Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2013-03-01
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| Series: | Frontiers in Molecular Neuroscience |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fnmol.2013.00002/full |
| _version_ | 1818700362355310592 |
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| author | Jasper eAkerboom Nicole eCarreras Calderón Nicole eCarreras Calderón Lin eTian Lin eTian Sebastian eWabnig Matthias ePrigge Johan eTolö Andrew eGordus Michael B Orger Michael B Orger Kristen E Severi John J Macklin Ronak ePatel Stefan R Pulver Trevor J Wardill Trevor J Wardill Elisabeth eFischer Christina eSchüler Tsai-Wen eChen Karen S Sarkisyan Jonathan S Marvin Cornelia I Bargmann Douglas S Kim Sebastian eKügler Leon eLagnado Peter eHegemann Alexander eGottschalk Eric R Schreiter Loren L Looger |
| author_facet | Jasper eAkerboom Nicole eCarreras Calderón Nicole eCarreras Calderón Lin eTian Lin eTian Sebastian eWabnig Matthias ePrigge Johan eTolö Andrew eGordus Michael B Orger Michael B Orger Kristen E Severi John J Macklin Ronak ePatel Stefan R Pulver Trevor J Wardill Trevor J Wardill Elisabeth eFischer Christina eSchüler Tsai-Wen eChen Karen S Sarkisyan Jonathan S Marvin Cornelia I Bargmann Douglas S Kim Sebastian eKügler Leon eLagnado Peter eHegemann Alexander eGottschalk Eric R Schreiter Loren L Looger |
| author_sort | Jasper eAkerboom |
| collection | DOAJ |
| description | Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics. |
| first_indexed | 2024-12-17T15:03:44Z |
| format | Article |
| id | doaj.art-46e541bc41ce4dd2bdcb29ec64a7b4d7 |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-12-17T15:03:44Z |
| publishDate | 2013-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-46e541bc41ce4dd2bdcb29ec64a7b4d72022-12-21T21:43:49ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992013-03-01610.3389/fnmol.2013.0000243920Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogeneticsJasper eAkerboom0Nicole eCarreras Calderón1Nicole eCarreras Calderón2Lin eTian3Lin eTian4Sebastian eWabnig5Matthias ePrigge6Johan eTolö7Andrew eGordus8Michael B Orger9Michael B Orger10Kristen E Severi11John J Macklin12Ronak ePatel13Stefan R Pulver14Trevor J Wardill15Trevor J Wardill16Elisabeth eFischer17Christina eSchüler18Tsai-Wen eChen19Karen S Sarkisyan20Jonathan S Marvin21Cornelia I Bargmann22Douglas S Kim23Sebastian eKügler24Leon eLagnado25Peter eHegemann26Alexander eGottschalk27Eric R Schreiter28Loren L Looger29Howard Hughes Medical InstituteHoward Hughes Medical InstituteMedical Research Council Laboratory of Molecular BiologyHoward Hughes Medical InstituteUniversity of California Davis School of MedicineJohann Wolfgang Goethe-University FrankfurtHumboldt Universität zu BerlinUniversity Medicine GöttingenThe Rockefeller UniversityHarvard UniversityChampalimaud Centre for the UnknownHarvard UniversityHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteMarine Biology LaboratoryJohann Wolfgang Goethe-University FrankfurtJohann Wolfgang Goethe-University FrankfurtHoward Hughes Medical InstituteHoward Hughes Medical InstituteHoward Hughes Medical InstituteThe Rockefeller UniversityHoward Hughes Medical InstituteUniversity Medicine GöttingenMedical Research Council Laboratory of Molecular BiologyHumboldt Universität zu BerlinJohann Wolfgang Goethe-University FrankfurtHoward Hughes Medical InstituteHoward Hughes Medical InstituteGenetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.http://journal.frontiersin.org/Journal/10.3389/fnmol.2013.00002/fullProtein Structure, Tertiaryfunctional imagingoptogeneticsGenetically encoded calcium indicators (GECIs)Optogenetic Neuroimaging |
| spellingShingle | Jasper eAkerboom Nicole eCarreras Calderón Nicole eCarreras Calderón Lin eTian Lin eTian Sebastian eWabnig Matthias ePrigge Johan eTolö Andrew eGordus Michael B Orger Michael B Orger Kristen E Severi John J Macklin Ronak ePatel Stefan R Pulver Trevor J Wardill Trevor J Wardill Elisabeth eFischer Christina eSchüler Tsai-Wen eChen Karen S Sarkisyan Jonathan S Marvin Cornelia I Bargmann Douglas S Kim Sebastian eKügler Leon eLagnado Peter eHegemann Alexander eGottschalk Eric R Schreiter Loren L Looger Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics Frontiers in Molecular Neuroscience Protein Structure, Tertiary functional imaging optogenetics Genetically encoded calcium indicators (GECIs) Optogenetic Neuroimaging |
| title | Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics |
| title_full | Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics |
| title_fullStr | Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics |
| title_full_unstemmed | Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics |
| title_short | Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics |
| title_sort | genetically encoded calcium indicators for multi color neural activity imaging and combination with optogenetics |
| topic | Protein Structure, Tertiary functional imaging optogenetics Genetically encoded calcium indicators (GECIs) Optogenetic Neuroimaging |
| url | http://journal.frontiersin.org/Journal/10.3389/fnmol.2013.00002/full |
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