFP, TaRZFP34-cYFP, TaAP2-15-cYFP, TaRZFP34-nYFP; TaAP2-15-nYFP+cYFP and TaRZFP34-cYFP+nYFP were then introduced into A. tumefaciens strain GV3101. ...
AP2 transcription factors play a crucial role in plant development and reproductive growth, as well as response to biotic and abiotic stress. However, the role of TaAP2-15, in the interaction between wheat and the stripe fungus, Puccinia striiformis f. sp. tritici (Pst), remains elusive. In this study, we isolated TaAP2-15 and characterized its function during the interaction. TaAP2-15 was localized in the nucleus of wheat and N. benthamiana. Silencing of TaAP2-15 by barley stripe mosaic virus (BSMV)-mediated VIGS (virus-induced gene silencing) increased the susceptibility of wheat to Pst accompanied by enhanced growth of the pathogen (number of haustoria, haustorial mother cells and hyphal length). We confirmed by quantitative real-time PCR that the transcript levels of pathogenesis-related genes (TaPR1 and TaPR2) were down-regulated, while reactive oxygen species (ROS)-scavenging genes (TaCAT3 andTaFSOD3D) were induced accompanied by reduced accumulation of H2O2. Furthermore, we found that TaAP2-15 interacted with a zinc finger protein (TaRZFP34) that is a homolog of OsRZFP34 in rice. Together our findings demonstrate that TaAP2-15 is positively involved in resistance of wheat to the stripe rust fungus and provides new insights into the roles of AP2 in the host-pathogen interaction.
... Size exclusion-fast protein liquid chromatography fractionation of a mouse testis extract in physiological (nondenaturing) conditions revealed that endogenous Liat1 partitioned into two distinct fractions: one <60 kDa and another between 60 and 440 kDa (Fig. 3A). A common signature of proteins that participate in nuclear bodies is their ability to selfassociate (32)(33)(34)(35)(36). Therefore, to determine if Liat1 self-associates, we used BiFC, which is based on the reconstitution of fluorescence when two complementary, nonfluorescent fragments of the yellow fluorescent protein (YFP) are joined through a pair of interacting proteins (37,38). For this, we generated four fusion constructs: 1) YN-Liat1, YFP amino acids 1 to 154 fused to the N terminus of Liat1; 2) YC-Liat1, YFP amino acids 154 to 239 fused to the N terminus of Liat1; 3) Liat1-YN, YFP amino acids 1 to 154 fused to the C terminus of Liat1; and 4) Liat1-YC, YFP amino acids 154 to 239 fused to the C terminus of Liat1. ...
Significance
Due to the difficulty in their biochemical characterization, until recently intrinsically disordered proteins have gone largely unannotated. However, an appreciation for their biological significance is emerging, as they are now known to facilitate liquid–liquid phase separation (LLPS), which can produce membrane-lacking organelles that enable the spatial regulation of important cellular processes. This report characterizes the Ligand of Ate1 (Liat1) as an intrinsically disordered protein that participates in LLPS in the nucleolus, a prominent membrane-lacking organelle specialized in ribosome biogenesis. Furthermore, it shows that the lysyl-hydroxylase activity of Jumonji Domain Containing 6 modifies Liat1 and inhibits its nucleolar targeting and potential functions.
... FRET is an assay that detects the distance of two molecules within 10 nm (Algar et al., 2019); the fluorescence signals captured by a confocal microscope measure FRET efficiency and indicate the strength of protein-protein interactions. The combination of FRET and BiFC is widely used for visualization and identification of protein interactions in living cells (Kerppola, 2008;Shyu et al., 2008). We employed BiFC to identify host proteins that bind to EspF, and then applied FRET and CO-IP to validate the interactions, thus proofing the interaction between EspF and ANXA6. ...
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in humans. EspF is one of the best-characterized effector proteins secreted from the type three secretion system to hijack host cell functions. However, the crucial pathogen-host interactions and the basis for the intestinal barrier disruption during infections remain elusive. Our previous study screened and verified the interaction between host protein ANXA6 and EspF protein. Here, by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (CO-IP), we verified that EspF interacts with ANXA6 through its C-terminal domain. Furthermore, we found that both the constitutive expression of EspF or ANXA6 and the co-expression of EspF-ANXA6 could decrease the levels of tight junction (TJ) proteins ZO-1 and occludin, and disrupt the distribution of ZO-1. Moreover, we showed that EspF-ANXA6 activated myosin light chain kinase (MLCK), induced the phosphorylation of myosin light chain (MLC) and PKCα, and down-regulated the expression level of Calmodulin protein. Collectively, this study revealed a novel interaction between the host protein (ANXA6) and EspF. The binding of EspF to ANXA6 may perturb TJs in an MLCK-MLC-dependent manner, and thus may be involved in EHEC pathogenic function.
... In the log-log plot, a linear fit to this relationship gave a slope of almost exactly 1 (0.99) (Fig 3A), validating the use of P2A site to produce proportional concentrations of mNG2 and TagBFP across the range of expression levels resulted from transient transfection. For the split FP 1-10/11 systems, on the other hand, it has been previously shown that the complementation process begins with a dynamic binding equilibrium between the two fragments [4,14]. When the effective K D is much higher than the concentrations of cotransfected FP 1-10 and FP 11 fragments, this equilibrium cause the complemented FP signal to depend linearly on the expression reference with a slope of 2 on a log-log plot, while lowering the K D reduces this slope to 1 as the complementation becomes more efficient and approaches saturation [4]. ...
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP 1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP 11 tags. To improve the FP 1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A 1-10/11 and mNG3K 1-10/11 ) and one mClover-based split FP (CloGFP 1-10/11 ). mNG3A 1-10/11 and mNG3K 1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG2 11 fragments in mammalian cells.
... In the log-log plot, a linear fit to this relationship gave a slope of almost exactly 1 (0.99) (Fig 3A), validating the use of P2A site to produce proportional concentrations of mNG2 and TagBFP across the range of expression levels resulted from transient transfection. For the split FP 1-10/11 systems, on the other hand, it has been previously shown that the complementation process begins with a dynamic binding equilibrium between the two fragments (4,14). When the effective K D is much higher than the concentrations of co-transfected FP 1-10 and FP 11 fragments, this equilibrium cause the complemented FP signal to depend linearly on the expression reference with a slope of 2 on a log-log plot, while lowering the K D reduces this slope to 1 as the complementation becomes more efficient and approaches saturation (4). ...
The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP 1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP 11 tags. To improve the FP 1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A 1-10/11 and mNG3K 1-10/11 ) and one mClover-based split FP (CloGFP 1-10/11 ). mNG3A 1-10/11 and mNG3K 1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG2 11 fragments in mammalian cells.
... BiFC assays require the identification of disruptive mutations or deletions to corroborate protein-protein interaction specificity (32). We searched for "hot spots" in the TMD sequence that could form the packing surface. ...
Significance
Key members of the Bcl-2 family of proteins that control cell death possess a carboxy-terminal transmembrane segment that contributes to subcellular localization and modulates apoptotic functions. The role of Mcl-1 and specially Bok proteins remains elusive. We reveal that the coexpression of Mcl-1 and Bok TMDs produces an increase in the number of ER mitochondrial-associated membranes (MAMs). Furthermore, our findings provide mechanistic insight into the interaction between Mcl-1 and Bok transmembrane domains that can be envisioned as a target for therapeutic intervention.
... We performed bimolecular fluorescence complementation (BiFC) assay (Kerppola, 2006) to investigate the role of GSK-3b in FBXL21-TCAP and FBXL21-SCF complex formation ( Figures 6 and S5). Specifically, VenN-FBXL21 (Venus N terminus fused with Fbxl21) and VenC-TCAP (Venus C terminus fused with TCAP) interaction was measured at the Venus emission wavelength (528 nm). ...
