Fluorescent Protein Labeling Research Articles

Genetic Transformation and Green Fluorescent Protein Labeling in Ceratocystis paradoxa from Coconut.

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a ‘reporter’ gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.

Establishing Novel Bacteriophage Detection Method Based on Imagining Flow Cytometry

Bacteriophage ΦX174 specifically infects E.coli cells. It shares the similar biological characteristics with pathogenic virus. Thus, it is widely used as the “model virus” in the evaluation of virus control effect due to its safe operation. In this study, we have established a new bacteriophage detection method based on imagining flow cytometry. Combining the nucleic acid dyeing and capsid protein fluorescence labeling, the method based on imagining flow cytometry shows good linearity and specificity within its detection range. The establishment of this method is of great importance in fast and accurate detection for bacteriophage.

Imaging and Tracking Stem Cell Engraftment in Ischemic Hearts by Near-Infrared Fluorescent Protein (iRFP) Labeling.

Stem cell-based therapies hold great promise as alternative therapeutic strategies for various chronic diseases, including ischemic cardiomyopathy. Tracking the engraftment of transplanted stem cells is critical to the assessment of donor cell survival in the host environment. Fluorescent proteins, such as green fluorescent protein (GFP), have been widely used to track the fate of donor cells; however, GFP labeling has limitations with regard to noninvasively measuring cell engraftment in vivo. Our research indicates that near-infrared fluorescent protein (iRFP) labeling offers advantages for noninvasive in vivo imaging and histological assessment. Here, we present a protocol for using the lentiviral vector-mediated iRFP vector system to label and track donor stem cells in ischemic mouse hearts.

On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates.

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.

Live Cell Imaging of Hepatitis C Virus Trafficking in Hepatocytes.

Standard fixed cell confocal microscopy is inherently limited in visualizing dynamic processes involving two- and three-dimensional movement. To overcome these limitations, live cell imaging approaches have been developed to study hepatitis C virus (HCV) entry, replicase protein trafficking, virion assembly, and egress. These studies have relied on fluorescent labeling of viral proteins by epitope tag insertion, genome labeling via nucleophilic dyes, or using lipophilic dyes to label the virion envelope. In this method review, we describe two approaches to study HCV virion trafficking in live cells. Lipophilic labeling of the envelope allows for study of the early events (through virion uncoating/fusion) in the HCV lifecycle. Tetracysteine (TC) tag insertion into the capsid protein permits study of virion assembly and capsid trafficking via binding of a fluorogenic biarsenical dye.

Sortase-mediated fluorescent labeling of CRISPR complexes.

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.

Potential Long-Term Treatment of Hemophilia a By Early Postnatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stem Cells

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Both mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been used independently to correct the HA phenotype, but poor survival and insufficient retention of transplanted cells in the long term limit their application. In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) with placental mesenchymal stem cells (PMSCs) can achieve stable, long-term engraftment. To investigate stem cell engraftment and therapeutic potential, ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector. To distinguish between PMSCs and ECFCs in the co-transplantation group, a Td-Tomato containing lentiviral vector was used to label PMSCs while a green fluorescent protein (GFP) labeling vector was used to label ECFCs. They were then transplanted intramuscularly either as ECFC only group, PMSC only group or co-transplantation of ECFCs and PMSCs group into neonatal (3 days) or adult (12 weeks) immune deficient NOD scid gamma (NSG) mice. Using an in vivo bioluminescence imaging system, we investigated cell engraftment for up to 26 weeks after transplantation. ECFC only and the co-transplantation groups achieved better engraftment than PMSCs only group in both neonatal and adult mice during the entire study period. The co-transplantation group showed higher engraftment rate than the ECFC only group at 16 weeks and 20 weeks post-transplantation. In addition, transplantation of ECFCs only or in combination with PMSCs in neonatal period achieved higher engraftment than transplantation in adult period. Immunohistochemical analyses were further employed to determine the fate and functionality of the engrafted cells. We found that engrafted ECFCs maintained endothelial phenotype and expressed high level of FVIII at 26 weeks post-transplantation. The presence of ECFCs in the co-transplantation group also promoted formation of functional vasculature after transplantation. To study the functional reversal of HA phenotype we co-transplanted ECFCs and PMSCs into F8 knock-out HA mice. The transplanted cells reduced the blood loss volume from 562.13±19.84µl of blood to 155.78±44.93µl in a tail clip assay in these HA mice. This work demonstrates that co-transplantation of ECFCs with PMSCs at the neonatal period is a promising strategy to achieve stable, long-term engraftment of transplanted cells, and thus holds great promise for the treatment of HA and other genetic diseases and tissue regeneration applications. DisclosuresNo relevant conflicts of interest to declare.

Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.

Label-free dynamic segmentation and morphological analysis of subcellular optical scatterers.

Imaging without fluorescent protein labels or dyes presents significant advantages for studying living cells without confounding staining artifacts and with minimal sample preparation. Here, we combine label-free optical scatter imaging with digital segmentation and processing to create dynamic subcellular masks, which highlight significantly scattering objects within the cells' cytoplasms. The technique is tested by quantifying organelle morphology and redistribution during cell injury induced by calcium overload. Objects within the subcellular mask are first analyzed individually. We show that the objects' aspect ratio and degree of orientation ("orientedness") decrease in response to calcium overload, while they remain unchanged in untreated control cells. These changes are concurrent with mitochondrial fission and rounding observed by fluorescence, and are consistent with our previously published data demonstrating scattering changes associated with mitochondrial rounding during calcium injury. In addition, we show that the magnitude of the textural features associated with the spatial distribution of the masked objects' orientedness values, changes by more than 30% in the calcium-treated cells compared with no change or changes of less than 10% in untreated controls, reflecting dynamic changes in the overall spatial distribution and arrangement of subcellular scatterers in response to injury. Taken together, our results suggest that our method successfully provides label-free morphological signatures associated with cellular injury. Thus, we propose that dynamically segmenting and analyzing the morphology and organizational patterns of subcellular scatterers as a function of time can be utilized to quantify changes in a given cellular condition or state.

Extending Single Molecule Imaging to Proteome Analysis by Quantitation of Fluorescent Labeling Homogeneity in Complex Protein Samples.

Fluorescence-based electrophoresis has been widely used for proteome analysis in which every protein species in cells is labeled with a fluorescent dye, separated by electric migration, and quantified using fluorescence detection. The ultimate limit of sensitivity for this approach could be reached by single-molecule fluorescence imaging and counting individual proteins, requiring exhaustive fluorescent labeling of proteins across molecular populations and species. However, it remains unclear how homogeneous the fluorescence labeling of individual protein molecules of each species is across the proteome. To address this question, we developed a method to measure the labeling homogeneity based on a single-molecule fluorescence counting assay. Our results reveal that the proportion of proteins labeled with at least one dye, called labeling occupancy (LO), was 35% for fluorescently labeled BSA using existing protocols. We then found that the LO could be improved to 82% under high pH and surfactant-rich conditions. Furthermore, when a proteome sample from a human cell lysate was analyzed, the total LO was 71%, whereby the values varied between 50 and 90% for low and high molecular weight proteome fractions, respectively. The results support the possibility of sensitive detection of proteins using single-molecule counting with fluorescent labeling at the proteome scale.

Tetrafluorination of Aromatic Azide Yields a Highly Efficient Staudinger Reaction: Kinetics and Biolabeling.

The development of highly efficient bioorthogonal reactions is of paramount importance for the research fields of biomaterials and chemical biology. We found that the o,o'-difluorinated aromatic azide was able to react with triphenylphosphine to produce water-stable phosphanimine. To further improve the efficiency of this kind of nonhydrolysis Staudinger reaction, a tetrafluorinated aromatic azide was employed to develop a faster nonhydrolysis Staudinger reaction with a rate of up to 51 m-1 s-1 , as revealed by high-performance liquid chromatography (HPLC) analysis and fluorescence kinetics. As a proof-of-concept study, the highly efficient Staudinger reaction was successfully used for chemoselective fluorescence labeling of proteins and nucleic acids (DNA and RNA) as well as for protein polyethyleneglycol (PEG)ylation. We believe that this bioorthogonal reaction can provide a broadly useful tool for various bioconjugations.

Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI.

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein’s activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Fluorogenic Probing of Membrane Protein Trafficking.

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an efficient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fluorescent marker FAST (fluorescence-activating and absorption-shifting tag). Cell-surface FAST-tagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

Zika Virus Induces Autophagy in Human Umbilical Vein Endothelial Cells.

Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the interaction between ZIKV and target cells during the early stages of infection, is still unclear. In this study, we demonstrate that infecting human umbilical vein endothelial cells (HUVEC) with ZIKV triggers cellular autophagy. We observed both an increase in the conversion of LC3-I to LC3-II and increased accumulation of fluorescent cells with LC3 dots, which are considered to be the two key indicators of autophagy. The ratio of LC3-II/GAPDH in each group was significantly increased at different times after ZIKV infection at different MOIs, indicating that the production of lipidated LC3-II increased. Moreover, both the ratio of LC3-II/GAPDH and the expression of viral NS3 protein increased with increasing time of viral infection. The expression level of p62 decreased gradually from 12 h post-infection. Expression profile of double fluorescent protein labelling LC3 indicated that the autophagy induced by ZIKV infection was a complete process. We further investigated the role of autophagy in ZIKV replication. We demonstrated that either the treatment with inhibitors of autophagosomes formation or short hairpin RNA targeting the Beclin-1 gene, which is critical for the formation of autophagosomes, significantly reduced viral production. Taken together, our results indicate that ZIKV infection induces autophagy of HUVEC, and inhibition of ZIKV-induced autophagy restrains viral replication.

A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.

The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap, and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap, and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels. © 2018 International Society for Advancement of Cytometry.

Crosstalk-free multicolor RICS using spectral weighting.

Raster image cross-correlation spectroscopy (ccRICS) can be used to quantify the interaction affinities between diffusing molecules by analyzing the fluctuations between two-color confocal images. Spectral crosstalk compromises the quantitative analysis of ccRICS experiments, limiting multicolor implementations to dyes with well-separated emission spectra. Here, we remove this restriction by introducing raster spectral image correlation spectroscopy (RSICS), which employs statistical filtering based on spectral information to quantitatively separate signals of fluorophores during spatial correlation analysis. We investigate the performance of RSICS by testing how different levels of spectral overlap or different relative signal intensities affect the correlation function and analyze the influence of statistical filter quality. We apply RSICS in vitro to resolve dyes with very similar emission spectra, and carry out RSICS in live cells to simultaneously analyze the diffusion of molecules carrying three different fluorescent protein labels (eGFP, Venus and mCherry). Finally, we successfully apply statistical weighting to data that was recorded with only a single detection channel per fluorophore, highlighting the general applicability of this method to data acquired with any type of multicolor detection. In conclusion, RSICS enables artifact-free quantitative analysis of concentrations, mobility and interactions of multiple species labeled with different fluorophores. It can be performed on commercial laser scanning microscopes, and the algorithm can be easily extended to other image correlation methods. Thus, RSICS opens the door to quantitative multicolor fluctuation analyses of complex (bio-) molecular systems.

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells.

In vivo imaging of adeno-associated viral vector labelled retinal ganglion cells

A defining characteristic of optic neuropathies, such as glaucoma, is progressive loss of retinal ganglion cells (RGCs). Current clinical tests only provide weak surrogates of RGC loss, but the possibility of optically visualizing RGCs and quantifying their rate of loss could represent a radical advance in the management of optic neuropathies. In this study we injected two different adeno-associated viral (AAV) vector serotypes in the vitreous to enable green fluorescent protein (GFP) labelling of RGCs in wild-type mice for in vivo and non-invasive imaging. GFP-labelled cells were detected by confocal scanning laser ophthalmoscopy 1-week post-injection and plateaued in density at 4 weeks. Immunohistochemical analysis 5-weeks post-injection revealed labelling specificity to RGCs to be significantly higher with the AAV2-DCX-GFP vector compared to the AAV2-CAG-GFP vector. There were no adverse functional or structural effects of the labelling method as determined with electroretinography and optical coherence tomography, respectively. The RGC-specific positive and negative scotopic threshold responses had similar amplitudes between control and experimental eyes, while inner retinal thickness was also unchanged after injection. As a positive control experiment, optic nerve transection resulted in a progressive loss of labelled RGCs. AAV vectors provide strong and long-lasting GFP labelling of RGCs without detectable adverse effects.

Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.

The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells without overexpression of fusion proteins or genetic tagging has not been routinely possible. Here we describe a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells. The labeling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies or antibody fragments (Fabs) into living cells and the specific binding of these antibodies to the target protein inside of the cell. Our protocol describes step by step the procedure from testing of the suitability of the desired antibody, over the digestion of the antibody to Fabs until the labeling and the delivery by electroporation of the antibody or Fab into the cells. VANIMA can be adapted to any monoclonal antibody, self-produced or commercial, and many different metazoan cell lines. Additionally, our method is simple to implement and can be used not only to visualize and track endogenous factors, but also to specifically label posttranslational modifications, which cannot be achieved by any other labeling technique so far.

Live Imaging of Xist RNA.

Live imaging gives additional layers of information such as physical dynamics of a molecule of your interest. Aptamer-based green fluorescent protein (GFP) labeling is suitable for visualization of RNA molecules. Here we describe a method to visualize Xist RNA using the Bgl aptamer system.