title: Reversible optogenetic control of kinase activity during differentiation and embryonic development.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Vishnu V
authors.identifier: 0000-0001-9905-5965
authors.initials: VV
authors.lastname: Krishnamurthy
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: John S
authors.identifier: 0000-0001-7179-0589
authors.initials: JS
authors.lastname: Khamo
authors.affiliation: Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
authors.forename: Wenyan
authors.initials: W
authors.lastname: Mei
authors.affiliation: Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
authors.forename: Aurora J
authors.initials: AJ
authors.lastname: Turgeon
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Humza M
authors.identifier: 0000-0001-7042-312X
authors.initials: HM
authors.lastname: Ashraf
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Payel
authors.initials: P
authors.lastname: Mondal
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Dil B
authors.identifier: 0000-0003-4567-8774
authors.initials: DB
authors.lastname: Patel
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Risner
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Ellen E
authors.identifier: 0000-0001-7953-2736
authors.initials: EE
authors.lastname: Cho
authors.affiliation: Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA yangj@illinois.edu kaizkaiz@illinois.edu.
authors.forename: Jing
authors.identifier: 0000-0002-4983-1660
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA yangj@illinois.edu kaizkaiz@illinois.edu.|Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.|Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Kai
authors.identifier: 0000-0002-6687-4558
authors.initials: K
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D056931:Cryptochromes / Q000737:chemistry / Q000235:genetics; D047108:Embryonic Development / Q000235:genetics*; D008027:Light; D020935:MAP Kinase Signaling System; D062308:Optogenetics / Q000379:methods*; D016716:PC12 Cells; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D019076:Transgenes; D014981:Xenopus; D048490:raf Kinases / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C103618:CRY2 protein, Arabidopsis; D056931:Cryptochromes; D010770:Phosphotransferases; D048490:raf Kinases
keywords: Bicistronic gene expression; Cryptochrome; Differentiation; Light-controlled protein-protein interaction; Optogenetics; Xenopus