Use of Kaede and Kikume Green–Red Fusions for Live Cell Imaging of G Protein-Coupled Receptors

2014 ◽  
pp. 139-156 ◽  
Author(s):  
Antje Schmidt ◽  
Burkhard Wiesner ◽  
Ralf Schülein ◽  
Anke Teichmann
Keyword(s):  
G Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
G Protein Coupled Receptors ◽  
Live Cell ◽  
G Protein Coupled

Live Cell Imaging of G Protein-Coupled Receptors

2012 ◽  
pp. 139-169 ◽  
Author(s):  
Anke Teichmann ◽  
Antje Schmidt ◽  
Burkhard Wiesner ◽  
Alexander Oksche ◽  
Ralf Schülein
Keyword(s):  
G Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
G Protein Coupled Receptors ◽  
Live Cell ◽  
G Protein Coupled

scholarly journals Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging

2020 ◽  
Vol 21 (21) ◽  
pp. 7880
Author(s):  
Leonore Mensching ◽  
Sebastian Rading ◽  
Viacheslav Nikolaev ◽  
Meliha Karsak
Keyword(s):  
Single Cell ◽  
G Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Single Cell Level ◽  
Cb2 Receptor ◽  
Cell Level ◽  
Cb2 Receptors ◽  
G Protein Coupled

G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3′,5′-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.

scholarly journals Comparison of Calcium Dynamics and Specific Features for G Protein–Coupled Receptor–Targeting Drugs Using Live Cell Imaging and Automated Analysis

2017 ◽  
Vol 22 (7) ◽  
pp. 848-858 ◽  
Author(s):  
Rishikesh Kumar Gupta ◽  
Sarpras Swain ◽  
Dinesh Kankanamge ◽  
Pantula Devi Priyanka ◽  
Ranjana Singh ◽  
...  
Keyword(s):  
Feature Extraction ◽  
G Protein ◽  
Drug Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Calcium Dynamics ◽  
A Cell ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are targets for designing a large fraction of the drugs in the pharmaceutical industry. For GPCR-targeting drug screening using cell-based assays, measurement of cytosolic calcium has been widely used to obtain dose–response profiles. However, it remains challenging to obtain drug-specific features due to cell-to-cell heterogeneity in drug–cell responses obtained from live cell imaging. Here, we present a framework combining live cell imaging of a cell population and a feature extraction method for classification of responses of drugs targeting GPCRs CXCR4 and α2AR. We measured the calcium dynamics using confocal microscopy and compared the responses for SDF-1α and norepinephrine. The results clearly show that the clustering patterns of responses for the two GPCRs are significantly different. Additionally, we show that different drugs targeting the same GPCR induce different calcium response signatures. We also implemented principal component analysis and k means for feature extraction and used nondominated (ND) sorting for ranking a group of drugs at various doses. The presented approach can be used to model a cell population as a mixture of subpopulations. It also offers specific advantages, such as higher spatial resolution, classification of responses, and ranking of drugs, potentially providing a platform for high-content drug screening.

scholarly journals Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist

2021 ◽  
Author(s):  
Stephanie E. Crilly ◽  
Wooree Ko ◽  
Zara Y. Weinberg ◽  
Manojkumar A. Puthenveedu
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Opioid Receptors ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Subcellular Location ◽  
Unanswered Question ◽  
Receptor Coupling ◽  
Δ Opioid Receptor ◽  
G Protein Coupled

AbstractThe prevailing model for the variety in drug responses is that they stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand can produce different GPCR active states based on the environment of receptors in cells is a fundamental unanswered question. Here we address this question using live cell imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neurons. We show that, although Golgi and surface pools of DOR regulated cAMP, the two pools engaged distinct conformational biosensors in response to the same ligand. Further, DOR recruited arrestin on the plasma membrane but not the Golgi. Our results suggest that the same agonist drives different conformations of a GPCR at different locations, allowing receptor coupling to distinct effectors at different locations.

scholarly journals Simple methods for quantifying super-resolved cortical actin

2021 ◽  
Author(s):  
Evelyn Garlick ◽  
Emma L Faulkner ◽  
Stephen J Briddon ◽  
Steven G Thomas
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Movement ◽  
G Protein Coupled Receptors ◽  
Cortical Actin ◽  
Surface Receptors ◽  
Actin Networks ◽  
Development And Validation ◽  
G Protein Coupled

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or corrals, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by both Super Resolved Radial Fluctuations (SRRF) and expansion microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 μm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

scholarly journals ARL3 activation requires the co-GEF BART and effector-mediated turnover

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yasmin ElMaghloob ◽  
Begoña Sot ◽  
Michael J McIlwraith ◽  
Esther Garcia ◽  
Tamas Yelland ◽  
...  
Keyword(s):  
G Protein ◽  
Primary Cilium ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Guanine Exchange Factor ◽  
Bona Fide ◽  
Modified Proteins ◽  
Adp Ribosylation Factor

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

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