Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

Abstract

Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM.

Genetically Encoded FRET‐Based Tension Sensors

“…Tension sensor studies using FRET pairs with yellow/red (Austen et al., ; Price et al., ; Ringer et al., ), green/red (Kumar et al., ; LaCroix, Lynch, Berginski, & Hoffman, ), or blue/yellow (Borghi et al., ; Price et al., ) emission spectra have been published. For the special case of tension sensor multiplexing (i.e., the simultaneous measurement of two tension sensors) the two orthogonal pairs mTFP1/ShadowG and LSSmOrange/mKate2 are available (Demeautis et al., ; Ringer et al., ).…”

FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells

“…Recently, our team has developed a similar method by taking advantages of the LSSmOrange (Large Stoke Shift) and the dark fluorophore ShadowG [54]. We modified two substrate kinase biosensors, EKAR2G and AKAR4 (E-Kinase Activity Reporter type 2G for ERK and A-Kinase Activity Reporter type 4 for PKA), with a new pair of fluorophores mTFP1/ShadowG and LSSmOrange/mKate2, respectively.…”

Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis

“…The capability of sensing the activation of kinases, the activity of caspases, or the transport of second messengers as Ca 2+ or cAMP opened up the possibility of following biochemical reactions in real time and with a spatiotemporal resolution (Sizaire and Tramier, 2017;Greenwald et al, 2018;Palmer et al, 2011). The novel frontier of these probes consists in combining two or more FRET biosensors at a time to unravel the interdependence of signal transduction pathways, an approach known as multiplex FRET (Piljic and Schultz, 2008;Carlson and Campbell, 2009;Ai et al, 2008;Ding et al, 2011;Su et al, 2013;Demeautis et al, 2017;Ross et al, 2018).…”