Fluorescent Protein Tags Research Articles

Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD.

The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.

A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo.

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are sub-optimal in vivo due to poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled non-invasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol-or CCl4- induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

BAC Recombineering and Transgenesis to Study Cell Polarity and Polarized Tissue Morphogenesis in Mice.

Planar cell polarity (PCP) signaling plays a critical role in coordinating cell polarity during various organogenesis processes in mammals, and its disruption is causal to numerous congenital disorders in humans. To elucidate its actions in mammals, mouse genetics is an indispensable approach. Given that both gain- and loss-of-function of many PCP genes often cause similar defects, the standard mouse transgenic approach may not always be ideal for studying PCP genes in their wild-type and mutant forms. Here we describe using BAC (bacterial artificial chromosomes) transgenes as a versatile and effective alternative. Transgenes made from BACs, which are genomic clones 100-200kb in size, can more faithful recapitulate endogenous gene expression levels and patterns. Bacterial based recombination system can be used to efficiently introduce mutations, fluorescent protein tags, and LoxP sites for conditional expressions. Cre can also be inserted into BACs to map the contribution of cells expressing any PCP gene of interest, and study PCP mediated tissue morphogenesis.

Methods for Investigating Cell Division Mechanisms in C. elegans.

The nematode Caenorhabditis elegans is a widely used model organism for the study of mitotic and meiotic cell division. These self-fertilizing worms are particularly advantageous for such studies because they rapidly reproduce (each worm lays ~250 eggs in only 3-4days) and the cell division machinery is highly conserved between worms and humans. Worms are also genetically tractable and proteins can be readily depleted using RNA interference (RNAi), allowing for the characterization of protein function in vivo. To assess phenotypes, spindles can be directly visualized within the worm using fluorescent protein tags or embryos can be dissected out of the worm and immunostained. A combination of these techniques allows comprehensive characterization of a protein's function in a relatively short time span. Here, we describe methods for each of these techniques: RNA interference through feeding, in utero live imaging, in utero fixed imaging, and immunofluorescence.

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines.

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

Fluorescent Labeling of Small Extracellular Vesicles (EVs) Isolated from Conditioned Media.

Extracellular vesicles (EVs), such as exosomes, are produced by all known eukaryotic cells, and constitute essential means of intercellular communication. Recent studies have unraveled the important roles of EVs in migrating to specific sites and cells. Functional studies of EVs using in vivo and in vitro systems require tracking these organelles using fluorescent dyes or, alternatively, transfected and fluorescent-tagged proteins, located either intravesicularly or anchored to the EV bilayer membrane. Due to design simplicity, the fluorescent dye might be a preferred method if the cells are difficult to modify by transfection or when the genetic alteration of the mother cells is not desired. This protocol describes techniques to label cultured cell-derived EVs, using lipophilic DiR [DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide)] fluorophore. This technique can be used to study the cellular uptake and intracellular localization of EVs, and their biodistribution in vivo , which are crucial evaluations of any isolated EVs.

Fluorescently-tagged magnetic protein nanoparticles for high-resolution optical and ultra-high field magnetic resonance dual-modal cerebral angiography.

Extremely small paramagnetic iron oxide nanoparticles (FeMNPs) (<5 nm) can enhance positive magnetic resonance imaging (MRI) contrast by shortening the longitudinal relaxation time of water (T1), but these nanoparticles experience rapid renal clearance. Here, magnetic protein nanoparticles (MPNPs) are synthesized from protein-conjugated citric acid coated FeMNPs (c-FeMNPs) without loss of the T1 MRI properties and tagged with fluorescent dye (f-MPNPs) for optical cerebrovascular imaging. The c-FeMNPs shows average size 3.8 ± 0.7 nm with T1 relaxivity (r1) of 1.86 mM-1 s-1 and transverse/longitudinal relaxivity ratio (r2/r1) of 2.53 at 11.7 T. The f-MPNPs show a higher r1 value of 2.18 mM-1 s-1 and r2/r1 ratio of 2.88 at 11.7 T, which generates excellent positive MRI contrast. In vivo cerebral angiography with f-MPNPs enables detailed microvascular contrast enhancement for differentiation of major blood vessels of murine brain, which corresponds well with whole brain three-dimensional time-of-flight MRI angiograms (17 min imaging time with 60 ms repetition time and 40 μm isotropic voxels). The real-time fluorescence angiography enables unambiguous detection of brain capillaries with diameter < 40 μm. Biodistribution examination revealed that f-MPNPs were safely cleared by the organs like the liver, spleen, and kidneys within a day after injection. Blood biochemical assays demonstrated no risk of iron overload in both rats and mice. With hybrid neuroimaging technologies (e.g., MRI-optical) on the rise, f-MPNPs built on this platform can generate exciting neuroscience applications.

Plant Gene Modification by BAC Recombineering.

Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second "turbo"-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.

Studying Iridoid Transport in Catharanthus roseus by Grafting.