FBXL21 is a clock-controlled E3 ligase modulating circadian periodicity via subcellular-specific CRYPTOCHROME degradation. How FBXL21 regulates tissue-specific circadian physiology and what mechanism operates upstream is poorly understood. Here we report the sarcomere component TCAP as a cytoplasmic substrate of FBXL21. FBXL21 interacts with TCAP in a circadian manner antiphasic to TCAP accumulation in skeletal muscle, and circadian TCAP oscillation is disrupted in Psttm mice with an Fbxl21 hypomorph mutation. GSK-3β phosphorylates FBXL21 and TCAP to activate FBXL21-mediated, phosphodegron-dependent TCAP degradation. GSK-3β inhibition or knockdown diminishes FBXL21-Cul1 complex formation and delays FBXL21-mediated TCAP degradation. Finally, Psttm mice show significant skeletal muscle defects, including impaired fiber size, exercise tolerance, grip strength, and response to glucocorticoid-induced atrophy, in conjunction with cardiac dysfunction. These data highlight a circadian regulatory pathway where a GSK-3β-FBXL21 functional axis controls TCAP degradation via SCF complex formation and regulates skeletal muscle function.
... OsNINJA1 has already been reported to form a complex with OsMYC2 through OsJAZ8 ). Thus, it is possible that the interaction between OsSRO1a and OsNINJA1 in plant cells is a false positive: fluorescence complementation could occur if OsNINJA1 is part of an OsSRO1a-OsMYC2 complex even if OsNINJA1 and OsSRO1a do not directly interact with one another (Kerppola 2006). Nevertheless, our results indicate that OsSRO1a is a component of the OsMYC2-regulating complex. ...
Key message
OsNINJA1-interacting protein, OsSRO1a, acts as a mediator that suppresses OsMYC2 activity in response to JA.
Abstract
Jasmonic acid (JA) is an important plant hormone for the stable growth and development of higher plants. The rice gene NOVEL INTERACTOR OF JAZ1 (OsNINJA1) interacts with Jasmonate ZIM-domain (JAZ) proteins and is a repressor of JA signaling. In this study, we identified several OsNINJA1-interacting proteins in rice from a yeast two-hybrid screen. Among the newly identified genes, we focused on SIMILAR TO RCD ONE1a (OsSRO1a) and investigated its role in JA signaling. Full-length OsSRO1a interacted with OsNINJA1 in plant cells but not in yeast cells. OsSRO1a also interacted with OsMYC2, a positive transcription factor in JA signaling, in both plant and yeast cells. The expression of OsSRO1a was upregulated at a late phase after JA treatment. Transgenic rice plants overexpressing OsSRO1a exhibited JA-insensitive phenotypes. In wild-type plants, JA induces resistance against rice bacterial blight, but this phenotype was suppressed in the OsSRO1a-overexpressing plants. Furthermore, the degradation of chlorophyll under dark-induced senescence conditions and the JA-induced upregulation of OsMYC2-responsive genes were suppressed in the OsSRO1a-overexpressing plants. These results suggest that OsSRO1a is a negative regulator of the OsMYC2-mediated JA signaling pathway in rice.
... In our study, we adapted an in vitro BiFC assay system to detect the direct interactions between P1 and JEV E proteins in living cells that ultimately block JEV infections (Fig. 4). The BiFC method is based on the reassembly of a fluorescent protein from its two complementary non-fluorescent fragments, whose association is facilitated by interaction between the two proteins fused to each fragment (Hu et al., 2002;Kerppola, 2006). To investigate the functional roles and the regulatory mechanisms of protein-protein interaction, several other methods also have been developed for visualizing protein interactions in living cells, including fluorescence resonance energy transfer (FRET) (Miyawaki, 2003(Miyawaki, , 2011 and fluorescence cross-correlation spectroscopy (FCCS) (Bacia et al., 2006). ...
Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that is the most significant etiological agent of childhood viral neurological infections. However, no specific antiviral drug is currently available to treat JEV infections. The JEV envelope (E) protein is a class II viral fusion protein that mediates host cell entry, making interference with the interaction between the E protein of JEV and its cognate receptors an attractive strategy for anti-JEV drug development. In this study, we identified a peptide derived from a phage display peptide library against the E protein of JEV, designated P1, that potentially inhibits in vitro and in vivo JEV infections. P1 inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 μM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1. We adapted an in vitro BiFC assay system and demonstrated that P1 interacts with JEV E proteins and blocks their entry into cells. We also evaluated the therapeutic efficacy of P1 in a lethal JEV mouse model exhibiting systemic and brain infections. Interestingly, P1 treatment protected C57BL/6 mice against mortality, markedly reduced the viral loads in blood and brain, and diminished the histopathological lesions in the brain cells. In addition to controlling systemic infection, P1 has a very low level of cytotoxicity and acts in a sequence-specific manner, as scrambled peptide sP1 does not show any antiviral activity. In conclusion, our in vitro and in vivo experimental findings show that P1 possesses antiviral activity against JEV infections, is safe to use, and has potential for further development as an antiviral treatment against JEV infections.
... For these reasons, fluorescence methods are also the most extensively used for dynamics and interaction studies of GPCRs and other membrane proteins. Molecular co-localizations in membranes can be determined, using statistical analyses of dual color fluorescence microscopy images 11 , Förster resonance energy transfer (FRET) 12,13 , and bimolecular fluorescence complementation (BiFC) assays 14 . Interactions can also be studied via altered diffusion behavior of the interacting molecules in the membranes using several different fluorescence techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single-particle tracking (SPT) (see refs. ...
Protein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable. We introduce transient state imaging (TRAST), combining sensitive fluorescence detection of fluorophore markers with monitoring of their dark triplet state transitions, allowing imaging of such protein-lipid interactions. We first determined the dark state kinetics of the biomembrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) in lipid vesicles, and how its triplet state is quenched by spin-labels in the same membranes. We then monitored collisional quenching of NBD-lipid derivatives by spin-labelled stearic acids in live cell plasma membranes, and of NBD-lipid derivatives by spin-labelled G-Protein Coupled Receptors (GPCRs). We could then resolve transient interactions between the GPCRs and different lipids, how these interactions changed upon GPCR activation, thereby demonstrating a widely applicable means to image and characterize transient molecular interactions in live cell membranes in general, not within reach via traditional fluorescence readouts.
... Human EWSR1 cDNA (1,971 bp) and MAZ cDNA (1,482 bp) were subcloned into BiFC vectors pBiFC-VN173 and pBiFC-VC155 (Addgene), and co-transfected into tumor cells for 24 h. The fluorescence emission was observed under a confocal microscope, with excitation and emission wavelengths of 488 and 500 nm, respectively (Kerppola, 2008). ...
Aerobic glycolysis is a hallmark of metabolic reprogramming in tumor progression. However, the mechanisms regulating glycolytic gene expression remain elusive in neuroblastoma (NB), the most common extracranial malignancy in childhood. Herein, we identify that CUT-like homeobox 1 (CUX1) and CUX1-generated circular RNA (circ-CUX1) contribute to aerobic glycolysis and NB progression. Mechanistically, p110 CUX1, a transcription factor generated by proteolytic processing of p200 CUX1, promotes the expression of enolase 1, glucose-6-phosphate isomerase, and phosphoglycerate kinase 1, while circ-CUX1 binds to EWS RNA-binding protein 1 (EWSR1) to facilitate its interaction with MYC-associated zinc finger protein (MAZ), resulting in transactivation of MAZ and transcriptional alteration of CUX1 and other genes associated with tumor progression. Administration of an inhibitory peptide blocking circ-CUX1-EWSR1 interaction or lentivirus mediating circ-CUX1 knockdown suppresses aerobic glycolysis, growth, and aggressiveness of NB cells. In clinical NB cases, CUX1 is an independent prognostic factor for unfavorable outcome, and patients with high circ-CUX1 expression have lower survival probability. These results indicate circ-CUX1/EWSR1/MAZ axis as a therapeutic target for aerobic glycolysis and NB progression.
... Of interest, we found COA3, a COX assembly factor, as a potential interacting partner of EGFL9 within the mitochondria. To functionally validate EGFL9 localization in the mitochondria, we examined EGFL9 and COA3 interaction with a Bimolecular Fluorescence Complementation (BiFC) assay 20,21 . The results showed that only cells co-transfected with split-Venus EGFL9 and COA3 showed specific reconstituted yellow fluorescence protein (YFP) signals (Fig. 7a, Supplementary Fig. 9, and Supplementary Movie 3). ...
