Energy and wavelength requirement for PIP2 solubilization by photoexcited retinal. (A) UV-VIS absorption spectra for both 11-cis retinal (11CR) and all trans retinal (ATR). Note that 445 nm blue light spectrally overlaps with both absorption spectra. (B) The absorption spectra of melanopsin and retinal (left), (εATR = 44180.0 M−1cm−1). (C) The energy level diagram and the population (pop.) of energy levels of free retinal (blue) and melanopsin (red) according to their respective absorption maxima (right). Note that blue light (445 nm) can highly populate melanopsin compared to that of free retinal. (D–F) Images of HeLa cells expressing PIP2 sensor (mCherry-PH). (D) Cells were incubated with ATR (50 µM) for 5 minutes. A substantial PIP2 sensor translocation was observed upon exposing cells to short pulses of blue light (4.86 µW of 445 nm). The plot shows the dynamics of PIP2 sensor translocation to cytosol. (E) In the absence of retinal, cells did not show a detectable PIP2 sensor translocation when exposed to blue light or other wavelengths. (F) Both blue light excited ATR (50 µM) and 11CR (50 µM) exhibited a permanent accumulation of PIP2 sensor cytosol. Compared to exposed cell (yellow arrow), control cell without blue light (BL) exposure (white arrow) did not show any detectable PIP2 response. The plots show the dynamics of PIP2 sensor translocation in cells shown in F (mean ± S.E.M., n = 6 cells). (G) All trans retinal and blue light induce PIP2 sensor translocation in cells with distinct origins. Images of RAW264.7, NIH3t3, ARPE-19, MDA-MB-468, BT-20, HCT116 and HEK293 cells expressing mCherry-PH (PIP2 sensor). ATR (50 µM) was incubated in cells for 5 minutes followed by continuous exposure of blue light for 5 minutes. Blue light exposure induced PIP2 sensor translocation from PM in all the cell types tested while cells that were not exposed to blue light did not respond. Mean and S.E.M. are from 3 < independent experiments. (blue light (BL) = blue box). Scale = 5 µm.