Diverse Applications of Intermolecular FRET in Voltage Imaging

Abstract

Genetically encoded voltage indicators (GEVIs) enables the optical measurement of changes in membrane potential by a change in the fluorescence of the fluorescent protein (FP). ArcLight, one of the most popular GEVIs, is structurally composed of three primary domains: four transmembrane segments that constitutes the voltage-sensing domain (VSD), a cytoplasmic linker region that fuses a FP to the VSD, and the cytoplasmic FP, which functions as the optical reporter. Modifications to any of the domains results in an alteration of the GEVI optical signal. Intermolecular Förster Resonance Energy Transfer (FRET) was used to study the interactions between GEVI proteins when modifications were made to the above-mentioned domains. Robust voltage-dependent signals were observed from GEVIs utilizing either CFP-YFP or GFP-RFP FRET pairs. Mutations to the FPs results in changes of the voltage-dependent FRET signal, suggesting a change in interaction, which may result in a change of the distance and/or orientation of the donor and acceptor FPs. Attempts were also made to identify the optimal linker length for the best FRET interaction between the GEVIs. Intermolecular FRET also enables for the study of specific areas of the neuron. For instance, an axon targeted FRET donor can be co-expressed with a GEVI fused with the FRET acceptor. Only specific areas that expresses both the FRET donor and acceptor FP will result in a FRET signal. The many applications of intermolecular FRET create a wide range of possibilities ranging from better cell-type specific expression to better 2-photon probes.