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Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case...

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Autores principales: Vandame, Pauline, Spriet, Corentin, Riquet, Franck, Trinel, Dave, Cailliau-Maggio, Katia, Bodart, Jean-François
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Diversity Preservation International (MDPI) 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926607/
https://www.ncbi.nlm.nih.gov/pubmed/24434874
http://dx.doi.org/10.3390/s140101140
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author Vandame, Pauline
Spriet, Corentin
Riquet, Franck
Trinel, Dave
Cailliau-Maggio, Katia
Bodart, Jean-François
author_facet Vandame, Pauline
Spriet, Corentin
Riquet, Franck
Trinel, Dave
Cailliau-Maggio, Katia
Bodart, Jean-François
author_sort Vandame, Pauline
collection PubMed
description Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.
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spelling pubmed-39266072014-02-18 Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements Vandame, Pauline Spriet, Corentin Riquet, Franck Trinel, Dave Cailliau-Maggio, Katia Bodart, Jean-François Sensors (Basel) Article Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties. Molecular Diversity Preservation International (MDPI) 2014-01-10 /pmc/articles/PMC3926607/ /pubmed/24434874 http://dx.doi.org/10.3390/s140101140 Text en © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Vandame, Pauline
Spriet, Corentin
Riquet, Franck
Trinel, Dave
Cailliau-Maggio, Katia
Bodart, Jean-François
Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title_full Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title_fullStr Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title_full_unstemmed Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title_short Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements
title_sort optimization of erk activity biosensors for both ratiometric and lifetime fret measurements
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926607/
https://www.ncbi.nlm.nih.gov/pubmed/24434874
http://dx.doi.org/10.3390/s140101140
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