The molecular mechanisms driving metastatic progression in triple-negative breast cancer (TNBC) patients are poorly understood. In this study, we demonstrate that epidermal growth factor-like 9 (EGFL9) is significantly upregulated in basal-like breast cancer cells and associated with metastatic progression in breast tumor samples. Functionally, EGFL9 is both necessary and sufficient to enhance cancer cell migration and invasion, as well as distant metastasis. Mechanistically, we demonstrate that EGFL9 binds cMET, activating cMET-mediated downstream signaling. EGFL9 and cMET co-localize at both the cell membrane and within the mitochondria. We further identify an interaction between EGFL9 and the cytochrome c oxidase (COX) assembly factor COA3. Consequently, EGFL9 regulates COX activity and modulates cell metabolism, promoting a Warburg-like metabolic phenotype. Finally, we show that combined pharmacological inhibition of cMET and glycolysis reverses EGFL9-driven stemness. Our results identify EGFL9 as a therapeutic target for combating metastatic progression in TNBC.
... Recently, a split FP approach, which was developed to study protein-protein interactions (Kerppola 2006), was adopted to eliminate the background signal in RNA imaging. In the bimolecular fluorescence complementation (BiFC) assay, nonfluorescent FP fragments reconstitute a complete fluorescent protein when they are brought into close proximity. ...
The MS2 system, with an MS2 binding site (MBS) and an MS2 coat protein fused to a fluorescent protein (MCP-FP), has been widely used to fluorescently label mRNA in live cells. However, one of its limitations is the constant background fluorescence signal generated from free MCP-FPs. To overcome this obstacle, we used a superfolder GFP (sfGFP) split into two or three nonfluorescent fragments that reassemble and emit fluorescence only when bound to the target mRNA. Using the high-affinity interactions of bacteriophage coat proteins with their corresponding RNA binding motifs, we showed that the nonfluorescent sfGFP fragments were successfully brought close to each other to reconstitute a complete sfGFP. Furthermore, real-time mRNA dynamics inside the nucleus as well as the cytoplasm were observed by using the split sfGFPs with the MS2-PP7 hybrid system. Our results demonstrate that the split sfGFP systems are useful tools for background-free imaging of mRNA with high spatiotemporal resolution.
... The proposed two-step model, consisting of a dynamic association/dissociation equilibrium followed by an irreversible folding/maturation process, can be generalized to other split fluorescent proteins, including the non-self-associating ones used to monitor protein-protein interaction in bimolecular fluorescence complementation (BiFC) assays 25,26 . In fact, there is no definitive boundary between the non-self-associating and selfassociating split FPs. ...
Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.
... Notably, both technologies face severe limitations when trying to examine the interaction profiles of membrane-bound proteins. In recent years, development of the protein fragment complementation assay (PCA), including the biomolecular fluorescence complementation (BiFC) approach, have reformed the study of protein-protein interactions in vivo (Hu et al. 2002;Remy and Michnick 2004;Kerppola 2008b). These assays are based on the observation that the two halves of a rationally dissected protein can reconstitute in vivo to create a functional protein. ...
A central focus of systems biology is the functional mapping of protein-protein interactions under physiological conditions. Here we describe MaGiCaL-BiFC, a lentivirus-based bimolecular fluorescence protein-fragment complementation approach for the high-throughput, genome-scale identification of protein-protein interactions in mammalian cells. After developing and validating this methodology using known protein-protein interaction pairs, we constructed genome-scale pooled BiFC libraries using the human ORFeome cDNA collection. These pooled libraries, containing ~ 12,000 unique human cDNAs, were used to screen for candidate interaction partners of the mitochondrial transmembrane protein TOMM22. Following infection of cells with the TOMM22 bait and the pooled cDNA libraries, cells harboring candidate TOMM22 interacting proteins were isolated from the cell pool via fluorescence activated cell sorting, and identified via microarray analysis. This approach identified several known interaction partners of TOMM22, as well as novel physical and functional partners that link the mitochondrial network to proteins involved in diverse cellular processes. Notably, protein kinase CK2 was identified as a novel physical interaction partner of human TOMM22. We found that this association occurs preferentially during mitosis and involves direct phosphorylation of TOMM22, an event that may lead to attenuation of mitochondrial protein import. Together, this data contributes to the growing body of evidence suggesting eloquent coordination between cell cycle progression and mitochondrial physiology. Importantly, through high-throughput screening and focused validation, our study demonstrates the power of the MaGiCaL-BiFC approach to uncover novel functional protein-protein interactions, including those involving proteins with membrane-spanning domains, or of a transient nature, all within their native cellular environment.
... for validation of interaction of two protein molecules (a and b) in living cells (Kerppola, 2008). BiFC ( Figure 3b) and colocalization ( Figure 3c) results illustrate that FadD13 can interact with human eEF1A1 in HEK293T cells. ...
Mycobacterium tuberculosis (Mtb) manipulates multiple host defense pathways to survive and persist in host cells. Understanding Mtb‐host cell interaction is crucial to develop an efficient means to control the disease. Here, we applied the Mtb proteome chip, through separately interacting with H37Ra and H37Rv stimulated macrophage lysates, screened 283 Mtb differential proteins. Through primary screening, we focused on fatty acylcoA synthetase FadD13. Mtb FadD13 is a potential drug target, but its role in infection remains unclear. Deletion of FadD13 in Mtb reduced the production of proinflammatory cytokines IL‐1β, IL‐18 and IL‐6. Bimolecular fluorescence complementation and colocalization showed that the binding partner of FadD13 in macrophage was eEF1A1 (a translation elongation factor). Knockdown eEF1A1 expression in macrophage abrogated the promotion of proinflammatory cytokines induced by FadD13. In addition, ΔfadD13 mutant decreased the expression of the NF‐κB signaling pathway related proteins p50 and p65, so did the eEF1A1 knockdown macrophage infected with H37Rv. Meanwhile, we found that deletion of FadD13 reduced Mtb survival in macrophages during Mtb infection, and purified FadD13 proteins induced broken of macrophage membrane. Taken together, FadD13 is crucial for Mtb proliferation in macrophages, and it plays a key role in the production of proinflammatory cytokines during Mtb infection.
... Methods 161 (2019) [16][17][18][19][20][21][22][23] (BiFC), a FP-based technique for the analysis of protein-protein interactions that relies on the reunion of two components each containing half of the fluorescent protein [65,66]. In AiFC, Broccoli and iSpinach, both are optimized variants of Spinach, were chosen for the construction of genetically encoded probes. ...
mRNAs play essential role in gene expression and regulation. Direct visualization of specific intracellular mRNAs can provide key information about mRNA distribution and dynamics that accelerating study of their functionality. This highlights the necessity of characterizing mRNA in mammalian cells with good spatial and temporal resolution. Live cell imaging and tracking of mRNA remains a challenge due to their relatively short half-life and lack of versatile imaging probes. This review provides an overview of current methods of live-cell mRNA imaging, including exogenous hybridization probes and genetically encoded strategies. It then focuses on the development and application of aptamer initiated fluorescence complementation (AiFC) method for real-time imaging of mammalian mRNAs and includes a detailed protocol for observing β-actin mRNA in HeLa cells.
... Here we studied whether VirD5 interacts not only with Spt4, but also with other proteins at the kinetochore. To this end, we performed BIFC experiments (Kerppola, 2008) with a set of candidate proteins (Table S4) from the inner and outer kinetochore of yeast, many of which are conserved in animals and plants. These were fused with the N-terminal part of the improved yellow fluorescent protein (YFP) called Venus (VN173), and introduced into yeast cells together with VirD5 fused with the C-terminal part of Venus (VC173). ...
Aided by translocated virulence proteins, Agrobacterium tumefaciens transforms plant cells with oncogenic T‐DNA. In the host cells the virulence protein VirD5 moves to the nucleus, where it becomes localized at the kinetochores, and disturbs faithful chromosome segregation, but the molecular mechanism underlying this remains unknown.
To gain more insight, we screened amongst the kinetochore proteins for VirD5 interactors using bimolecular fluorescence complementation assays, and tested chromosome segregation in yeast cells.