The plant Catharanthus roseus is well known for its spatial separation of iridoid and monoterpenoid indole alkaloid (MIA) biosynthesis at both intracellular and intercellular levels, collectively suggested by RNA in situ hybridization, enzymatic and transcriptomic studies using leaf epidermis, and fluorescent protein tagging studies. Although documented in other plant species, the long-distance transport of iridoid glycosides, such as secologanin, has not been known in C. roseus until a recent study suggested that secologanin is transported from root to shoot, by grafting low iridoid/MIA mutant scions onto wild-type stock plants. This chapter describes the in vitro cultivation of C. roseus plants and grafting techniques to enable studies concerning iridoid/MIA transport between organs. The iridoid and MIA analysis methods are also provided.

Drosophila Trachea as a Novel Model of COPD.

COPD, a chronic obstructive pulmonary disease, is one of the leading causes of death worldwide. Clinical studies and research in rodent models demonstrated that failure of repair mechanisms to cope with increased ROS and inflammation in the lung leads to COPD. Despite this progress, the molecular mechanisms underlying the development of COPD remain poorly understood, resulting in a lack of effective treatments. Thus, an informative, simple model is highly valued and desired. Recently, the cigarette smoke-induced Drosophila COPD model showed a complex set of pathological phenotypes that resemble those seen in human COPD patients. The Drosophila trachea has been used as a premier model to reveal the mechanisms of tube morphogenesis. The association of these mechanisms to structural changes in COPD can be analyzed by using Drosophila trachea. Additionally, the timeline of structural damage, ROS, and inflammation can be studied in live organisms using fluorescently-tagged proteins. The related function of human COPD genes identified by GWAS can be screened using respective fly homologs. Finally, the Drosophila trachea can be used as a high-throughput drug screening platform to identify novel treatments for COPD. Therefore, Drosophila trachea is an excellent model that is complementary to rodent COPD models.

Microinjection Method for Analyzing Zebrafish Early Stage Oocytes.

Maternal factors which accumulate and establish oocyte polarity during the early stages of oogenesis play key roles in embryonic development, as well as germ cell formation. However, vertebrate oogenesis, especially early stages of oogenesis, is not well understood due to the difficulty of accessing these oocytes and the lack of analytical methods. Here, we report on a microinjection method for analyzing zebrafish early-stage oocytes and some artifacts to be aware of when performing oocyte injections or analyzing oocytes. Using this method, we successfully injected mRNAs encoding fluorescent-tagged proteins into early-stage oocytes and observed subcellular localization in the live oocytes. This method is expected to advance the functional analysis of genes involved in oogenesis.

Chemical Biology Toolbox to Visualize Protein Aggregation in Live Cells.

Protein misfolding and aggregation is a complex biochemical process and has been associated with numerous human degenerative diseases. Developing novel chemical and biological tools and approaches to visualize aggregated proteins in live cells is in high demand for mechanistic studies, diagnostics, and therapeutics. In this review, we summarize the recent developments in the chemical biology toolbox applied to protein aggregation studies in live cells. These methods exploited fluorescent protein tags, fluorescent chemical tags, and small-molecule probes to visualize the protein-aggregation process, detect proteome stresses, and quantify the protein homeostasis network capacity. Inspired by these seminal works, we have generalized design principles for the development of new detection methods and probes in the future that will illuminate this important biological process.

Tau seeding in chronic traumatic encephalopathy parallels disease severity.

Chronic traumatic encephalopathy (CTE), a neurodegenerative tauopathy, is associated with behavioral, mood and cognitive impairment, including dementia. Tauopathies are neurodegenerative diseases whose neuropathological phenotypes are characterized by distinct histopathologic features of tau pathology, which progressively deposit throughout the brain. In certain tauopathies, especially Alzheimer's disease (AD), tau deposition appears to follow brain network connections. Experimental evidence suggests that the progression of tau pathology in humans, mouse and cell models could be explained by tau seeds that adopt distinct conformations and serve as templates for their own amplification to mediate transcellular propagation of pathology. Tau seeds are efficiently detected by the induction of aggregation in cell-based "biosensors" that express tau repeat domain (RD) with a disease-associated mutation (P301S) fused to complementary fluorescent protein tags (cyan and yellow fluorescent protein). Biosensors enable quantification of tau seeding in fixed and fresh-frozen brain tissue. Phospho-tau deposition in CTE follows progressive stages (I-IV), but the relationship of seeding to this deposition is unclear. We have used an established biosensor assay to independently quantify tau seeding as compared to AT8 phospho-tau histopathology in thin sections of fixed tissues of 11 brain regions from 27 patients with CTE, 5 with other tauopathies, and 5 negative controls. In contrast to prior studies of AD, we detected tau seeding late in the course of CTE (predominantly stages III and IV). It was less anatomically prevalent than AT8-positive inclusions, which were relatively widespread. We especially observed seeding in the limbic system (amygdala, thalamus, basal ganglia), which may explain the dominant cognitive and behavior impairments that characterize CTE.

Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus.

