Use Of Fluorescent Protein Research Articles

Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius.

Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins for live imaging in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. We expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Of the tested proteins, thermal green protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. These initial results in live protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.

Analysis of Fluorescent Proteins for Observing Single Gene Locus in a Live and Fixed Escherichia coli Cell.

Fluorescent proteins (FPs) are essential tools for advanced microscopy techniques such as super-resolution imaging, single-particle tracking, and quantitative single-molecule counting. Various FPs fused to DNA-binding proteins have been used to observe the subcellular location and movement of specific gene loci in living and fixed bacterial cells. However, quantitative assessments of the properties of FPs for gene locus measurements are still lacking. Here, we assessed various FPs to observe specific gene loci in live and fixed Escherichia coli cells using a fluorescent repressor-operator binding system (FROS), tet operator-Tet repressor proteins (TetR). Tsr-fused FPs were used to assess the intensity and photostability of various FPs (five red FPs: mCherry2, FusionRed, mRFP, mCrimson3, and dKatushka; and seven yellow FPs: SYFP2, Venus, mCitrine, YPet, mClover3, mTopaz, and EYFP) at the single-molecule level in living cells. These FPs were then used for gene locus measurements using FROS. Our results indicate that TetR-mCrimson3 (red) and TetR-EYFP (yellow) had better properties for visualizing gene loci than the other TetR-FPs. Furthermore, fixation procedures affected the clustering of diffusing TetR-FPs and altered the locations of the TetR-FP foci. Fixation with formaldehyde consistently disrupted proper DNA locus observations using TetR-FPs. Notably, the foci measured using TetR-mCrimson3 remained close to their original positions in live cells after glyoxal fixation. This in vivo study provides a cell-imaging guide for the use of FPs for gene-locus observation in E. coli and a scheme for evaluating the use of FPs for other cell-imaging purposes.

Fluorescent tools for the standardized work in Gram-negative bacteria

Standardized and thoroughly characterized genetic tools are a prerequisite for studying cellular processes to ensure the reusability and consistency of experimental results. The discovery of fluorescent proteins (FPs) represents a milestone in the development of genetic reporters for monitoring transcription or protein localization in vivo. FPs have revolutionized our understanding of cellular dynamics by enabling the real-time visualization and tracking of biological processes. Despite these advancements, challenges remain in the appropriate use of FPs, specifically regarding their proper application, protein turnover dynamics, and the undesired disruption of cellular functions. Here, we systematically compared a comprehensive set of 15 FPs and assessed their performance in vivo by focusing on key parameters, such as signal over background ratios and protein stability rates, using the Gram-negative model organism Salmonella enterica as a representative host. We evaluated four protein degradation tags in both plasmid- and genome-based systems and our findings highlight the necessity of introducing degradation tags to analyze time-sensitive cellular processes. We demonstrate that the gain of dynamics mediated by the addition of degradation tags impacts the cell-to-cell heterogeneity of plasmid-based but not genome-based reporters. Finally, we probe the applicability of FPs for protein localization studies in living cells using standard and super-resolution fluorescence microscopy. In summary, our study underscores the importance of careful FP selection and paves the way for the development of improved genetic reporters to enhance the reproducibility and reliability of fluorescence-based research in Gram-negative bacteria and beyond.

EYFP fusions to HD-Zip IV transcription factors enhance their stability and lead to phenotypic changes in Arabidopsis

ABSTRACT Green fluorescent protein (GFP) and its derivatives are extensively used for labeling cells, monitoring gene expression and/or tracking the localization or interactions of proteins. Previous reports of detrimental effects of fluorescent protein (FP) expression include cytotoxicity and interference with fusion protein function or localization. Only a few studies have documented the fluorescent tag-specific effects in plants. Here, we show that placing an enhanced yellow FP (EYFP) tag on the amino-terminus of GLABRA2 (GL2) and PROTODERMAL FACTOR2 (PDF2), two developmentally important HD-Zip IV transcription factors from Arabidopsis, enhances their protein stability. Additionally, expression of EYFP:GL2 not only rescued the gl2 null mutant but also resulted in the abnormal development of abaxially curled leaves associated with EYFP-tag induced GL2 overexpression. Our study raises concerns on the use of FPs regarding their effects on the native properties of target proteins as well as biological consequences of fusion protein expression on morphology.

Live-Cell Imaging of RNA Transport in Axons of Cultured Primary Neurons.

The use of fluorescent proteins has revolutionized the study of protein localization and transport. However, the visualization of other molecules and specifically RNA during live-cell imaging remains challenging. In this chapter, we provide guidance to the available methods, their advantages and drawbacks as well as provide a detailed protocol for the detection of RNA transport using the MS2/PP7-split-Venus system for background-free RNA imaging.

Indirect Methods To Measure Unfolded Proteins In Living Cells Using Fluorescent Proteins.

In the study of the unfolded protein response pathway, it is essential to determine the amount of unfolded proteins that the cell is accumulating. Besides being essential it is one of the most challenging technique because of the difficulty to detect unfolded proteins without producing protein denaturation with the method itself. Thus, indirect methods became very useful as the use of fluorescent proteins. In this chapter, we present some of the most used methods to indirectly measure protein folding in living cells using fluorescent proteins.

Color-Coded Imaging of Cancer and Stromal-Cell Interaction in the Pancreatic-Cancer Tumor Microenvironment (TME).

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor (Yang et al., Cancer Res 64:8651-8656, 2004; Yang et al. J Cell Biochem 106: 279-284, 2009). The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or non-colored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Color-Coded Imaging of the Tumor Microenvironment (TME) in Human Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally distinct fluorescent proteins can be used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored nude mice or noncolored nude mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Using this model, specific cancer cell or stromal cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment.

