scholarly journals mCherry contains a fluorescent protein isoform that interferes with its reporter function

2021 ◽  
Author(s):  
Maxime Fages-Lartaud ◽  
Lisa Tietze ◽  
Florence Elie ◽  
Rahmi Lale ◽  
Martin Frank Hohmann-Marriott
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Red Fluorescent Protein ◽  
Functional Protein ◽  
Red Fluorescent Proteins ◽  
Expression Studies ◽  
Protein Isoform

AbstractFluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

ESTABLISHMENT OF VISIBLE ANIMAL METASTASIS MODELS FOR HUMAN NASOPHARYNGEAL CARCINOMA BASED ON A FAR-RED FLUORESCENT PROTEIN

2012 ◽  
Vol 05 (03) ◽  
pp. 1250019 ◽  
Author(s):  
YING ZHENG ◽  
CHUAN HUANG ◽  
ZHIYONG CHENG ◽  
MIN CHEN
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Animal Models ◽  
Tumor Growth ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Vectors ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Metastasis Model

Background and aims: The spectral properties of enhanced green fluorescent protein (EGFP) used in current visualizable animal models for nasopharyngeal carcinoma (NPC) result in a limited imaging depth. Far-red fluorescent proteins have optimal spectral wavelengths that allow deep tissue penetration, thus are well-suited for the imaging of tumor growth and metastases in live animals. This study aims to establish an imageable animal model of NPC using far-red fluorescent proteins. Methods: Eukaryotic expression vectors of far-red fluorescent proteins, mLumin and Katushka S158A, were separately transfected into 5-8F NPC cells, and cell lines stably expressing the far-red fluorescent proteins were obtained. These cells were intraperitoneally or intravenously injected into mice, and their tumorigenic and metastatic potential were examined through fluorescence imaging. Finally, factors affecting their tumorigenic ability were further assessed through testing side population (SP) cells proportion by flow cytometry. Results: NPC cell line with high tumorigenicity and metastasis (5-8F-mL2) was screened out, which stably expressed far-red fluorescent protein. Intraperitoneal and intravenous injection of 5-8F-mL2 cells resulted in an abdomen metastasis model and a lung metastasis model. In addition, NPC cell line without tumorigenicity (5-8F-Katushka S158A) was screened out. The percentage of SP cells between 5-8F-mL2 and 5-8F-Katushka S158A was found different, suggesting that the SP cell proportion may play a key role in the determination of cell tumorigenic ability. Conclusion: We successfully established animal models for NPC with high tumorigenicity and metastasis using a super-bright far-red fluorescent protein. Owing to the super-brightness and excellent wavelength parameters, these models may be applied as useful tools for intuitive and efficient monitoring of tumor growth and metastasis, as well as assessing the efficacy of nasopharyngeal cancer drugs.

scholarly journals mBeRFP, a versatile fluorescent tool to enhance multichannel live imaging and its applications

2022 ◽  
Author(s):  
Emmanuel Martin ◽  
Magali Suzanne
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Live Imaging ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Living Organisms ◽  
Illumination Source

Cell and developmental biology increasingly require live imaging of protein dynamics in cells, tissues or living organisms. Thanks to the discovery and the development of a panel of fluorescent proteins over the last decades, live imaging has become a powerful and commonly used approach. However, multicolor live imaging remains challenging. The generation of long Stokes shift red fluorescent proteins, such as mBeRFP, offers interesting new perspectives to bypass this limitation. Here, we constructed a set of mBeRFP-expressing vectors and provided a detailed characterization of this fluorescent protein for in vivo live imaging and its applications in Drosophila. Briefly, we showed that a single illumination source is sufficient to simultaneously stimulate mBeRFP and GFP. We demonstrated that mBeRFP can be easily combined with classical green and red fluorescent protein without any crosstalk. We also showed that the low photobleaching of mBeRFP is suitable for live imaging, and that this protein can be used for quantitative applications such as FRAP or laser ablation. Finally, we believe that this fluorescent protein, with the set of new possibilities it offers, constitutes an important tool for cell, developmental and mechano biologists in their current research.

scholarly journals A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

2020 ◽  
Author(s):  
Benjamin B. Kim ◽  
Haodi Wu ◽  
Yukun A. Hao ◽  
Michael Pan ◽  
Mariya Chavarha ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
High Gain ◽  
Motion Artifacts ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
Transmembrane Voltage ◽  
Site Mutagenesis ◽  
Green Fluorescent

AbstractA ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP3. Among red fluorescent proteins tested, mCyRFP3 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP3 (ASAP3-R3) provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-R3 to visualize membrane voltage changes throughout the cell cycle of motile cells.

scholarly journals Optimization of the DsRed fluorescent protein for use in Mycobacterium tuberculosis

2018 ◽  
Author(s):  
Paul Carroll ◽  
Julian Muwanguzi-Karugaba ◽  
Tanya Parish
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Full Length ◽  
Red Fluorescent Protein ◽  
Red Fluorescent Proteins ◽  
N Terminus

