Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level 英文参考文献.docVIP

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Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level 英文参考文献.doc

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Auto-LuminescentGenetically-EncodedRatiometricIndicatorforReal-TimeCa2ImagingattheSingleCellLevel英文参考文献

Auto-LuminescentGenetically-EncodedRatiometric IndicatorforReal-TimeCa ImagingattheSingleCell Level 2+ KentaSaito1,NoriyukiHatsugai1,2,KazukiHorikawa1,KentaroKobayashi1,ToruMatsu-ura3 ,Katsuhiko Mikoshiba3,TakeharuNagai1,4* 1ResearchInstituteforElectronicScience,HokkaidoUniversity,Kita-20,Nishi-10Kita-ku,Sapporo,Hokkaido,Japan,2ResearchCenterforCooperativeProjects,Hokkaido University, Kita-15, Nishi-7 Kita-ku, Sapporo, Hokkaido, Japan, 3Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako City, Saitama,Japan,4PrecursoryResearchforEmbryonicScience,JapanScienceandTechnologyAgency,Sanbancho,Chiyoda-ku,Tokyo,Japan Abstract Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to afluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaginginnearrealtimewithoutexternallightillumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca -binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent 2+ protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescentprotein’schromophoresimprovedthedynamicrangeofBRETsignalchangeinBRACupto60%,whichisthe largestdynamicrangeamongBRET-basedindicatorsreportedsofar.UsingBRAC,wedemonstratedsuccessfulvisualization 2+ of Ca dynamics at the single-cell level with temporal resolution at 1Hz. Moreover, BRAC signals were acquired by ratiometricimagingcapableofcancelingoutCa -independentsignaldriftsduetochangeincellshape,focusshift,etc. 2+ Conclusions/Significance:ThebrightnessandlargedynamicrangeofBRACshouldfacilitatehigh-sensitiveCa2+ notonlyinsinglelivecellsbutalsoinsmalllivingsubjects. imaging Citation:SaitoK,HatsugaiN,HorikawaK,KobayashiK,Matsu-uraT,etal.(2010)Auto-LuminescentGenetically-Encod