Monitoring Gene Expression during a Galleria mellonella Bacterial Infection.

Galleria mellonella larvae are an alternative in vivo model that has been extensively used to study the virulence and pathogenicity of different bacteria due to its practicality and lack of ethical constraints. However, the larvae possess intrinsic autofluorescence that obstructs the use of fluorescent proteins to study bacterial infections, hence better methodologies are needed. Here, we report the construction of a promoter probe vector with bioluminescence expression as well as the optimization of a total bacterial RNA extraction protocol to enhance the monitoring of in vivo infections. By employing the vector to construct different gene promoter fusions, variable gene expression levels were efficiently measured in G. mellonella larvae at various time points during the course of infection and without much manipulation of the larvae. Additionally, our optimized RNA extraction protocol facilitates the study of transcriptional gene levels during an in vivo infection. The proposed methodologies will greatly benefit bacterial infection studies as they can contribute to a better understanding of the in vivo infection processes and pathogen–mammalian host interactions.

Expression of a Malassezia Codon Optimized mCherry Fluorescent Protein in a Bicistronic Vector.

The use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labeling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

Back to the Future: Genetically Encoded Fluorescent Proteins as Inert Tracers of the Intracellular Environment.

Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.

Presenting a codon-optimized palette of fluorescent proteins for use in Candida albicans

Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. These fluorophores are often developed for use in mammalian systems, with incremental enhancements or new versions published frequently. However, the successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms. In this contribution, we present a palette of Candida-optimized fluorescent proteins ranging from cyan to red and assess their application potential. We also compare a range of reported expression optimization techniques, and find that none of these strategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. In addition to reporting new fluorescent protein variants for applications in Candida albicans, our work highlights the ongoing challenges in optimizing protein expression in heterologous systems.

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes.

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Light Microscopy in Trypanosomes: Use of Fluorescent Proteins and Tags.

Fluorescence microscopy enables the localization of proteins to specific structures within a cell which have either been fused to a fluorescence protein or detected by immunofluorescence. Here, we describe the various procedures that can be used to prepare both the procyclic form and bloodstream form of the human pathogen Trypanosoma brucei for fluorescence microscopy. The choice of procedure to be used is determined by various parameters, including protein characteristics and the scientific question being investigated.

Self-assembling as regular nanoparticles dramatically minimizes photobleaching of tumour-targeted GFP

Fluorescent proteins are useful imaging and theranostic agents, but their potential superiority over alternative dyes is weakened by substantial photobleaching under irradiation. Enhancing protein photostability has been attempted through diverse strategies, with irregular results and limited applicability. In this context, we wondered if the controlled oligomerization of Green Fluorescent Protein (GFP) as nanoscale supramolecular complexes could stabilize the fluorophore through the newly formed protein-protein contacts, and thus, enhance its global photostability. For that, we have here analyzed the photobleaching profile of several GFP versions, engineered to self-assemble as tumour-homing nanoparticles with different targeting, size and structural stability. This has been done under prolonged irradiation in confocal laser scanning microscopy and by small-angle X-ray scattering. The results show that the oligomerization of GFP at the nanoscale enhances, by more than seven-fold, the stability of fluorescence emission. Interestingly, GFP nanoparticles are much more resistant to X-ray damage than the building block counterparts, indicating that the gained photostability is linked to enhanced structural resistance to radiation. Therefore, the controlled oligomerization of self-assembling fluorescent proteins as protein nanoparticles is a simple, versatile and powerful method to enhance their photostability for uses in precision imaging and therapy. Statement of significanceFluorescent protein assembly into regular and highly symmetric nanoscale structures has been identified to confer enhanced structural stability against radiation stresses dramatically reducing their photobleaching. Being this the main bottleneck in the use of fluorescent proteins for imaging and theranostics, this protein architecture engineering principle appears as a powerful method to enhance their photostability for a broad applicability in precision imaging, drug delivery and theranostics.

Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil.

The plant immune system is made up of a complex response network that involves several lines of defense to fight invading pathogens. Fungal plant pathogens on the other hand, have evolved a range of ways to infect their host. The interaction between Ascochyta lentis and two lentil genotypes was explored to investigate the progression of ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation system for A. lentis by constructing a new binary vector, pATMT-GpdGFP, for the constitutive expression of green fluorescent protein (EGFP). Green fluorescence was used as a highly efficient vital marker to study the developmental changes in A. lentis during AB disease progression on the susceptible and resistant lentil accessions, ILL6002 and ILL7537, respectively. The initial infection stages were similar in both the resistant and susceptible accessions where A. lentis uses infection structures such as germ tubes and appressoria to gain entry into the host while the host uses defense mechanisms to prevent pathogen entry. Penetration was observed at the junctions between neighbouring epidermal cells and occasionally, through the stomata. The pathogen attempted to penetrate and colonize ILL7537, but further fungal advancement appeared to be halted, and A. lentis did not enter the mesophyll. Successful entry and colonization of ILL6002 coincided with structural changes in A. lentis and the onset of necrotic lesions 5–7 days post inoculation. Once inside the leaf, A. lentis continued to grow, colonizing all parts of the leaf followed by plant cell collapse. Pycnidia-bearing spores appeared 14 days post inoculation, which marks the completion of the infection cycle. The use of fluorescent proteins in plant pathogenic fungi together with confocal laser scanning microscopy, provide a valuable tool to study the intracellular dynamics, colonization strategy and infection mechanisms during plant-pathogen interaction.

A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)

ABSTRACTEukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that “late Golgi” and “early endosomes,” two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities.

Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

Cell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individual Escherichia coli cells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae.

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.