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

被引:39
作者
Demeautis, Claire [1 ,2 ]
Sipieter, Francois [3 ,4 ,5 ]
Roul, Julien [1 ,2 ,10 ]
Chapuis, Catherine [1 ,2 ]
Padilla-Parra, Sergi [6 ,7 ]
Riquet, Franck B. [3 ,4 ,8 ]
Tramier, Marc [1 ,2 ,9 ]
机构
[1] CNRS, UMR 6290, Rennes, France
[2] Univ Rennes 1, Inst Genet & Dev Rennes, Rennes, France
[3] Univ Ghent, Dept Biomed Mol Biol, Mol Signaling & Cell Death Unit, Ghent, Belgium
[4] VIB, Mol Signaling & Cell Death Unit, Inflammat Res Ctr, Ghent, Belgium
[5] CNRS, UMR 8523, Equipe Biophoton Cellulaire Fonct, Lab Phys Lasers Atomes & Mol PhLAM, Villeneuve Dascq, France
[6] Univ Oxford, Div Struct Biol, Henry Wellcome Bldg Genom Med, Oxford OX3 7BN, England
[7] Univ Oxford, Wellcome Trust Ctr Human Genet, Cellular Imaging Core, Oxford, England
[8] Univ Lille 1, CNRS, UMR 8576, Unite Glycobiol Struct & Fonct, Villeneuve Dascq, France
[9] Univ Rennes 1, Biosit, Microscopy Rennes Imaging Ctr, Rennes, France
[10] Univ Toulouse, Lab Anal & Architecture Syst, 7 Ave Colonel Roche BP 54200, F-31031 Toulouse, France
关键词
GROWTH-FACTOR RECEPTOR; ACTIVATED PROTEIN-KINASE; SIGNAL-REGULATED KINASE; FLUORESCENT PROTEIN; MAP KINASE; A ACTIVITY; CYCLIC-AMP; CAMP; PATHWAYS; RAF;
D O I
10.1038/srep41026
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
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页数:14
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