Multiplexed imaging of G-proteins and ERK activity upon activation of CaSR

We have previously shown that the calcium sensing receptor (CaSR) activates different G proteins and second messengers in single cells, and that GPCR and G protein-mediated ERK activity can be highly dynamic and heterogeneous at the single cell level. Here, we attempted to investigate the CaSR-Gi-ERK signaling pathway in single cells with previously characterized biosensors. We developed a strategy to simultaneously track G-protein and ERK activities in a single cell, using FRET-based and translocation sensors, a novel large-Stokes-shift green fluorescent protein, and an imaging setup with a single detector and a single wavelength for excitation. Extensive characterization and optimization to unmix the signals showed that the differences in dynamic range and type of read-out obtained from these biosensors made it very challenging to obtain robust FRET-measurements in the developed setup. When focusing solely on ERK, we found signs of possible ERK activity in response to calcium stimulation via CaSR, as well as milder changes in the absence of calcium treatment. Further optimization of the model, probes, and image processing will be necessary to develop a setup to robustly measure FRET and translocation simultaneously in single cells. We present the challenges we encountered and discuss future developments that can overcome them.