We found that VirD5 interacts with the conserved mitotic Aurora kinase Ipl1 in yeast and likewise with plant Aurora kinases. In vitro VirD5 was found to stimulate the activity of Ipl1. Phosphorylation of substrates by Ipl1 in vivo is known to result in the detachment between kinetochore and spindle microtubule. This is necessary for error correction, but increased Ipl1/Aurora kinase activity is known to cause spindle instability, explaining enhanced chromosome mis‐segregation seen in the presence of VirD5. That activation of the Ipl1/Aurora kinase at least partially underlies the toxicity of VirD5 became apparent by artificial boosting the activity of the specific counteracting phosphatase Glc7 in vivo, which relieved the toxicity.
These findings reveal a novel mechanism by which a pathogenic bacterium manipulates host cells.
... The proposed 2-step model, consisting of a dynamic association/dissociation equilibrium followed by an irreversible folding/maturation process, can be generalized to other split fluorescent proteins, including the non-self-associating ones used to monitor protein-protein interaction in bimolecular fluorescence complementation (BiFC) assays (24) (25). In fact, there is no definitive boundary between the non-self-associating and self-associating split FPs. ...
Self-associating split fluorescent proteins (FPs) have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Newly developed self-associating split FPs, however, have suffered from suboptimal fluorescence signal. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living animals, demonstrating its broad applications.
... This information is necessary in order to understand protein function at the cellular, tissue and organism levels. In recent years, the Bimolecular Fluorescent Complementation assay (Citovsky et al., 2006;Kerppola, 2008) has gained momentum in evaluating PPI in vivo. When combined with fluorescently labeled cellular marker proteins, BiFC offers the advantage of precisely determining the subcellular localization of PPI. ...
Plant pathogenic single strand positive-sense RNA viruses with the tripartite genome are classified into two families: Bromoviridae and Virgaviridae. Family Bromoviridae contains four genera Bromo, Cucumo, Alfamo, and Ilarviruses characterized by icosahedral particles. By contrast family Virgaviridae contains only one genus, Hordeivirus, with tripartite genome and characterized by helical particles. Unlike in monopartite plant viruses, packaging in tripartite RNA viruses requires a well-orchestrated process to ensure that viral progeny is selectively encapsidated and distributed optimally into three or four different viral capsids. Among the tripartite RNA viruses mentioned above, brome mosaic virus (BMV), the type member of the genus bromovirus, has been extensively used as a model system to unravel the mechanism of genome packaging. Using the available research data on BMV, this review is focused in updating the readers on how various macromolecular interactions (e.g. packaging signals) and biological factors (i.e. type of host plant) modulate genome packaging. The review also offers new directions of research to further our knowledge on the genome packaging in tripartite viruses.
... The Protein Analysis through Evolutionary Relationships (PANTHER) over-representation test and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to determine the biological function of the B. diazoef f icens and G. max proteins predicted in the PPI network. We analyzed the subnetworks of the protein interactome to identify the candidate proteins possibly related to the soybean−rhizobium symbiosis and used a luciferase complementation image (LCI) assay 29,30 to confirm a subnetwork with two 14-3-3 proteins. In addition, we discuss how these predicted PPIs can help us to better understand this process. ...
Although the legume-rhizobium symbiosis is a most important biological process, there is a limited knowledge about the protein interaction network between host and symbiont. Using interolog and domain-based approaches, we constructed an inter-species protein interactome containing 5,115 protein-protein interactions between 2,291 Glycine max and 290 Bradyrhizobium diazoefficiens USDA 110 proteins. The interactome was further validated by expression pattern analysis in nodules, GO term semantic similarity, co-expression analysis and luciferase complementation image assay. In G. max-B. diazoefficiens interactome, bacterial proteins are mainly ion channel and transporters of carbohydrates and cations, while G. max proteins are mainly involved in the processes of metabolism, signal transduction and transport. We also identified the top ten highly interacting proteins (hubs) for each species. KEGG pathway analysis for each hub showed that two 14-3-3 proteins (SGF14g and SGF14k) and five heat shock proteins in G. max are possibly involved in symbiosis, and ten hubs in B. diazoefficiens may be important symbiotic effectors. Subnetwork analysis showed that 18 symbiosis-related SNARE proteins may play roles in regulating bacterial ion channels, and SGF14g and SGF14k possibly regulate the rhizobium dicarboxylate transport protein DctA. The predicted interactome provide a valuable basis for understanding the molecular mechanism of nodulation in soybean.
... Bimolecular fluorescence complementation experiments (Bi-FC) were performed to verify direct interaction of SclB and RcoA in vivo. Strains were constructed for these experiments, which express fusion proteins, where one half of a split YFP (cYFP) was fused to SclB and the other half (nYFP) to RcoA [102]. Two additional strains, expressing either SclB-cYFP and free nYFP or RcoA-nYFP and free cYFP, served as controls (S9D Fig). ...
The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. Bfunction is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.
... The full-length coding sequences of TaCIPK5 and TaCIPK5Δ were cloned into a pUC-pSPYNE vector and fused with the N-terminal fragment of yellow fluorescent protein (YFP) to form YFP N -TaCIPK5 and YFP N -TaCIPK5Δ constructs. The full-length coding sequence of TaCBL4 was cloned into a pUC-pSPYCE vector as a fusion with the N-terminal fragment of YFP to form YFP C -TaCBL4 (Kerppola, 2008) (Supplementary Table S2). Plasmids of YFP N -TaCIPK5/YFP C -TaCBL4 or YFP N -TaCIPK5Δ/YFP C -TaCBL4 were then co-transformed into wheat protoplasts following the PEG-mediated transformation methods as previously described by Walter et al. (2004). ...
Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). Recent research has demonstrated that the CBL-CIPK complex is not only required for abiotic stress signaling, but also is likely involved in biotic stress perception. However, the role of this complex in immune signaling, including pathogen perception, is lacking. In this study, we isolated one signaling component of the TaCBL-TaCIPK complex (TaCBL4-TaCIPK5) and characterized the role in the interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). Among all TaCBLs in wheat, TaCBL4 mRNA accumulation markedly increased after infection by Pst. Silencing of TaCBL4 resulted in enhanced susceptibility to the avirulent Pst infection. In addition, TaCIPK5 was screened out to physically interact with TaCBL4 in planta and positively contributed to wheat resistance to Pst. Moreover, the disease resistance phenotype of TaCBL4 and TaCIPK5 co-silenced plants was consistent with that of single knockdown plants. The accumulation of reactive oxygen species (ROS) was significantly altered in all silencing plants during Pst infection. Together these findings demonstrate that the TaCBL4-TaCIPK5 complex positively modulates wheat resistance in a ROS-dependent manner. Our work provides new insights for understanding the roles of CBL-CIPK in wheat.
... The BiFC technique reduces the size of tagging by using a fluorescence protein split into 2 non-fluorescent fragments, subsequently tagged to the proteins of interest. Only when those proteins of interest are associated together is fluorescence measured (Outeiro et al. 2008, Kerppola 2006, Chen et al. 2006). Johnson's group applied a split green fluorescent protein (GFP) complementation technique to quantify tau aggregation by BiFC in situ (Chun, Waldo & Johnson 2011, Chun, Waldo & Johnson 2007; fulllength tau protein was directly fused to a smaller GFP fragment (GFP11), and co-expressed in cells with a larger GFP fragment (GFP1-10). ...
