Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylat ion in cells based on fluorescence resonance energy transfer (FRET), EGFR p hosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-base d imaging method, the information about phosphorylation is contained only i n the (donor) GFP fluorescence lifetime and is independent of the antibody- derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell af ter photobleaching of the acceptor. We show that this calibration, by accep tor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows th e full quantitation of FRET efficiencies, and therefore the degree of expos ed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is rec eptor dimerisation [1-4] and concomitant activation of its intracellular ty rosine kinase domain [5-7]. Trans autophosphorylation of the receptor [8,9] on specific tyrosine residues couples the activated dimer to the intracell ular signal transduction machinery as these phosphorylated residues serve a s docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. T he time-course and extent of EGFR phosphorylation are therefore important d eterminants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in s ignal transduction.