Abstract
We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.
Highlights
With increasing knowledge of intracellular signalling networks, it becomes more evident that molecules can be involved in processes occurring in multiple pathways
For the first time to our knowledge, we report the application of automated fluorescence lifetime imaging (FLIM) Förster resonance energy transfer (FRET) to screen for protein binding partners within cells – here shown to identify interactions between the Ras-association domain family (RASSF) and mammalian sterile 20-like kinases (MST) – and the estimation of the KD for these interactions
We investigated the differences in the binding characteristics between the isolated SARAH domain and the full length MST1 when interacting with those RASSF proteins that are able to dimerise (RASSF1-6)
Summary
With increasing knowledge of intracellular signalling networks, it becomes more evident that molecules can be involved in processes occurring in multiple pathways. One approach to detect FRET is to measure the fluorescence intensity ratio of the acceptor and the donor fluorophores observing the increase of the fluorescence intensity in the acceptor channel with the simultaneous decrease of the intensity in the donor channel This spectral ratiometric imaging acquisition is fast but requires additional control samples to correct for spectral cross-talk between the fluorophores and to calibrate the spectral response of the specific optical set-up (instrument and sample corrections), making comparison between different samples difficult. It is possible to utilise the depolarisation of the acceptor fluorescence as a FRET readout Polarisation-based measurements can achieve similar acquisition speeds as spectral ratiometric readouts and are highly sensitive to detect the occurrence of FRET, but it is again difficult to quantify FRET efficiencies and population fractions of interacting donors[7,8]. Polarisation has been applied as a first step to screen for possible interaction partners that were subsequently investigated using fluorescence lifetime[9]
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