Abnormal folding and hyperphosporylation of tau protein leads to the generation of paired helical filaments (PHFs) and neurofibrillary tangles (NFTs), a key neuropathological hallmark of Alzheimer’s disease and other tauopathies. Cellular models able to recapitulate tau pathology are useful for understanding disease mechanisms and screening and profiling compounds that interfere with tau aggregation. We have established a HEK T-REx cell culture model where the inducible expression of mutant tau, accompanied by the introduction of aggregated mutant tau extracted from transgenic mouse brain, leads to endogenous tau aggregation and filament assembly, suggesting a seeding process as a likely mechanism underlying NFT formation. We found that substantial aggregation of soluble tau into Triton X-100-insoluble tau can be induced by spontaneous uptake of mutant tau aggregates, which are internalised through an endocytic mechanism that is temperature-, time- and ATP-dependent, can be potentiated by transfection reagents and impaired by pharmacological agents inhibiting macropinocytosis, suggesting a potential mechanism for the propagation of tau pathology in tauopathy brains. We also found that seed-competent tau species are sarkosly-insoluble, tagged by AT8 and MC1 antibodies, and are present in conditioned media from seeded cells. Finally, we established a more physiologically relevant model of seeded tau aggregation in rodent neurons. In summary, our study establishes cell-based tauopathy models that not only provide mechanistic insights into the pathogenesis of tau aggregation, but also offer a robust system for identifying therapeutic strategies to prevent propagation and spreading of tau pathology.
Nitric oxide (˙NO) is a free radical that induces nitrosative stress, which can jeopardize cell viability. Yeasts have evolved diverse detoxification mechanisms to effectively counteract ˙NO‐mediated cytotoxicity. One mechanism relies on the flavohemoglobin Yhb1, whereas a second one requires the S‐nitrosoglutathione reductase Fmd2. To investigate heme‐dependent activation of Yhb1 in response to ˙NO, we use hem1Δ‐derivative Schizosaccharomyces pombe strains lacking the initial enzyme in heme biosynthesis, forcing cells to assimilate heme from external sources. Under these conditions, yhb1⁺ mRNA levels are repressed in the presence of iron through a mechanism involving the GATA‐type transcriptional repressor Fep1. In contrast, when iron levels are low, the transcription of yhb1⁺ is derepressed and further induced in the presence of the ˙NO donor DETANONOate. Cells lacking Yhb1 or expressing inactive forms of Yhb1 fail to grow in a hemin‐dependent manner when exposed to DETANONOate. Similarly, the loss of function of the heme transporter Str3 phenocopies the effects of Yhb1 disruption by causing hypersensitivity to DETANONOate under hemin‐dependent culture conditions. Coimmunoprecipitation and bimolecular fluorescence complementation assays demonstrate the interaction between Yhb1 and the heme transporter Str3. Collectively, our findings unveil a novel pathway for activating Yhb1, fortifying yeast cells against nitrosative stress.
Ralstonia Solanacearum is a soilborn beta-proteobacterium responsible of bacterial wilt on Solanaceaous crops. This bacterium is considered as one of the most harmful plant disease worldwide. This bacterium possesses the ability to infect more than 250 different species, including crops with major economic importance (tomato, potato, tobacco, eucalyptus…). R. solanacearum is divided into four phylotypes originated from different areas: I (Asian), IIA and IIB (American), III (African), IV (Indonesian). Among these phylotype, phylotype I is currently in demographic expansion, is highly recombinogenic and has a wide hosts range. Thus, altogether, these characteristics demonstrated that this phylotype has a high evolutionary potential (sensu McDonald and Linde, 2002).
In order to control this bacterium, genetic plant resistance seems to be the most promising method. This method consists in using cultivars with different source of resistance such as resistance genes and/or resistant QTLs. The AG91-25 (E6), an eggplant cultivar possessing a major resistance gene (ERs1), is capable
to control some of phylotype I strains of R. solanacearum. However, in order to optimize the management of this resistance and to avoid its fast breakdown, we need to deeply investigate the durability of this resistant gene. Durability can be estimated by studying the evolutionary potential of our pathogen faced to E6 source of resistance and by understanding the molecular mechanisms underlying the interaction between the host (R gene) and its pathogene (Type III Effector – T3E).
In order to study R. solanacearum evolutionary dynamics under selective pressure from E6 resistant cultivar, we set up an experimental evolution trial in the field. This trial consisted of three couples of resistant (E6) and susceptible eggplants (E8) microplots, implanted twice a year during three years, hence consisting of 5 cycles. A Multi-Locus VNTR Analysis (MLVA) scheme, consisting of 8 minisatellite loci, was developed in order to characterize the strains extracted from these crop cycles. These VNTRs were specific to R. solanacearum phylotype I strains, they were highly polymorphic and discriminatory at different scale: globally, regionally and locally.
Our results showed no breakdown of E6 resistance by R. solanacearum populations, which confirms that this resistance is durable. It seemed that this cultivar reduced the soil bacterial population, preventing bacterial population to infest the resistant host. At the same time, 100% of the E8 plants have died, starting at cycle 2. Bacterial wilt seemed to spread with a “plant-to-plant” dynamics within each microplot. Genetic diversity reduction was also observed during the successive cycle of susceptible eggplant, associated with the increase of frequency of two main haplotypes. However, we failed to identify a clear genetic structuration, neither at the plot scale nor at the
microplot scale. Nevertheless, isolation-by-distance data seemed to show that a spatial structure is currently establishing. Altogether, our results suggested that our plot populations appeared to have a clonal epidemic structure.
We also looked into 10 T3Es’ involvement in the interaction between R. solanacearum and the resistant eggplant (E6). Their distribution was completely different within a collection of phylogenetically diverse strains (91 strains): ripAJ and ripE1 are the most shared T3Es whereas ripP1 and ripP2 were the less common T3E whithin our collection of strains. Some T3Es showed few (ripAJ) or no length polymorphism at all (ripE1 and ripP2) whereas some other (ripAU) are extremely polymorphic. Nevertheless, the T3E effector repertoire did not seemed to be correlated to a specific phenotype on E6 eggplant. Yet, we identified the effector gene ripAX2 as having avirulence function
on E6 resistant eggplant. Its recognition by E6 seemed to occur in the hypocotyle region rather than in the mesophyll, highlighting a possible organ-specificity of the interaction between ERs1 and ripAX2. However, this effector variability and its genomic environment did not seem to be associated with the R. solanacearum strains virulence on E6.
Combining elements of biology, chemistry, physics, and medicine, the science of human physiology is complex and multifaceted. In this report, we offer a broad and multiscale perspective on key developments and challenges in visualization for physiology. Our literature search process combined standard methods with a state‐of‐the‐art visual analysis search tool to identify surveys and representative individual approaches for physiology. Our resulting taxonomy sorts literature on two levels. The first level categorizes literature according to organizational complexity and ranges from molecule to organ. A second level identifies any of three high‐level visualization tasks within a given work: exploration, analysis, and communication. The findings of this report may be used by visualization researchers to understand the overarching trends, challenges, and opportunities in visualization for physiology and to provide a foundation for discussion and future research directions in this area.
Although the legume - rhizobium symbiosis is a most important biological process, there is a limited knowledge about the protein interaction network between host and symbiont. Using interolog and domain-based approaches, we constructed an inter-species protein interactome with 5115 protein-protein interactions between 2291 Glycine max and 290 Bradyrhizobium diazoefficiens USDA 110 proteins. The interactome was validated by expression pattern analysis in nodules, GO term semantic similarity, and co-expression analysis. One sub-network was further confirmed using luciferase complementation image assay. In the G. max-B. diazoefficiens interactome, bacterial proteins are mainly ion channel and transporters of carbohydrates and cations, while G. max proteins are mainly involved in the processes of metabolism, signal transduction, and transport. We also identified the top ten highly interacting proteins (hubs) for each of the two species. KEGG pathway analysis for each hub showed that two 14-3-3 proteins (SGF14g and SGF14k) and five heat shock proteins in G. max are possibly involved in symbiosis, and ten hubs in B. diazoefficiens may be important symbiotic effectors. Subnetwork analysis showed that 18 symbiosis-related SNARE proteins may play roles in regulating bacterial ion channels, and SGF14g and SGF14k possibly regulate the rhizobium dicarboxylate transport protein DctA. The predicted interactome and symbiosis proteins provide a valuable basis for understanding the molecular mechanism of root nodule symbiosis in soybean.
