scholarly journals Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real time

2021 ◽  
Vol 134 (7) ◽  
Author(s):  
Kévin Leguay ◽  
Barbara Decelle ◽  
Yu Yan He ◽  
Anthony Pagniez ◽  
Mireille Hogue ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Process ◽  
Cancer Therapies ◽  
Erm Proteins ◽  
Metastatic Cells ◽  
Precise Understanding ◽  
The Impact

ABSTRACT Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

scholarly journals Development of conformational BRET biosensors that monitor Ezrin, Radixin and Moesin activation in real-time

2020 ◽  
Author(s):  
Kévin Leguay ◽  
Barbara Decelle ◽  
Yu Yan He ◽  
Mireille Hogue ◽  
Hiroyuki Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Throughput Screening ◽  
Activation Process ◽  
Cancer Therapies ◽  
Erm Proteins ◽  
Metastatic Cells ◽  
Precise Understanding ◽  
The Family ◽  
The Impact ◽  
Upstream Regulators

SUMMARYEzrin, Radixin and Moesin compose the family of ERM proteins. They link actin and microtubule filaments to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape as well as providing new opportunities for the development of innovative cancer therapies. ERMs are believed to exist in two main conformational states: a close-inactive conformation in the cytoplasm that is recruited at the plasma membrane to convert into an open-active conformation. Here, we developed and characterized novel BRET-based conformational biosensors, compatible with high throughput screening, that monitor Ezrin, Radixin or Moesin individual activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to uncover a novel aspect of ERM activation process involving a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

scholarly journals Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase

2018 ◽  
Vol 23 (9) ◽  
pp. 974-981
Author(s):  
Yu-Chi Juang ◽  
Xavier Fradera ◽  
Yongxin Han ◽  
Anthony William Partridge
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Histidine Decarboxylase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gastric Ulcers ◽  
Statistical Parameters ◽  
Time Resolved ◽  
Neurological Response

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z′ score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure–activity relationships.

High-throughput screening of TRPV1 ligands in the light of the Bioluminescence Resonance Energy Transfer technique

2021 ◽  
pp. MOLPHARM-AR-2021-000271
Author(s):  
Yann Chappe ◽  
Pauline Michel ◽  
Alexandre Joushomme ◽  
Solène Barbeau ◽  
Sandra Pierredon ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer

scholarly journals GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

2020 ◽  
Vol 117 (25) ◽  
pp. 14522-14531
Author(s):  
Allison Anderson ◽  
Ikuo Masuho ◽  
Ezequiel Marron Fernandez de Velasco ◽  
Atsushi Nakano ◽  
Lutz Birnbaumer ◽  
...  
Keyword(s):  
G Protein ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Cardiac Pacemaker ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Protein Isoforms ◽  
Minimal Impact ◽  
Signaling Dynamics ◽  
The Impact

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+(GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαoover Gαias a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαoor Gαi2was consistent with these findings. Gαi2ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαoablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαosubstrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Dynamic receptor superstructures at the plasma membrane

1997 ◽  
Vol 30 (1) ◽  
pp. 67-106 ◽  
Author(s):  
S. DAMJANOVICH ◽  
R. GÁSPÁR, Jr. ◽  
C. PIERI
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Physical Parameters ◽  
Conformational States ◽  
Lipid Domain ◽  
Receptor Clusters

1. INTRODUCTION 681.1 Receptor patterns in the plasma membrane 681.2 Different types of receptor patterns 712. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 732.1 Fluorescence resonance energy transfer 732.2 Flow cytometric energy transfer measurement 782.3 Fluorescence anisotropy and energy transfer 792.4 Photobleaching energy transfer on single cells 812.5 Two-dimensional mapping of receptor superstructures 822.6 Detecting single receptor molecules 852.7 Chemical identification of receptor clusters 862.8 Electron microscopy 872.9 Scanning force microscopy 883. CONFORMATIONAL STATES OF RECEPTORS 903.1 Multi-subunit receptor structures 903.2 Physical parameters influencing conformational states 913.3 Chemical interactions and receptor conformations 924. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 934.1 Synthesis of receptors and their localization in the plasma membrane 934.2 Lipid domain structure of the plasma membrane 944.3 The validity of the Singer–Nicolson model 945. CONCLUSIONS 966. ACKNOWLEDGEMENTS 967. REFERENCES 97

scholarly journals Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

2008 ◽  
Vol 19 (10) ◽  
pp. 4366-4373 ◽  
Author(s):  
Xinxin Gao ◽  
Jin Zhang
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Lipid Rafts ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Analysis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

scholarly journals Tracking the sarcoplasmic reticulum membrane voltage in muscle with a FRET biosensor

2018 ◽  
Vol 150 (8) ◽  
pp. 1163-1177 ◽  
Author(s):  
Colline Sanchez ◽  
Christine Berthier ◽  
Bruno Allard ◽  
Jimmy Perrot ◽  
Clément Bouvard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ryanodine Receptor ◽  
Striated Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Voltage ◽  
Voltage Sensitivity ◽  
Cell Functions

Ion channel activity in the plasma membrane of living cells generates voltage changes that are critical for numerous biological functions. The membrane of the endoplasmic/sarcoplasmic reticulum (ER/SR) is also endowed with ion channels, but whether changes in its voltage occur during cellular activity has remained ambiguous. This issue is critical for cell functions that depend on a Ca2+ flux across the reticulum membrane. This is the case for contraction of striated muscle, which is triggered by opening of ryanodine receptor Ca2+ release channels in the SR membrane in response to depolarization of the transverse invaginations of the plasma membrane (the t-tubules). Here, we use targeted expression of voltage-sensitive fluorescence resonance energy transfer (FRET) probes of the Mermaid family in differentiated muscle fibers to determine whether changes in SR membrane voltage occur during depolarization–contraction coupling. In the absence of an SR targeting sequence, FRET signals from probes present in the t-tubule membrane allow calibration of the voltage sensitivity and amplitude of the response to voltage-clamp pulses. Successful SR targeting of the probes was achieved using an N-terminal domain of triadin, which completely eliminates voltage-clamp–activated FRET signals from the t-tubule membrane of transfected fibers. In fibers expressing SR-targeted Mermaid probes, activation of SR Ca2+ release in the presence of intracellular ethyleneglycol-bis(β-amino-ethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) results in an accompanying FRET signal. We find that this signal results from pH sensitivity of the probe, which detects cytosolic acidification because of the release of protons upon Ca2+ binding to EGTA. When EGTA is substituted with either 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or the contraction blocker N-benzyl-p-toluene sulfonamide, we find no indication of a substantial change in the FRET response caused by a voltage change. These results suggest that the ryanodine receptor–mediated SR Ca2+ efflux is well balanced by concomitant counterion currents across the SR membrane.

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