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Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells
journal contribution
posted on 2017-07-04, 00:00 authored by Nur P. Damayanti, Kevin Buno, Yi Cui, Sherry L. Voytik-Harbin, Roberto Pili, Jennifer Freeman, Joseph M. K. IrudayarajPhosphorylation is an important post-translational
modification
implicated in cellular signaling and regulation. However, current
methods to study protein phosphorylation by various kinases lack spatiotemporal
resolution or the ability to simultaneously observe in real time the
activity of multiple kinases in live cells. We present a peptide biosensor
strategy with time correlated single photon counting-fluorescence
lifetime imaging (TCSPC-FLIM) to interrogate the spatial and temporal
dynamics of VEGFR-2 and AKT phosphorylation activity in real time
in live 2D and 3D cell culture models at single cell resolution. By
recording the increase in fluorescence lifetime due to a change in
the solvatochromic environment of the sensor upon phosphorylation,
we demonstrate that spatiotemporal maps of protein kinase activity
can be obtained. Our results suggest that fluorescence lifetime imaging
of peptide biosensors can be effectively and specifically used to
monitor and quantify phosphorylation of multiple kinases in live cells.
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TCSPC-FLIMfluorescence lifetimeReal-Time Multiplex Kinase Phosphorylation SensorsVEGFR -2spatiotemporal mapssolvatochromic environmentprotein kinase activitypeptide biosensorsphoton counting-fluorescence lifetime imagingcell resolutionkinases lack spatiotemporal resolutionAKT phosphorylation activitystudy protein phosphorylationfluorescence lifetime imaging2 Dpeptide biosensor strategy3 D cell culture modelspost-translational modificationLiving Cells Phosphorylation
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