Bei der Zelltodinduktion spielt Wasserstoffperoxid sowohl in Pflanzen, als auch in Tieren eine wichtige Rolle. Das Signalmolekül Wasserstoffperoxid muss dabei über verschiedene Membranen an seinen Zielort transportiert werden. Dieser Transport geschieht in Pflanzen und Tieren durch Aquaporine. Diese integralen Membranproteine ermöglichen den Transport von Wasserstoffperoxid über die Membranen zwischen Zellkompartimenten. Die pflanzlichen Aquaporine werden durch die Proteine Aquaporin Interactor 1 und 2 (AQI1 und AQI2) reguliert. AQI2 ist dabei ein pflanzliches Homolog von AQI1. Beide Proteine sind in der Lage an das Aquaporin zu binden und den Kanal zu verschließen. Der Einstrom von Wasser und Wasserstoffperoxid kann dadurch verhindert werden. Aquaporin Interactor 1 bindet dabei bevorzugt an das im Tonoplasten lokalisierte Aquaporin TIP1.1, während Aquaporin Interactor 2 stärker an das in der Plasmamembran lokalisierte Aquaporin PIP2.2 bindet. Aquaporin Interactor 1 ist innerhalb der Vakuole oder an der Tonoplastenmembran lokalisiert. Im Gegensatz dazu wird für Aquaporin Interactor 2 eine Lokalisation im Apoplasten angenommen. So kann AQI1 die Aquaporine der Tonoplastenmembran und AQI2 die Aquaporine der Plasmamembran regulieren. Bekannt ist die humane Aminoacylase 1 für ihre Fähigkeit zur Hydrolyse von N-acetylierten Aminosäuren. Dabei hat das Zink-bindende Metalloenzym ein weites Substratspektrum. Das bevorzugte Substrat ist N-acetyl-Methionin. Auch das pflanzliche Homolog AQI1 ist zur Hydrolyse dieses Substrats fähig und für die Funktion ebenfalls auf Metallionen angewiesen. Aquaporin Interactor 2 besitzt keine solche Aminoacylase Aktivität. aqi1 knock-out Mutanten in Arabidopsis thaliana und Nicotiana tabacum zeigen eindeutig, dass die Hydrolyse von N-acetyl-Methionin ausschließlich von AQI1 bewerkstelligt werden kann. Diese Aminoacylase-Aktivität wird für die Bindung an die Aquaporine aber nicht benötigt. Die Aminoacylase-Aktivität und die Fähigkeit zur Bindung an die Aquaporine sind zwei unabhängige Funktionen von Aquaporin Interactor 1. Nach bisherigem Kenntnisstand muss davon ausgegangen werden, dass AQI2 ausschließlich als Aquaporin-regulierendes Protein fungiert. Nach Pathogenbefall ist im betroffenen Pflanzengewebe eine verstärkte Aminoacylase-Aktivität zu detektieren. Sowohl nach Agrobakterieninfiltration, als auch nach Infektion mit Tabakmosaikvirus, ist diese AQI1 Induktion zu beobachten. Dies demonstriert eine Rolle des Proteins bei der Pathogenabwehr. Ein weiteres Aquaporin-interagierendes Protein ist BHRF1, ein anti-apoptotisches Protein aus dem Epstein-Barr-Virus. Bisher konnte diese Interaktion nur für pflanzliche Aquaporine gezeigt werden. BHRF1-transgene N. tabacum Pflanzen zeigen spontan auftretende Zelltodereignisse. Diese werden vermutlich durch die Interaktion mit den Aquaporinen und mehreren Proteinen des G-Protein Signalwegs hervorgerufen. Durch die Bindung von BHRF1 an die Aquaporine kann es die endogenen Interaktionspartner AQI1 und AQI2 verdrängen. Eine korrekte Regulation der Aquaporine durch AQI1 und AQI2 ist somit nicht mehr gewährleistet. Zudem bindet BHRF1 an das pflanzliche AtRGS1 (regulator of G-protein signalling) Protein, einem Glukosesensor. AtRGS1 ist eine Kombination aus G-Protein gekoppeltem Rezeptor und RGS Protein. Die RGS Domäne bewirkt die Hydrolyse des an die Gα-Untereinheit gebunden GTPs. BHRF1 bindet auch an humane RGS Proteine. BHRF1 hat somit auch im Menschen einen möglichen Einfluss auf den G-Protein Signalweg. Eine genaue Regulation der Aquaporine für die Zelltodregulation ist von großer Wichtigkeit. Eine Fehlregulation, wie sie durch das virale BHRF1 zustande kommt, führt zu Zelltodereignissen. BHRF1 tritt dabei vermutlich in Konkurrenz mit den endogenen Interaktionspartnern der Aquaporine und den Proteinen des G-Protein Signalwegs und verursacht somit eine Fehlregulation verschiedener Signalwege.
In the last two decades, there have been enormous developments in the area of reporter gene imaging for various bioimaging applications, especially to track cellular events that are occurring in intact cells and cells within living animals. As part of this process, there has been a significant interest in identifying new reporters or developing new substrates that can allow us to image multiple cellular events simultaneously without any signal overlap between the targets. Even though chemical dyes are useful for some of these applications, reporter proteins which mimic biological properties of proteins when tagged directly with the target proteins are very useful. Although molecular imaging has significantly advanced through use of different imaging probes (radiolabeled ligands, MR contrast agents, CT contrast agents, fluorescent dyes, fluorescent and bioluminescent proteins) and techniques (PET, SPECT, MRI, CT, optical, ultrasound, and photoacoustic imaging), optical imaging, such as fluorescence and bioluminescence imaging, has shown promising applications in various preclinical settings, especially in imaging cellular pathways and studies involving drug development. This is mainly owing to its simple and easy nature in performing the assay and also its high-throughput and cost-effective applications. In this chapter, we review the evolution of optical imaging with specific emphasis on fluorescent proteins, as well as an introduction regarding the general approach of optical imaging in in vitro and in vivo applications. We explain this by briefly introducing different optical imaging methods and fluorescent assays developed based on fluorescent dyes and fluorescent proteins followed by a detailed review of different fluorescent proteins currently used for various assay developments and applications.
Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested "AND" gate design of SPARK2--in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest--yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.
Chemotaxis is based on ligand-receptor interactions that are transmitted via protein-protein interactions to the flagellar motors. Ligand-receptor interactions in chemotaxis can be deployed for the development of rapid biosensor assays, but there is no consensus as to what the best readout of such assays would have to be. Here we explore two potential fluorescent readouts of chemotactically active Escherichia coli cells. In the first, we probed interactions between the chemotaxis signaling proteins CheY and CheZ by fusing them individually with non-fluorescent parts of a ‘split’-Green Fluorescent Protein. Wild-type chemotactic cells but not mutants lacking the CheA kinase produced distinguishable fluorescence foci, two-thirds of which localize at the cell poles with the chemoreceptors and one-third at motor complexes. Cells expressing fusion proteins only were attracted to serine sources, demonstrating measurable functional interactions between CheY~P and CheZ. Fluorescent foci based on stable split-eGFP displayed small fluctuations in cells exposed to attractant or repellent, but those based on an unstable ASV-tagged eGFP showed a higher dynamic behaviour both in the foci intensity changes and the number of foci per cell. For the second readout, we expressed the pH-sensitive fluorophore pHluorin in the cyto- and periplasm of chemotactically active E. coli . Calibrations of pHluorin fluorescence as a function of pH demonstrated that cells accumulating near a chemo-attractant temporally increase cytoplasmic pH while decreasing periplasmic pH. Both readouts thus show promise as proxies for chemotaxis activity, but will have to be further optimized in order to deliver practical biosensor assays.
IMPORTANCE
Bacterial chemotaxis may be deployed for future biosensing purposes with the advantages of its chemoreceptor ligand-specificity and its minute-scale response time. On the downside, chemotaxis is ephemeral and more difficult to quantitatively read out than, e.g., reporter gene expression. It is thus important to investigate different alternative ways to interrogate chemotactic response of cells. Here we gauge the possibilities to measure dynamic response in the Escherichia coli chemotaxis pathway resulting from phosphorylated CheY-CheZ interactions by using (unstable) split-fluorescent proteins. We further test whether pH differences between cyto- and periplasm as a result of chemotactic activity can be measured with help of pH-sensitive fluorescent proteins. Our results show that both approaches conceptually function, but will need further improvement in terms of detection and assay types to be practical for biosensing.