Biotin is an important enzyme cofactor that plays a key role in all three domains. The classical bifunctional enzyme BioDA in eukaryotes (such as Aspergillus flavus and Arabidopsis thaliana) is involved in the antepenultimate and penultimate steps of biotin biosynthesis. In this study, we identified a A. flavus bifunctional gene bioDA which could complement both Escherichia coli ΔEcbioD and ΔEcbioA mutants. Interestingly, the separated domain of AfBioD and AfBioA could, respectively, fuse with EcBioA and EcBioD well and work together. What is more, we found that BioDA was almost localized to the mitochondria in A. flavus, as shown by N-terminal red fluorescent protein tag fusion. Noteworthy, the subcellular localization of AfBioDA is never affected by common environmental stresses (such as hyperosmotic stress or oxidative stress). The knockout strategy demonstrated that the deletion of AfbioDA gene from the chromosome impaired the biotin de novo synthesis pathway in A. flavus. Importantly, this A. flavus mutant blocked biotin production and decreased its pathogenicity to infect peanuts. Based on the structural comparison, we found that two inhibitors (amiclenomycin and gemcitabine) could be candidates for antifungal drugs. Taken together, our findings identified the bifunctional AfbioDA gene and shed light on biotin biosynthesis in A. flavus.

TRIM28 is a transcriptional activator of the mutant TERT promoter in human bladder cancer

Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.

Bacillus siamensis CNE6- a multifaceted plant growth promoting endophyte of Cicer arietinum L. having broad spectrum antifungal activities and host colonizing potential

Exploration of endophytic bacteria with multiple plant growth promoting (PGP) attributes is considered as an eco-friendly and cost-effective alternative to agricultural chemicals for increasing crop productivity. In the present endeavor, healthy chickpea plants (Cicer arietinum L.) collected from district Birbhum, West Bengal, India were subjected for the isolation of endophytic bacteria having multifarious PGP properties. One potent endophytic Gram positive bacterial strain CNE6 was isolated from the nodule of chickpea and was identified as Bacillus siamensis based on 16S rDNA sequence homologies. The isolate showed a number of PGP properties like phosphate solubilization, IAA production, nitrogen fixation, hydroxamate type of siderophore production and ACC deaminase activities. The isolate CNE6 produced 33.27 ± 2.16 μg/mL of IAA in the presence of tryptophan. Production of IAA was also confirmed by HPLC analysis and it was found effective for inducing lateral root branching in chickpea. In addition, the isolate displayed significant antagonistic activity against a number of plant pathogenic fungi when tested by dual culture overlay and agar well diffusion assay. 50 % cell free supernatant of CNE6 was found effective for 60–80 % inhibition of radial growth of pathogenic fungi tested. Scanning electron microscopic observation revealed massive degradation of pathogenic fungal mycelia by the antifungal metabolites of CNE6. LC–MS analysis of bacterial lipopeptides suggested the production of antifungal antibiotics like surfactin, fengycin and iturin by the isolate. The presence of genes encoding antifungal lipopeptides was also confirmed by PCR amplification using specific primers. Green fluorescent protein (GFP) tagging of CNE6 using broad host range plasmid vector (pDSK-GFPuv) followed by colonization study indicated very good host colonization potential of the isolate and its probable movement through xylem vessels. Enhanced shoot and root length and chlorophyll content upon treatment with CNE6 as observed in in vivo pot experiments also supported the positive role of the endophytic isolate on overall development and growth of the chickpea plants. This is the first report of Bacillus siamensis as an endophyte of Cicer arietinum L. which can be successfully applied for improving the productivity of this crop plant.

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila.

In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM. However, this technique still requires the generation of new molecular constructions. Thus, we decided to develop a versatile method that would take advantage of the numerous GFP-tagged proteins already existing and create a tool combining a nanobody anti-GFP (GBP) with APEX. This GBP-APEX tool allows a simple and efficient detection of any GFP fusion proteins without the needs of specific antibodies nor the generation of additional constructions. We have shown the feasibility and efficiency of this method to detect various proteins in Drosophila ovarian follicles such as nuclear proteins, proteins associated with endocytic vesicles, plasma membranes or nuclear envelopes. Lastly, we expressed this tool in Drosophila with the UAS/GAL4 system that enables spatiotemporal control of the protein detection.

Translation of Cellular Protein Localization Using Convolutional Networks

Protein localization in cells has been analyzed by fluorescent labeling using indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localization of different proteins had not been analyzed using artificial intelligence. Here, we applied convolutional networks for the prediction of localization of the cytoskeletal proteins from the localization of the other proteins. Lamellipodia are one of the actin-dependent subcellular structures involved in cell migration and are mainly generated by the Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 2 (WAVE2) and the membrane remodeling I-BAR domain protein IRSp53. Focal adhesion is another actin-based structure that contains vinculin protein and promotes lamellipodia formation and cell migration. In contrast, microtubules are not directly related to actin filaments. The convolutional network was trained using images of actin filaments paired with WAVE2, IRSp53, vinculin, and microtubules. The generated images of WAVE2, IRSp53, and vinculin were highly similar to their real images. In contrast, the microtubule images generated from actin filament images were inferior without the generation of filamentous structures, suggesting that microscopic images of actin filaments provide more information about actin-related protein localization. Collectively, this study suggests that image translation by the convolutional network can predict the localization of functionally related proteins, and the convolutional network might be used to describe the relationships between the proteins by their localization.

Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata.

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.