scholarly journals Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Leigh A. Stoddart ◽  
Nicholas D. Kindon ◽  
Omolade Otun ◽  
Clare R. Harwood ◽  
Foteini Patera ◽  
...  
Keyword(s):  
Rational Design ◽  
Live Cells ◽  
Fluorescent Labelling ◽  
Non Invasive ◽  
Cellular Environment ◽  
Selective Approach ◽  
Fluorescent Ligands ◽  
Fluorescent Moiety ◽  
Fluorescent Tag ◽  
G Protein Coupled

AbstractTo study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

scholarly journals Ligand-directed covalent labelling of a GPCR with a fluorescent tag

2020 ◽  
Author(s):  
Leigh A Stoddart ◽  
Nicholas D Kindon ◽  
Omolade Otun ◽  
Clare R. Harwood ◽  
Foteini Patera ◽  
...  
Keyword(s):  
Rational Design ◽  
Living Cells ◽  
Model System ◽  
Fluorescent Labelling ◽  
Wild Type ◽  
Non Invasive ◽  
Selective Approach ◽  
Fluorescent Moiety ◽  
Fluorescent Tag ◽  
G Protein Coupled

AbstractHere, we describe rational design of a compound that covalently and selectively labels a G protein-coupled receptor (GPCR) in living cells with a fluorescent moiety. Using wild-type adenosine A2A receptor as a model system, we show fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

scholarly journals Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3575
Author(s):  
Shenggang Wang ◽  
Yue Huang ◽  
Xiangming Guan
Keyword(s):  
Fluorescent Probes ◽  
Biological System ◽  
High Sensitivity ◽  
Intracellular Distribution ◽  
Live Cell ◽  
Live Cells ◽  
High Demand ◽  
Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

scholarly journals Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

scholarly journals Holographic optical trapping Raman micro-spectroscopy for non-invasive measurement and manipulation of live cells

2018 ◽  
Vol 26 (19) ◽  
pp. 25211 ◽  
Author(s):  
Faris Sinjab ◽  
Dennis Awuah ◽  
Graham Gibson ◽  
Miles Padgett ◽  
Amir M. Ghaemmaghami ◽  
...  
Keyword(s):  
Optical Trapping ◽  
Live Cells ◽  
Non Invasive ◽  
Invasive Measurement

scholarly journals Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

2018 ◽  
Vol 293 (19) ◽  
pp. 7466-7473 ◽  
Author(s):  
Qingwen Wan ◽  
Najeah Okashah ◽  
Asuka Inoue ◽  
Rony Nehmé ◽  
Byron Carpenter ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Live Cells ◽  
G Protein Coupled

Rational design of an ESIPT-based fluorescent probe for selectively monitoring glutathione in live cells and zebrafish

2020 ◽  
Vol 238 ◽  
pp. 118429 ◽  
Author(s):  
Ya-Long Zheng ◽  
Han-Chen Zhang ◽  
Di-Hua Tian ◽  
De-Chen Duan ◽  
Fang Dai ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Rational Design ◽  
Live Cells

Rational design of a lysosomal-targeted ratiometric two-photon fluorescent probe for imaging hydrogen polysulfides in live cells

2020 ◽  
Vol 173 ◽  
pp. 107877 ◽  
Author(s):  
Qingxin Han ◽  
Xuan Liu ◽  
Xuechuan Wang ◽  
Ruojun Yin ◽  
Huie Jiang ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Rational Design ◽  
Live Cells ◽  
Two Photon

scholarly journals Targeted Molecular Imaging Using Aptamers in Cancer

2018 ◽  
Vol 11 (3) ◽  
pp. 71 ◽  
Author(s):  
Sorah Yoon ◽  
John Rossi
Keyword(s):  
Wide Spectrum ◽  
Rna Aptamers ◽  
Tissue Imaging ◽  
Live Cells ◽  
Target Cells ◽  
New Class ◽  
Non Invasive ◽  
Resolution Imaging ◽  
Tissue Specimens

Imaging is not only seeing, but also believing. For targeted imaging modalities, nucleic acid aptamers have features such as superior recognition of structural epitopes and quick uptake in target cells. This explains the emergence of an evolved new class of aptamers into a wide spectrum of imaging applications over the last decade. Genetically encoded biosensors tagged with fluorescent RNA aptamers have been developed as intracellular imaging tools to understand cellular signaling and physiology in live cells. Cancer-specific aptamers labeled with fluorescence have been used for assessment of clinical tissue specimens. Aptamers conjugated with gold nanoparticles have been employed to develop innovative mass spectrometry tissue imaging. Also, use of chemically conjugated cancer-specific aptamers as probes for non-invasive and high-resolution imaging has been transformative for in vivo imaging in multiple cancers.

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