The bimolecular fluorescent complementation (BiFC) is a fluorescent complementation method largely used to investigate protein–protein interaction in living cells. This method is based on the ability of two nonfluorescent fragments to assemble forming a native fluorescent reporter with the same spectral properties of the native reporter. Such fragments are fused to putative protein partners that in case of interaction will bring the two halves in close proximity, allowing for the reconstitution of an active fluorescent reporter. The BiFC has been used to investigate protein–protein interaction in a number of different organisms, including plants. In plant cells, many essential pathways of protein trafficking and subcellular localization necessitate the intervention of several protein units organized in multisubunit complexes. It is well known that vacuolar sorting of many secretory soluble proteins require the intervention of specific transmembrane cargo receptors able to interact forming dimers. In this chapter we describe a BiFC method for the efficient visualization of RMR (Receptor Membrane RING-H2) type 2 dimerization in agro-infiltrated Nicotiana benthamiana leaves. Furthermore, this relatively simple method represents an optimal strategy to test protein–protein interaction using any other putative protein partners of interest in plant cells.
ATF2 and c-Jun are key components of activating protein-1 and function as homodimers or heterodimers. c-Jun-ATF2 heterodimers activate the expression of many target genes, including c-jun, in response to a variety of cellular and environmental signals. Although it has been believed that c-Jun and ATF2 are constitutively localized in the nucleus, where they are phosphorylated and activated by mitogen-activated protein kinases, the molecular mechanisms underlying the regulation of their transcriptional activities remain to be defined. Here we show that ATF2 possesses a nuclear export signal in its leucine zipper region and two nuclear localization signals in its basic region, resulting in continuous shuttling between the cytoplasm and the nucleus. Dimerization with c-Jun in the nucleus prevents the export of ATF2 and is essential for the transcriptional activation of the c-jun promoter. Importantly, c-Jun-dependent nuclear localization of ATF2 occurs during retinoic acid-induced differentiation and UV-induced cell death in F9 cells. Together, these findings demonstrate that ATF2 and c-Jun mutually regulate each other by altering the dynamics of subcellular localization and by positively impacting transcriptional activity.
We present an approach for monitoring protein-protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the beta-gal activity resulting from the forced interaction of nonfunctional weakly complementing beta-gal peptides (Deltaalpha and Deltaomega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras. To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Deltaalpha and Deltaomega, respectively. Enzymatic beta-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.
Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.
We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over
time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent
expression. We used in vivo labeling with E5 to measure expression from the heat shock–dependent promoter in Caenorhabditis elegansand from the Otx-2 promoter in developing Xenopusembryos. Thus, E5 is a “fluorescent timer” that can be used to monitor both activation and down-regulation of target promoters
on the whole-organism scale.
To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.
Protein-protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the approximately 6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623-627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.
Networks of protein interactions coordinate cellular functions. We describe a bimolecular fluorescence complementation (BiFC) assay for determination of the locations of protein interactions in living cells. This approach is based on complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are brought together by interactions between proteins fused to each fragment. BiFC analysis was used to investigate interactions among bZIP and Rel family transcription factors. Regions outside the bZIP domains determined the locations of bZIP protein interactions. The subcellular sites of protein interactions were regulated by signaling. Cross-family interactions between bZIP and Rel proteins affected their subcellular localization and modulated transcription activation. These results attest to the general applicability of the BiFC assay for studies of protein interactions.
We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.
Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins. Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins. However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP. The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.
The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.
During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag-Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques-two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy-to examine Rous sarcoma virus Gag-Gag and -membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag-Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein-protein and -membrane interactions involved in the formation of complex macromolecular structures.
Drosophila melanogaster is a proven model system for many aspects of human biology. Here we present a two-hybrid–based protein-interaction map of
the fly proteome. A total of 10,623 predicted transcripts were isolated and screened against standard and normalized complementary
DNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. A computational method of rating two-hybrid
interaction confidence was developed to refine this draft map to a higher confidence map of 4679 proteins and 4780 interactions.
Statistical modeling of the network showed two levels of organization: a short-range organization, presumably corresponding
to multiprotein complexes, and a more global organization, presumably corresponding to intercomplex connections. The network
recapitulated known pathways, extended pathways, and uncovered previously unknown pathway components. This map serves as a
starting point for a systems biology modeling of multicellular organisms, including humans.
To initiate studies on how protein-protein interaction (or “interactome”) networks relate to multicellular functions, we have
mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from
high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the
overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains ∼5500 interactions. Topological and biological features of
this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological
hypotheses.
The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth,
glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future
advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein,
called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We
show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting
its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis
susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc
signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult
tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and
apoptosis in many cell types.
The pancreas consists of three main cell lineages (endocrine, exocrine, and duct) that develop from common primitive foregut
precursors. The transcriptional network responsible for endocrine cell development has been studied extensively, but much
less is known about the transcription factors that maintain the exocrine and duct cell lineages. One transcription factor
that may be important to exocrine cell function is Mist1, a basic helix-loop-helix (bHLH) factor that is expressed in acinar
cells. In order to perform a molecular characterization of this protein, we employed coimmunoprecipitation and bimolecular
fluorescence complementation assays, coupled with electrophoretic mobility shift assay studies, to show that Mist1 exists
in vivo as a homodimer complex. Analysis of transgenic mice expressing a dominant-negative Mist1 transgene (Mist1mutant basic [Mist1MB]) revealed the cell autonomous effect of inhibiting endogenous Mist1. Mist1MB cells become disorganized, exhibit a severe depletion of intercellular gap junctions, and express high levels of the glycoprotein
clusterin, which has been shown to demarcate immature acinar cells. Inhibition of Mist1 transcriptional activity also leads
to activation of duct-specific genes, such as cytokeratin 19 and cytokeratin 20, suggesting that alterations in the bHLH network produce a direct acinar-to-ductal phenotypic switch in mature cells. We
propose that Mist1 is a key transcriptional regulator of exocrine pancreatic cells and that in the absence of functional Mist1,
acinar cells do not maintain their normal identity.
Transforming growth factor beta (TGF-beta) has a major role in cell proliferation, differentiation and apoptosis in many cell types. Integration of the TGF-beta pathway with other signalling cascades that control the same cellular processes may modulate TGF-beta responses. Here we report the discovery of a new functional link between TGF-beta and growth factor signalling pathways, mediated by a physical interaction between the serine-threonine kinase PKB (protein kinase B)/Akt and the transcriptional activator Smad3. Formation of the complex is induced by insulin, but inhibited by TGF-beta stimulation, placing PKB-Smad3 at a point of convergence between these two pathways. PKB inhibits Smad3 by preventing its phosphorylation, binding to Smad4 and nuclear translocation. In contrast, Smad3 does not inhibit PKB. Inhibition of Smad3 by PKB occurs through a kinase-activity-independent mechanism, resulting in a decrease in Smad3-mediated transcription and protection of cells against TGF-beta-induced apoptosis. Consistently, knockdown of the endogenous PKB gene with small-interfering RNA (siRNA) has the opposite effect. Our results suggest a very simple mechanism for the integration of signals arising from growth-factor- and TGF-beta-mediated pathways.
Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.
Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.
To investigate the role of subcellular localization in regulating the specificity of G protein βγ signaling, we have applied
the strategy of bimolecular fluorescence complementation (BiFC) to visualize βγ dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to β and a carboxyl-terminal yellow fluorescent protein fragment
to γ. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was
not obtained with either subunit alone. Fluorescence was dependent on βγ assembly in that it was not obtained using β2 and γ1, which do not form a functional dimer. In addition to assembly, BiFC βγ complexes were functional as demonstrated by more
specific plasma membrane labeling than was obtained with individually tagged fluorescent β and γ subunits and by their abilities
to potentiate activation of adenylyl cyclase by αs in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise
combinations of three different β subunits with three different γ subunits were compared. BiFC βγ complexes containing either
β1 or β2 localized to the plasma membrane, whereas those containing β5 accumulated in the cytosol or on intracellular membranes. These results indicate that the β subunit can direct trafficking
of the γ subunit. Taken together with previous observations, these results show that the G protein α, β, and γ subunits all
play roles in targeting each other. This method of specifically visualizing βγ dimers will have many applications in sorting
out roles for particular βγ complexes in a wide variety of cell types.
