New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations 英文参考文献.docVIP

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New Alternately Colored FRET Sensors for Simultaneous Monitoring of Zn2+ in Multiple Cellular Locations 英文参考文献.doc

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NewAlternatelyColoredFRETSensorsforSimultaneousMonitoringofZn2inMultipleCellularLocations英文参考文献

NewAlternatelyColored2+ FRETSensorsforSimultaneous MonitoringofZn inMultipleCellularLocations JoseG.Miranda1,AmandaL.Weaver1,YanQin1,J.GenevievePark1,CaitlinI.Stoddard1,MichaelZ.Lin2, AmyE.Palmer1* 1DepartmentofChemistryandBiochemistryandBioFrontiersInstitute,UniversityofColorado,Boulder,Colorado,UnitedStatesofAmerica,2DepartmentofPediatrics andEngineering,StanfordMedicalSchool,Stanford,California,UnitedStatesofAmerics Abstract Geneticallyencodedsensorsbasedonfluorescenceresonanceenergytransfer(FRET)arepowerfultoolsforreportingon ions,moleculesandbiochemicalreactionsinlivingcells.HerewedescribethedevelopmentofnewsensorsforZn based 2+ 2+ on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn2+ levels and dynamicsincellswithhighspatialandtemporalresolution.HerewedevelopasuiteofsensorsusingalternateFRETpairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors weretargetedtoboththenucleusandcytosolandcharacterizedandvalidatedinlivingcells.Sensorsbasedonthenew FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors 2+ testedandwereoptimalformonitoringchangesincytosolicandnuclearZn .Usingagreen-redsensortargetedtothe nucleusandcyan-yellow sensor targetedtoeithertheER,Golgi,ormitochondria, wewereabletomonitor Zn uptake simultaneouslyintwocompartments,revealingthatnuclearZn risesquickly,whereastheER,Golgi,andmitochondriaall 2+ 2+ sequesterZn2+moreslowlyandwithadelayof600–700sec.Lastly,thesestudiesprovidethefirstglimpseofnuclearZn andrevealthatnuclearZn isbufferedatahigherlevelthancytosolicZn . 2+ 2+ 2+ Citation:MirandaJG,WeaverAL,QinY,ParkJG,StoddardCI,etal.(2012)NewAlternatelyColoredFRETSensorsforSimultaneousMonitoringofZn2+inMultiple CellularLocat