scholarly journals A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

2020 ◽  
Vol 21 (7) ◽  
pp. 2572 ◽  
Author(s):  
Urooj Fatima ◽  
Mohammad K. Okla ◽  
Mohd Mohsin ◽  
Ruphi Naz ◽  
Walid Soufan ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Real Time ◽  
Fluorescent Proteins ◽  
Essential Element ◽  
Resonance Energy ◽  
Primary Source ◽  
Essential Amino Acids ◽  
Living Cells ◽  
Live Cells ◽  
Non Invasive

Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N- and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

scholarly journals Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 430
Author(s):  
Amreen ◽  
Hayssam M. Ali ◽  
Mohammad Ahmad ◽  
Mohamed Z. M. Salem ◽  
Altaf Ahmad
Keyword(s):  
Hydrogen Peroxide ◽  
Real Time ◽  
Mammalian Cells ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Living Cells ◽  
Stress Conditions ◽  
Live Cells ◽  
Cell Physiology ◽  
Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

scholarly journals cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins

Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 39
Author(s):  
Namdoo Kim ◽  
Seunghan Shin ◽  
Se Won Bae
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Intracellular Signaling ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Early Stage ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adenosine Monophosphate ◽  
Non Invasive

Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.

scholarly journals A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1541 ◽  
Author(s):  
Masoud Ramuz ◽  
Alveera Hasan ◽  
Lena Gruscheski ◽  
Ivan Diakonov ◽  
Nikoleta Pavlaki ◽  
...  
Keyword(s):  
Real Time ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Induced Pluripotent Stem Cell ◽  
Software Tool ◽  
Live Cells ◽  
Non Invasive ◽  
Significant Difference ◽  
Induced Pluripotent

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

A novel microfluidic microelectrode chip for a significantly enhanced monitoring of NPY-receptor activation in live mode

Lab on a Chip ◽  
2017 ◽  
Vol 17 (24) ◽  
pp. 4294-4302 ◽  
Author(s):  
Franziska D. Zitzmann ◽  
Heinz-Georg Jahnke ◽  
Felix Nitschke ◽  
Annette G. Beck-Sickinger ◽  
Bernd Abel ◽  
...  
Keyword(s):  
Real Time ◽  
Microfluidic Chip ◽  
Microelectrode Array ◽  
Fem Simulation ◽  
Receptor Activation ◽  
Living Cells ◽  
Real Time Monitoring ◽  
Non Invasive ◽  
Npy Receptor ◽  
Simulation Based

We present a FEM simulation based step-by-step development of a microelectrode array integrated into a microfluidic chip for the non-invasive real-time monitoring of living cells.

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 GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

scholarly journals AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

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.

scholarly journals FRET Microscopy in Yeast

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 122 ◽  
Author(s):  
Skruzny ◽  
Pohl ◽  
Abella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Bioactive Molecules ◽  
Biophysical Processes ◽  
Microscopy Method ◽  
Fret Biosensors

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

A supersensitive biosensor based on MoS2 nanosheet arrays for the real-time detection of H2O2 secreted from living cells

2019 ◽  
Vol 55 (65) ◽  
pp. 9653-9656 ◽  
Author(s):  
Huitong Du ◽  
Xinyue Zhang ◽  
Zhe Liu ◽  
Fengli Qu
Keyword(s):  
Real Time ◽  
Living Cells ◽  
Sensitive Detection ◽  
Live Cells ◽  
The Real ◽  
Highly Sensitive ◽  
Nanosheet Arrays ◽  
Mos2 Nanosheet ◽  
Real Time Detection ◽  
Highly Sensitive Detection

A self-supported MoS2 nanosheet biosensor for highly sensitive detection of H2O2 secreted from live cells.

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