Reversible optogenetic control of kinase activity during differentiation and embryonic development

Development. 2016 Nov 1;143(21):4085-4094. doi: 10.1242/dev.140889. Epub 2016 Oct 3.


A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically 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 for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of 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 with 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.

Keywords: Bicistronic gene expression; Cryptochrome; Differentiation; Light-controlled protein-protein interaction; Optogenetics; Xenopus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arabidopsis
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Cell Differentiation / genetics*
  • Cryptochromes / chemistry
  • Cryptochromes / genetics
  • Embryonic Development / genetics*
  • Light
  • MAP Kinase Signaling System
  • Optogenetics / methods*
  • PC12 Cells
  • Phosphorylation
  • Phosphotransferases / genetics
  • Phosphotransferases / metabolism*
  • Rats
  • Signal Transduction
  • Transgenes
  • Xenopus
  • raf Kinases / metabolism


  • Arabidopsis Proteins
  • CRY2 protein, Arabidopsis
  • Cryptochromes
  • Phosphotransferases
  • raf Kinases