ABSTRACTObjectiveWe have previously codon-optimized a number of red fluorescent proteins for use in Mycobacterium tuberculosis (mCherry, tdTomato, Turbo-635). We aimed to expand this repertoire to include DsRed, another widely used and flexible red fluorescent protein.ResultsWe generated expression constructs with a full length DsRed under the control of one of three strong, constitutive promoters (Phsp60, PrpsA or PG13) for use in mycobacteria. We confirmed that full length DsRed (225 amino acids) was expressed and fluoresced brightly. In contrast to mCherry, truncated versions of DsRed lacking several amino acids at the N-terminus were not functional. Thus, we have expanded the repertoire of optimized fluorescent proteins for mycobacteria.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals Acclimatization of symbiotic corals to mesophotic light environments through wavelength transformation by fluorescent protein pigments

2017 ◽  
Vol 284 (1858) ◽  
pp. 20170320 ◽  
Author(s):  
Edward G. Smith ◽  
Cecilia D'Angelo ◽  
Yoni Sharon ◽  
Dan Tchernov ◽  
Joerg Wiedenmann
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Environmental Gradients ◽  
Red Light ◽  
Spectral Composition ◽  
Light Environment ◽  
Homogeneous Distribution ◽  
Red Fluorescent Proteins ◽  
Ecological Importance ◽  
Colour Morphs

The depth distribution of reef-building corals exposes their photosynthetic symbionts of the genus Symbiodinium to extreme gradients in the intensity and spectral quality of the ambient light environment. Characterizing the mechanisms used by the coral holobiont to respond to the low intensity and reduced spectral composition of the light environment in deeper reefs (greater than 20 m) is fundamental to our understanding of the functioning and structure of reefs across depth gradients. Here, we demonstrate that host pigments, specifically photoconvertible red fluorescent proteins (pcRFPs), can promote coral adaptation/acclimatization to deeper-water light environments by transforming the prevalent blue light into orange-red light, which can penetrate deeper within zooxanthellae-containing tissues; this facilitates a more homogeneous distribution of photons across symbiont communities. The ecological importance of pcRFPs in deeper reefs is supported by the increasing proportion of red fluorescent corals with depth (measured down to 45 m) and increased survival of colour morphs with strong expression of pcRFPs in long-term light manipulation experiments. In addition to screening by host pigments from high light intensities in shallow water, the spectral transformation observed in deeper-water corals highlights the importance of GFP-like protein expression as an ecological mechanism to support the functioning of the coral– Symbiodinium association across steep environmental gradients.

Color morphs of the coral, Acropora tenuis, show different responses to environmental stress and different expression profiles of fluorescent-protein genes

2020 ◽  
Author(s):  
Noriyuki Satoh ◽  
Koji Kinjo ◽  
Kohei Shintaku ◽  
Daisuke Kezuka ◽  
Hiroo Ishimori ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Profiles ◽  
Biological Significance ◽  
Gene Expression Profiles ◽  
Gfp Expression ◽  
Red Fluorescent Proteins ◽  
Color Morphs ◽  
Acropora Tenuis ◽  
Green Fluorescent

ABSTRACTCorals of the family Acroporidae are key structural components of reefs that support the most diverse marine ecosystems. Due to increasing anthropogenic stresses, coral reefs are in decline. Along the coast of Okinawa, Japan, three different color morphs of Acropora tenuis have been recognized for decades. These include brown (N morph), yellow-green (G) and purple (P) forms. The tips of axial coral polyps exhibit specific fluorescence spectra. This attribute is inherited asexually, and color morphs do not change seasonally. In Okinawa Prefecture, during the summer of 2017, the N and P morphs experienced bleaching, in which some N morphs died while P morphs recovered. In contrast, G morphs successfully withstood the stress. Symbiotic dinoflagellates are essential symbiotic partners of scleractinian corals. Photosynthetic activity of symbionts was reduced in July in N and P morphs; however, the three color-morphs host similar sets of Clade-C zoothanthellae, suggesting that beaching of N and P morphs cannot be attributed to differences in symbiont clades. The decoded Acropora tenuis genome includes five genes for green fluorescent proteins (GFP), two for cyan fluorescent proteins (CFP), three for red fluorescent proteins (RFP), and seven genes for chromoprotein (ChrP). A summer survey of gene expression profiles demonstrated that (a) expression of CFP and REP was quite low in all three morphs, (b) P morphs expressed higher levels of ChrP, (c) both N and G morphs expressed GFP highly, and (d) GFP expression was reduced in N morphs, compared to G morphs, which maintained higher levels of GFP expression throughout the summer. Although further studies are required to understand the biological significance of these color morphs of Acropora tenuis, our results suggest that thermal stress resistance is modified by genetic mechanisms that coincidentally lead to diversification of color morphs.

scholarly journals Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

2015 ◽  
Vol 7 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Kevin M. Dean ◽  
Jennifer L. Lubbeck ◽  
Lloyd M. Davis ◽  
Chola K. Regmi ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Microfluidic Platform ◽  
Red Fluorescent Proteins ◽  
New Variant ◽  
Selection Of

We use a high-throughput microfluidic platform that sorts cells on the basis of fluorescent protein photostability to identify a new variant with improved photon output.

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