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Sensors 2014, 14(1), 1140-1154; doi:10.3390/s140101140
Article

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

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Received: 8 November 2013; in revised form: 6 December 2013 / Accepted: 19 December 2013 / Published: 10 January 2014
(This article belongs to the Special Issue Fluorescent Biosensors)
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Abstract: 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.
Keywords: genetically-encoded biosensor; ERK; FRET genetically-encoded biosensor; ERK; FRET
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Vandame, P.; Spriet, C.; Riquet, F.; Trinel, D.; Cailliau-Maggio, K.; Bodart, J.-F. Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements. Sensors 2014, 14, 1140-1154.

AMA Style

Vandame P, Spriet C, Riquet F, Trinel D, Cailliau-Maggio K, Bodart J-F. Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements. Sensors. 2014; 14(1):1140-1154.

Chicago/Turabian Style

Vandame, Pauline; Spriet, Corentin; Riquet, Franck; Trinel, Dave; Cailliau-Maggio, Katia; Bodart, Jean-François. 2014. "Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements." Sensors 14, no. 1: 1140-1154.


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