title: Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight.
abstract: Genetically encoded calcium indicators (GECIs) produce unprecedentedly large signals that have enabled routine optical recording of single neuron activity in vivo in rodent brain. Genetically encoded voltage indicators (GEVIs) offer a more direct measure of neuronal electrical status, however the signal-to-noise characteristics and signal polarity of the probes developed to date have precluded routine use in vivo. We applied directed evolution to target modulable areas of the fluorescent protein in GEVI ArcLight to create the first GFP-based GEVI (Marina) that exhibits a ΔF/ΔV with a positive slope relationship. We found that only three rounds of site-directed mutagenesis produced a family of "brightening" GEVIs with voltage sensitivities comparable to that seen in the parent probe ArcLight. This shift in signal polarity is an essential first step to producing voltage indicators with signal-to-noise characteristics comparable to GECIs to support widespread use in vivo.
authors.affiliation: The John B. Pierce Laboratory, Inc. , New Haven, Connecticut 06519, United States.
authors.forename: Jelena
authors.identifier: 0000-0003-4813-609X
authors.initials: J
authors.lastname: Platisa
authors.affiliation: The John B. Pierce Laboratory, Inc. , New Haven, Connecticut 06519, United States.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Vasan
authors.affiliation: The John B. Pierce Laboratory, Inc. , New Haven, Connecticut 06519, United States.
authors.forename: Amy
authors.initials: A
authors.lastname: Yang
authors.affiliation: The John B. Pierce Laboratory, Inc. , New Haven, Connecticut 06519, United States.
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Pieribone
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004558:Electric Stimulation; D004622:Embryo, Mammalian; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008967:Molecular Biology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D056969:Voltage-Sensitive Dye Imaging
chemical_list: C576393:Arclight fusion protein; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: ArcLight; Genetically encoded voltage indicators; biosensors; directed evolution; green fluorescent protein; mutagenesis