Crystallin genes are selectively expressed during lens development. Maf and Sox family proteins synergistically enhanced gammaF-crystallin promoter activity in a lens cell line. Mutational analysis of the gammaF-crystallin promoter identified a composite regulatory element containing nonconsensus Maf and Sox recognition sequences. Mutations in these recognition sequences or changes in their spacing eliminated synergistic transcription activation. The transcriptional synergy was also affected by changes in the orientation of the Maf recognition sequence that had no detectable effect on binding affinity. The interaction between Maf and Sox proteins was visualized in living cells by bimolecular fluorescence complementation analysis. The N-terminal region of Maf mediated the interaction with Sox proteins in cells. Synergistic transcription activation required the N-terminal region of Maf as well as the ancillary DNA binding domain and the unique portion of the basic region that mediate specific recognition of the gammaF-crystallin promoter element. A mutation in the ancillary DNA binding domain of Maf (R288P) that has been shown to cause cataract eliminated the transcriptional activity of Maf but had no detectable effect on DNA binding in vitro. Whereas wild-type Maf was uniformly distributed in the nucleoplasm, R288P Maf was enriched in nuclear foci. Cajal bodies and gemini of coiled bodies were closely associated with the foci occupied by R288P Maf. Wild-type Maf formed complexes with Sox proteins in the nucleoplasm, whereas R288P Maf recruited Sox proteins as well as other interaction partners to the nuclear foci. The mislocalization of normal cellular proteins to these foci provides a potential explanation for the dominant disease phenotype of the R288P mutation in Maf.
Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA-protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.
Figure 1. Strategy for antiparallel leucine zipper-directed protein reassembly of GFP.3 Both the ribbon and topographical structures are depicted: GFP is shown in green, NZGFP is shown in blue and CZGFP is shown in red. The sequences of the designed leucine zippers, NZ and CZ, are ALKKELQANKKELAQLKWELQALKKELAQ and EQLE- KKLQALEKKLAQLEWKNQALEKKLAQ, respectively.
LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein–protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set. J. Cell. Biochem. 80:293–303, 2001. © 2001 Wiley-Liss, Inc.
Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.
We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub. However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.
Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.
Confocal fluorescence correlation spectroscopy (FCS) and other confocal spectroscopic techniques are ideally suited for the analysis of molecular interactions at the subcellular level. However, one requires exact positioning in three dimensions within the cell. Our instrument integrates FCS with high sensitivity digital imaging microscopy and high precision positioning. We present first measurements of intracellular FCS, with specification of the instrumental requirements and methods of data analysis. We propose the term fluorescence correlation microscopy (FCM) for this extended modality of FCS.
The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells.
LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.
We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.
Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.
We report a photoactivatable variant of theAequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times
when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new
tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the
cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope
and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350-400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest.
The ability to make sensitive measurements of protein-protein interaction kinetics in single neurons is critical for understanding the molecular and cellular basis of neuronal function. We have developed a reporter technology based on the differential induction of Escherichia coli TEM-1 beta-lactamase (Bla) enzymatic activity that can function as a sensor of the interaction state of two target proteins within single neurons in vivo. To modulate Bla enzymatic activity, we first split the enzyme into two separate, complementary protein fragments that we identified by using a functional screening approach based on circular permutation of the Bla enzyme. The split enzyme was then brought together by the phosphorylation-dependent association of the kinase inducible domain of the cAMP response element binding protein (CREB) and the KIX domain of the CREB binding protein. Using an intracellular substrate whose fluorescence spectrum changes after hydrolysis by Bla, we performed time-lapse ratiometric imaging measurements of Bla enzymatic induction after association of the CREB and CREB binding protein interaction domains. This approach permits direct imaging of protein-protein interactions in single cells with high signal discrimination.
In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.
Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.
This chapter presents an overview of the fluorescence correlation spectroscopy (FCS) of green fluorescent protein (GFP) fusion proteins in living plant cells. FCS is mainly applied to well-defined in vitro systems. However, the possibility of monitoring molecular dynamics under equilibrium conditions at a single-molecule level makes FCS an attractive technique for intracellular studies. The fluorescence photons pass through a pinhole and are detected by a highly sensitive detector. The signal-to-noise ratio achieved by this method is very high, as signal interference from scattered laser light, background fluorescence, and Raman emission can be largely eliminated. The chapter also mentions the applications of fluorescence cross-correlation spectroscopy (FCCS), including enzyme kinetics, nucleotide hybridization, conformational dynamics in DNA, and protein-DNA interactions. From these studies it is clear that FCCS is an attractive technique to observe very specific molecular interactions. At present, it is a challenge to apply this technique to cellular systems to monitor molecular interactions.
In recent years, our ability to unravel the finer details of intracellular signaling has improved remarkably. Technological innovations resulting from the introduction of green fluorescent protein (GFP) have played a significant role in these advances. Fluorescent indicators allow us to visualize events within a cell in real-time and space. This review focuses on indicators that use GFP-based fluorescence resonance energy transfer (FRET) technologies and discusses how these methodologies have given insights into biological questions relating to the spatiotemporal patterns of signaling by cAMP, calcium, receptor tyrosine kinases, and other molecules.
In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.
The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S phase transition of the cell cycle in normal lymphocytes. Surprisingly, Skp2 enhances c-Myc-induced S phase transition and activates c-Myc target genes in a Myc-dependent manner. Further, Myc-induced transcription was shown to be Skp2 dependent, suggesting interdependence between c-Myc and Skp2 in activation of transcription. Moreover, Myc-dependent association of Skp2, ubiquitylated proteins, and subunits of the proteasome to a c-Myc target promoter was demonstrated in vivo. The results suggest that Skp2 is a transcriptional cofactor for c-Myc and indicates a close relationship between transcription activation and transcription factor ubiquitination.
Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection. Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine. In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A. tumefaciens. Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation. VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate. Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding. Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.
Imaging specific molecules and their interactions in space and time will be essential to understand how genomes create cells, how cells constitute organisms and how errant cells cause disease. Molecular imaging must be extended and applied from nanometre to metre scales and from milliseconds to days. This quest will require input from physics, chemistry, and the genetics and biochemistry of diverse organisms with useful talents.
Förster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.
The transcription factor NF-kappaB regulates genes involved in innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Proinflammatory cytokines induce the activation of NF-kappaB in both transient and persistent phases. We investigated the mechanism for this biphasic NF-kappaB activation. Our results show that MEKK3 is essential in the regulation of rapid activation of NF-kappaB, whereas MEKK2 is important in controlling the delayed activation of NF-kappaB in response to stimulation with the cytokines TNF-alpha and IL-1alpha. MEKK3 is involved in the formation of the IkappaBalpha:NF-kappaB/IKK complex, whereas MEKK2 participates in assembling the IkappaBbeta:NF-kappaB/IKK complex; these two distinct complexes regulate the proinflammatory cytokine-induced biphasic NF-kappaB activation. Thus, our study reveals a novel mechanism in which different MAP3K and IkappaB isoforms are involved in specific complex formation with IKK and NF-kappaB for regulating the biphasic NF-kappaB activation. These findings provide further insight into the regulation of cytokine-induced specific and temporal gene expression.
Acetylation and other modifications on histones comprise histone codes that govern transcriptional regulatory processes in chromatin. Yet little is known how different histone codes are translated and put into action. Using fluorescence resonance energy transfer, we show that bromodomain-containing proteins recognize different patterns of acetylated histones in intact nuclei of living cells. The bromodomain protein Brd2 selectively interacted with acetylated lysine 12 on histone H4, whereas TAF(II)250 and PCAF recognized H3 and other acetylated histones, indicating fine specificity of histone recognition by different bromodomains. This hierarchy of interactions was also seen in direct peptide binding assays. Interaction with acetylated histone was essential for Brd2 to amplify transcription. Moreover association of Brd2, but not other bromodomain proteins, with acetylated chromatin persisted on chromosomes during mitosis. Thus the recognition of histone acetylation code by bromodomains is selective, is involved in transcription, and potentially conveys transcriptional memory across cell divisions.