Reproducible and Sustained Regulation of Gαs Signalling Using a Metazoan Opsin as an Optogenetic Tool 英文参考文献.docVIP

ReproducibleandSustainedRegulationofGa sSignalling UsingaMetazoanOpsinasanOptogeneticTool HelenaJ.Bailes,Ling-YuZhuang,RobertJ.Lucas* FacultyofLifeSciences,TheUniversityofManchester,Manchester,UnitedKingdom 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,GiandGs)hasbeenachievedusingvariantsofmammalianrodopsin.Weshowherethatthelightresponse 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 Carybdearastonii,theboxjellyfish,(JellyOp)overcomesthislimitation.Visiblelightinducedhighamplitude,reversible,and reproducibleincreasesincAMPinmammaliancellsexpressingJellyOp.WhilesingleflashesproducedabriefcAMPspike, repeated stimulation could sustain elevated levels for 10s of minutes. JellyOp was more photosensitive than currently availableoptogenetictools,respondingtowhitelightatirradiances$1mW/cm2.WeconcludethatJellyOpisapromising newtoolformimickingtheactivityofGs-coupledGproteincoupledreceptorswithfinespatiotemporalresolution. Citation:BailesHJ,ZhuangL-Y,LucasRJ(2012)ReproducibleandSustainedRegulationofGasSignallingUsingaMetazoanOpsinasanOptogeneticTool.PLoS ONE7(1):e30774.doi:10.1371/journal.pone.0030774 Editor:PaulA.Bartell,PennsylvaniaStateUniversity,UnitedStatesofAmerica ReceivedNovember4,2011;AcceptedDecember26,2011;PublishedJanuary24,2012 Copyright: ? 2012 Bailes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwassupportedbyaMilsteinAward,MedicalResearchCouncilUKtoRJLandHJB(G0801731)andaBBSRCdoctoral