Work

Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight

Year: 2017

Type: article

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.

Source: ACS Chemical Neuroscience

DOI: 10.1021/acschemneuro.6b00234

Authors: Jelena Platisa, Ganesh Vasan, Amy Yang, Vincent A. Pieribone

Institutions: Yale University, John B. Pierce Laboratory

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funders: National Institutes of Health, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences