GFP Variants Research Articles

Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

BackgroundThe purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC.Materials and methodsBlood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay.ResultsRisk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1β, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects.ConclusionIn conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli.

The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and is not copied by host polymerases but is selectively copied by an orthogonal DNA polymerase (O-DNAP), which does not copy the genome. We designed mutant O-DNAPs that selectively increase the mutation rate of the orthogonal replicon by two to four orders of magnitude. We demonstrate the utility of our system for accelerated continuous evolution by evolving a 150-fold increase in resistance to tigecycline in 12 days. And, starting from a GFP variant, we evolved a 1000-fold increase in cellular fluorescence in 5 days.

Development of Responsive Promoters and their Utilization for Stable CHO Sensor Cell Lines.

Chinese hamster ovary (CHO) cells are the most important mammalian expression systems to produce recombinant proteins. To ensure a proper expression of the desired molecule, it is important to monitor and adjust bioprocess parameters like oxygen concentration as well as osmolality. However, the observation of crucial cultivation parameters can be an elaborate procedure requiring lots of hands-on work. In addition, for emerging modeling approaches for bioprocesses, a model cell line responding with a measurable signal to an external influence would be highly valuable. This protocol describes in detail the procedure to generate responsive promoters reacting to limiting conditions as well as the generation of stable sensor cell lines communicating with the operator. Thereby, hypoxia and osmolality sensing response elements established in CHO cells will be utilized to trigger the expression of a minimal CMV promoter. To assess the activity of the responsive promoter in close to real time, unstable variants of GFP and BFP will be expressed, which can be analyzed via flow cytometry. Finally, an automated sampling system coupled to a fluorescence microscope enables a continuous observation of CHO cells and reports emerging limiting conditions by detecting increasing amounts of a specific fluorescent protein.

Live cell transcription-coupled nucleotide excision repair dynamics revisited

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags.

Tet-Dependent Gene Expression in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is increasingly recognized as an important nosocomial pathogen among the Gram-negative bacteria. Intrinsic resistance to different classes of antibiotics makes treatment of infections challenging. A deeper understanding of S. maltophilia physiology and virulence requires molecular genetic tools. Here, we describe the implementation of tetracycline-dependent gene regulation (tet regulation) in this bacterium. The exploited tet regulatory sequence of transposon Tn10 contained the tetR gene and three intertwined promoters, one of which was required for regulated expression of a target gene or operon. The episomal tet architecture was tested with a gfp variant as a quantifiable reporter. Fluorescence intensity was directly correlated with the concentration of the inducer anhydrotetracycline (ATc) applied and the duration of induction. Also, the expression of the rmlBACD operon of S. maltophilia K279a was subjected to tet control. These genes code for the synthesis of dTDP-l-rhamnose, an activated nucleotide sugar precursor of lipopolysaccharide (LPS) formation. A ΔrmlBACD mutant was complemented with a plasmid carrying this operon downstream of the tet sequence. In the presence of ATc, the LPS pattern was similar to that of wild-type S. maltophilia, whereas without the inducer, fewer and apparently shorter O-antigen chains were detected. This underscores the functionality and usefulness of the tet system for gene regulation and, prospectively, the validation of targets for new anti-S. maltophilia drugs. IMPORTANCE Stenotrophomonas maltophilia is an emerging pathogen in hospital settings and poses a threat to immunocompromised patients. Due to a high level of resistance to different types of antibiotics, treatment options are limited. We here adapted a tool for inducible expression of genes of interest, known as the tet system, to S. maltophilia. Genes relevant to producing surface carbohydrate structures (lipopolysaccharide [LPS]) were placed under the control of the tet system. In the presence of an inducer, the LPS pattern was similar to that of wild-type S. maltophilia, whereas in the "off" state of the system (without inducer), fewer and apparently shorter versions of LPS were detected. The tet system is functional in S. maltophilia and may be helpful to reveal gene-function relationships to gain a deeper understanding of the bacterium's physiology and virulence.

Designed active-site library reveals thousands of functional GFP variants

Mutations in a protein active site can lead to dramatic and useful changes in protein activity. The active site, however, is sensitive to mutations due to a high density of molecular interactions, substantially reducing the likelihood of obtaining functional multipoint mutants. We introduce an atomistic and machine-learning-based approach, called high-throughput Functional Libraries (htFuncLib), that designs a sequence space in which mutations form low-energy combinations that mitigate the risk of incompatible interactions. We apply htFuncLib to the GFP chromophore-binding pocket, and, using fluorescence readout, recover >16,000 unique designs encoding as many as eight active-site mutations. Many designs exhibit substantial and useful diversity in functional thermostability (up to 96 °C), fluorescence lifetime, and quantum yield. By eliminating incompatible active-site mutations, htFuncLib generates a large diversity of functional sequences. We envision that htFuncLib will be used in one-shot optimization of activity in enzymes, binders, and other proteins.

Live measurement of organelle pH by plant pHluorins.

The regulation of intracellular pH homeostasis is essential for the survival of cells, but assessing the organellar pH in plants has remained challenging. The plant-compatible genetically encoded pH sensors, developed from pH-sensitive GFP variants pHluorins, are irreplaceable tools for noninvasive real-time tracking of pH in particular organelles. Plant ecliptic pHluorin (PEpHluorin) shows acidification-dependent fluorescence intensity changes, whereas the plant ratiometric pHluorin (PRpHluorin or RaVC) allows precise pH measurements in vivo.

Endogenous gpdh-1 transcriptional reporters as new tools for the study of the osmotic stress response.

In vivo monitoring of gpdh-1 gene expression using standard transcriptional reporters is a powerful and commonly used tool for genetic dissection of the osmotic stress response in C. elegans . Like all transgene reporters, these gpdh-1 reporters have important limitations that restrict their utility. To overcome these limitations, we created three different gpdh-1 reporters using CRISPR/Cas9 methods to insert several variants of GFP into the endogenous gpdh-1 locus. These new strains provide a more powerful and accurate tool for the analysis of gpdh-1 regulatory pathways.

Intracellular phase separation of globular proteins facilitated by short cationic peptides

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis

The influence of fusion tags to produce recombinant proteins in the cytoplasm of Bacillus subtilis is not well-studied as in E. coli. This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene (gfp+) and low expression gene (egfp) in the cytoplasm of B. subtilis cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the Pgrac212 in B. subtilis. The fusions of His-tags with GFP+ caused a reduction compared to the construct without His-tag. When three His-tags fused with egfp, the EGFP production levels were significantly increased up to 3.5-, 12-, and 15-fold. This study suggested that His-tag at the N-terminus could enhance the protein production for the low expression gene and reduce that of the high expression gene in B. subtilis.

Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development.

The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress.

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

Four-color single-molecule imaging with engineered tags resolves the molecular architecture of signaling complexes in the plasma membrane

SummaryLocalization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. We present a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. Two engineered, monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies, are employed for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enables us to quantitatively resolve the stoichiometry and dynamics of the interferon-γ (IFNγ) receptor signaling complex in the plasma membrane of living cells by multicolor single-molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we identify ligand-induced receptor homo- and heterodimerization. Multicolor single-molecule co-tracking and quantitative single-molecule Förster resonance energy transfer moreover reveals transient assembly of IFNγ receptor heterotetramers and confirms its structural architecture.

Perturbation of kinetochore function using GFP-binding protein in fission yeast.

Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems.

FGF1 Fusions with the Fc Fragment of IgG1 for the Assembly of GFPpolygons-Mediated Multivalent Complexes Recognizing FGFRs.

FGFRs are cell surface receptors that, when activated by specific FGFs ligands, transmit signals through the plasma membrane, regulating key cellular processes such as differentiation, division, motility, metabolism and death. We have recently shown that the modulation of the spatial distribution of FGFR1 at the cell surface constitutes an additional mechanism for fine-tuning cellular signaling. Depending on the multivalent, engineered ligand used, the clustering of FGFR1 into diverse supramolecular complexes enhances the efficiency and modifies the mechanism of receptor endocytosis, alters FGFR1 lifetime and modifies receptor signaling, ultimately determining cell fate. Here, we present a novel approach to generate multivalent FGFR1 ligands. We functionalized FGF1 for controlled oligomerization by developing N- and C-terminal fusions of FGF1 with the Fc fragment of human IgG1 (FGF1-Fc and Fc-FGF1). As oligomerization scaffolds, we employed GFPpolygons, engineered GFP variants capable of well-ordered multivalent display, fused to protein G to ensure binding of Fc fragment. The presented strategy allows efficient assembly of oligomeric FGFR1 ligands with up to twelve receptor binding sites. We show that multivalent FGFR1 ligands are biologically active and trigger receptor clustering on the cell surface. Importantly, the approach described in this study can be easily adapted to oligomerize alternative growth factors to control the activity of other cell surface receptors.

Engineering A Modular Protein Color Switch using An Entropy‐driven Beta Strand Exchange

Protein conformational switches are widely used in functional regulation and biosensing to visualize biological substances in vitro and in vivo. A major challenge in protein switch design strategies is coupling the target recognition domain to an output domain to produce a change in fluorescence wavelength or intensity. To overcome this challenge, we tested a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; (2) alternate frame folding (AFF), which allows a protein (GFP variants, in this case) to switch between two mutually exclusive folds. For proof-of-concept, we chose an unfolded, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain, and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein. Upon addition of ligand, cpFKBP folding effects a conformational change in which the 10th beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). Single point mutations and insertions were introduced to thermodynamically balance the protein switch for response within a reasonable timescale. We observe a shift in emission wavelength within an hour in response to ligand in vitro and in cellulo with a yellow:green color change of ~2.5-fold. This protein switch design has the advantages of genetic encodability, potential modularity and a ratiometric response. The design strategy further provides insight into protein engineering principles for application in molecular diagnostics, cellular biosensors and activatable proteins.

A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms

pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as “luminescent pH indicator protein” (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

Four-Color Single-Molecule Imaging with Engineered Tags Resolves the Molecular Architecture of Signaling Complexes in the Plasma Membrane

Localization and tracking of individual receptors by single-molecule imaging opens unique possibilities to unravel the assembly and dynamics of signaling complexes in the plasma membrane. For this purpose, we here developed a comprehensive workflow for imaging and analyzing receptor diffusion and interaction in live cells at single molecule level with up to four colors. We engineered two monomeric GFP variants, which are orthogonally recognized by anti-GFP nanobodies (NB) for efficient and selective labeling of target proteins in the plasma membrane with photostable fluorescence dyes. This labeling technique enabled us to quantitatively resolve the stoichiometry and dynamics of the interferon-g (IFNg) receptor signaling complex in the plasma membrane of living cells by multicolor single molecule imaging. Based on versatile spatial and spatiotemporal correlation analyses, we unambiguously identify ligand-induced receptor homo-and heterodimerization. Detailed analysis by multicolor single-molecule co-tracking and quantitative single-molecule FRET revealed transient assembly of IFNγ-receptor heterotetramers in the plasma membrane with a structural architecture as observed for the isolated ectodomains in vitro.