title: Green-to-Red Photoconversion of GCaMP.
abstract: Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs--including GCaMP3, GCaMP5 and GCaMP6--can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.
authors.affiliation: Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Minrong
authors.initials: M
authors.lastname: Ai
authors.affiliation: Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Holly
authors.initials: H
authors.lastname: Mills
authors.affiliation: Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kanai
authors.affiliation: Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Jason
authors.initials: J
authors.lastname: Lai
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Deng
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States of America.
authors.forename: Eric
authors.initials: E
authors.lastname: Schreiter
authors.affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States of America.
authors.forename: Loren
authors.initials: L
authors.lastname: Looger
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Thomas
authors.initials: T
authors.lastname: Neubert
authors.affiliation: Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University, School of Medicine, New York, New York, United States of America.
authors.forename: Greg
authors.initials: G
authors.lastname: Suh
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003116:Color; D004331:Drosophila melanogaster; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055668:Photochemical Processes; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009219:Myosin-Light-Chain Kinase; D002118:Calcium