Extended Data Fig. 8: cpLOV2-based optical dimerizer to photo-control protein localization and subcellular events.
From: Circularly permuted LOV2 as a modular photoswitch for optogenetic engineering
a, Photo-triggered interaction between PM-anchored ssrA–cpLOV2-CAAX and mCh-sspB fused to effector domains (5-ptase for PI(4,5)P2 to PI4P conversion; or iSH2 of PI3K for (PI(4,5)P2 to PI(3,4,5)P3 conversion) in HeLa cells. These manipulations allowed light-inducible reprogramming of phosphoinositide metabolism in the plasma membrane (PM). GFP-PHPLCδ and PHAKT-GFP were used as PI(4,5)P2 and PI(3,4,5)P3 sensors, respectively. Scale bar, 10 µm. b, cpLOV2 enables light-inducible protein diffusion into the primary cilia of NIH3T3 cells. (Left) a schematic depicting light-inducible recruitment of ssrA-cpLOV2-GFP into the cilia (Arl13b-mCh-sspB as the bait). (Right) representative confocal images of NIH3T3 cells co-expressing ssrA-cpLOV2-GFP (green) and Arl13b-mCh-sspB (red) before and after blue light illumination for 2 min. Scale bar, 10 µm. Also see Supplementary Video 5. c, Light-inducible recruitment of targets to the nuclear envelope. sspB-mEmerald-Lamin A (green) was anchored to the nuclear envelope and ssrA-cpLOV2-mCh-NES (red) remained smoothly distributed within the nucleoplasm in the dark. Upon photostimulation, ssrA-cpLOV2-mCh-NES translocated to the nuclear envelope. Scale bar, 10 µm. Also see Supplementary Video 6. d, Bioluminescence to report light-inducible transcriptional activation mediated by dCas9-ssrA-cpLOV2 and sspB-VPR. (left) Cartoon illustrating the design of photo-induced association of dCas9-ssrA-cpLOV2 with sspB-VPR to turn on luciferase reporter gene expression. (right) Quantification of luciferase activity in HEK293T cells co-expressing the indicated constructs (n = 3 independent biological replicates; mean ± s.e.m.).
