A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. Lack of tools to dynamically manipulate signaling pathways in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise in modulating intracellular signaling networks with high spatial and temporal resolution. Applications of the optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared to co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.
- Received June 8, 2016.
- Accepted September 19, 2016.