scholarly journals Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Alexander I. Kostyuk ◽  
Maria-Armineh Tossounian ◽  
Anastasiya S. Panova ◽  
Marion Thauvin ◽  
Roman I. Raevskii ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Live Cells ◽  
Redox Probe ◽  
Fluorescent Biosensor ◽  
Hypohalous Acids ◽  
Injury Model ◽  
Inflammatory Processes

AbstractThe lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

scholarly journals Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

2021 ◽  
Author(s):  
Alexander I. Kostyuk ◽  
Maria-Armineh Tossounian ◽  
Anastasiya S. Panova ◽  
Marion Thauvin ◽  
Khadija Wahni ◽  
...  
Keyword(s):  
Hypochlorous Acid ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Reaction Rates ◽  
Immune Defense ◽  
Live Cells ◽  
E Coli ◽  
Injury Model ◽  
Inflammatory Processes ◽  
Genetically Encoded Biosensor

AbstractHypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

scholarly journals Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

2001 ◽  
Vol 21 (13) ◽  
pp. 4404-4412 ◽  
Author(s):  
David L. Stenoien ◽  
Anne C. Nye ◽  
Maureen G. Mancini ◽  
Kavita Patel ◽  
Martin Dutertre ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Real Time ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Living Cells ◽  
Live Cells ◽  
Creb Binding Protein ◽  
High Accumulation

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

2004 ◽  
Vol 10 (4) ◽  
pp. 442-448 ◽  
Author(s):  
Yingpei Zhang ◽  
Catherine Haskins ◽  
Marisa Lopez-Cruzan ◽  
Jianhua Zhang ◽  
Victoria E. Centonze ◽  
...  
Keyword(s):  
Real Time ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Subcellular Location ◽  
Live Cells ◽  
Excitation Peak ◽  
Caspase Substrate

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

scholarly journals Localization and Expression of MreB in Vibrio parahaemolyticus under Different Stresses

2008 ◽  
Vol 74 (22) ◽  
pp. 7016-7022 ◽  
Author(s):  
Shen-Wen Chiu ◽  
Shau-Yan Chen ◽  
Hin-chung Wong
Keyword(s):  
Stationary Phase ◽  
Vibrio Parahaemolyticus ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cellular Stress Response ◽  
Vbnc State

ABSTRACT MreB, the homolog of eukaryotic actin, may play a vital role when prokaryotes cope with stress by altering their spatial organization, including their morphology, subcellular architecture, and localization of macromolecules. This study investigates the behavior of MreB in Vibrio parahaemolyticus under various stresses. The behavior of MreB was probed using a yellow fluorescent protein-MreB conjugate in merodiploid strain SC9. Under normal growth conditions, MreB formed helical filaments in exponential-phase cells. The shape of starved or stationary-phase cells changed from rods to small spheroids. The cells differentiated into the viable but nonculturable (VBNC) state with small spherical cells via a “swelling-waning” process. In all cases, drastic remodeling of the MreB cytoskeleton was observed. MreB helices typically were loosened and fragmented into short filaments, arcs, and spots in bacteria under these stresses. The disintegrated MreB exhibited a strong tendency to attach to the cytoplasmic membrane. The expression of mreB generally declined in bacteria in the stationary phase and under starvation but was upregulated during the initial periods of cold shock and VBNC state differentiation and decreased afterwards. Our findings demonstrated the behavior of MreB in the morphological changes of V. parahaemolyticus under intrinsic or extrinsic stresses and may have important implications for studying the cellular stress response and aging.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

scholarly journals Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

2020 ◽  
Author(s):  
Song Lu ◽  
Dianbing Wang ◽  
Yu Hou ◽  
Dongge Guo ◽  
Yulin Deng ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Gene Interaction ◽  
Live Cells ◽  
Fluorescent Intensity ◽  
High Background ◽  
Repeat Sequences ◽  
Nuclear Location ◽  
Labeling System

Abstract The tagging of genomic loci in living cells provide visual evidence for the study of genomic spatial organization and gene interaction. CRISPR/dCas9 (clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci. However, the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout. This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS (target module) and scFv-sfGFP-NLS (signal module). We removed the nuclear location sequence (NLS) of the signal module and inserted two copies of EGFP into the signal module. The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm (N/C ratio) was decreased by 71%, and the ratio of the signal to the background (S/B ratio) was increased by 1.6 times. The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.

scholarly journals A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells

Sensors ◽  
2020 ◽  
Vol 20 (21) ◽  
pp. 5980
Author(s):  
Mei CQ Houser ◽  
Steven S Hou ◽  
Florian Perrin ◽  
Yuliia Turchyna ◽  
Brian J Bacskai ◽  
...  
Keyword(s):  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Proteolytic Processing ◽  
Live Cells ◽  
Cellular Events ◽  
Secretase Activity ◽  
Donor And Acceptor ◽  
Multiplexed Imaging

Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

High-Resolution Imaging of Redox Signaling in Live Cells Through an Oxidation-Sensitive Yellow Fluorescent Protein

2008 ◽  
Vol 1 (43) ◽  
pp. pl3-pl3 ◽  
Author(s):  
G. Maulucci ◽  
V. Labate ◽  
M. Mele ◽  
E. Panieri ◽  
G. Arcovito ◽  
...  
Keyword(s):  
High Resolution ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Redox Signaling ◽  
Live Cells ◽  
High Resolution Imaging ◽  
Resolution Imaging

Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy

2006 ◽  
Vol 34 (5) ◽  
pp. 675-678 ◽  
Author(s):  
G.A. Rutter ◽  
M.K. Loder ◽  
M.A. Ravier
Keyword(s):  
Confocal Microscopy ◽  
Cell Surface ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Β Cells ◽  
Large Dense Core Vesicles ◽  
Cell Depolarization

Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events (‘transient’ versus ‘full fusion’) are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 β-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

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