title: Reproducible and sustained regulation of Gαs signalling using a metazoan opsin as an optogenetic tool.
abstract: Originally developed to regulate neuronal excitability, optogenetics is increasingly also used to control other cellular processes with unprecedented spatiotemporal resolution. Optogenetic modulation of all major G-protein signalling pathways (Gq, Gi and Gs) has been achieved using variants of mammalian rod opsin. We show here that the light response driven by such rod opsin-based tools dissipates under repeated exposure, consistent with the known bleaching characteristics of this photopigment. We continue to show that replacing rod opsin with a bleach resistant opsin from Carybdea rastonii, the box jellyfish, (JellyOp) overcomes this limitation. Visible light induced high amplitude, reversible, and reproducible increases in cAMP in mammalian cells expressing JellyOp. While single flashes produced a brief cAMP spike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently available optogenetic tools, responding to white light at irradiances ≥1 µW/cm(2). We conclude that JellyOp is a promising new tool for mimicking the activity of Gs-coupled G protein coupled receptors with fine spatiotemporal resolution.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Helena J
authors.initials: HJ
authors.lastname: Bailes
authors.forename: Ling-Yu
authors.initials: LY
authors.lastname: Zhuang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lucas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D037462:Cubozoa; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009488:Neurosciences / Q000379:methods*; D055355:Opsins / Q000378:metabolism / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism* / Q000502:physiology; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D054928:Validation Studies as Topic
chemical_list: D055355:Opsins; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP