Challenging kinases activities within single cells.

Jean-Francois Bodart, Universite des Sciences et Technologies de Lille, Villeneuve d'Ascq, France

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 MAPK/ ERK, whose activity is a key node in signal transduction pathways and can drive cells into various processes. There is a constant need for better tools to analyze kinases in vivo to overcome the shortcomings of classical methodologies, and to detect even the slightest variations of their activities. Here we report the optimization of 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: (1) fluorophores and (2) 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. We characterized those new biosensors, which exhibited differences and discussed properties of genetically encoded kinase reporters as well as opportunities for using those tools.