Comparison of M3-muscarnic receptor mediated PIP2 hydrolysis vs blue light excited retinal (BLE-retinal) induced PIP2 sensor translocation. Images of HeLa cells expressing M3-muscarinic receptor, mCherry-PH (PIP2 sensor), DBD-YFP (DAG sensor). (A) Without blue light exposure, retinal addition does not change PIP2 or DAG sensor distribution (left), while the addition of carbachol resulted in PIP2 hydrolysis and DAG formation (middle). The addition of Gq inhibitor, YM254890 (1 µM) to cells resulted in reverse translocation of PIP2 and DAG sensors. (mean ± S.E.M., n = 6 cells) (B) When cells were exposed to blue light (4.86 µW of 445 nm) in the presence of retinal, they only showed PIP2 sensor translocation while no change in DAG sensor was observed (left). The addition of carbachol to these cells exhibited an additional PIP2 sensor translocation to the cytosol with a mild DAG sensor translocation to the PM (left) (mean ± S.E.M., n = 4 cells). Interestingly, addition of YM254890 only reversed both PIP2 and DBD responses elicited by carbachol. (C) The field of vision of cells shown in A and B. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm.