Optogenetic activation of opto-Raf and opto-AKT protects PC12 cells against phototoxicity.

<p>(<b>A</b>) The design scheme of opto-Raf. The CIB1 domain is anchored to the plasma membrane via a CAAX motif. Upon light stimulation, CIB1-CRY2 interaction should recruit cytoplasmic CRY2-mCherry-Raf1 to the plasma membrane and subsequently activate the downstream ERK pathway. In the dark, CIB1-CRY2 should spontaneously dissociate and return Raf1 to the cytoplasm to inactivate the Raf/ERK pathway. (<b>B</b>) CIB1-GFP-CAAX was clearly located at the plasma membrane as indicated by the green fluorescence image. Blue light rapidly recruited cytosolic CRY2-mCherry-Raf1 to the plasma membrane within 15 seconds (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153487#pone.0153487.s010" target="_blank">S1 Movie</a>)</b>. Scale bar = 20 μm. (<b>C</b>) Western blot analysis shows phosphor-ERK upon 10, 30 and 60 minutes of blue light activation of opto-Raf. No band was observed when the culture was kept in the dark. (<b>D</b>) The design scheme of opto-AKT, similar to that of opto-Raf. Blue light should activate opto-AKT and the pathway. (<b>E</b>) Blue light illumination induced rapid recruitment of CRY2-mCherry-AKT from cytosol to the plasma membrane within 20 seconds (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153487#pone.0153487.s011" target="_blank">S2 Movie</a>)</b>. Scale bar = 20 μm. (<b>F</b>) Western blot analysis of phosphorylated AKT confirms light-induced activation of CRY2-mCherry-AKT at 160kDa. The endogenous AKT band at 60kDa is not affected by the blue light stimulation. (<b>G</b>) Cells transfected with CIB1-GFP-CAAX show phototoxicity at high blue light intensities. Comparable cell death could be seen when the intensities were at 0 and 0.2 mW/cm², while significant cell death was observed when the cells were subjected to 1.5 mW/cm² of blue light. Scale bar = 100 μm. (<b>H-I</b>) Quantification of cell death rates for cells transfected with CIB1-GFP-CAAX only (<b>H</b>) or CIB1-GFP-CAAX and CRY2-mCherry (<b>I</b>) that were exposed to blue light at 0, 0.2, 0.8 and 1.5 mW/cm² for 24 hours. Untransfected cells in the same culture were used as controls. Each set of data comprises of 5 sets of experiments with 2000–5000 cells each. Data is represented as mean +/- standard deviation.