Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.
Red light is useful for optogenetic control of various protein activities in mammalian deep tissues due to its high tissue penetration, low invasiveness, and low light scattering. However, technology that enables for the optogenetic manipulation using red light remains elusive. Here we develop a red light-activatable, semi-synthetic photoswitch, named MagRed. MagRed is composed of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin, and its photo-state-specific de novo synthetic binder. MagRed allows us to reassemble split-proteins using red light and thereby develop a red light-activatable Cre recombinase, which is applicable for mammalian deep tissues. Additionally, we take advantage of MagRed to develop red light-inducible transcription system based on the CRISPR-Cas9 system, enabling for high induction (up to 378-fold) of multiple user-defined endogenous target genes. With high versatility and regulatability, MagRed provides a powerful technology that easily facilitates optogenetics applications for a variety of biological research areas.
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