A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3 [article]

Benjamin B Kim, Haodi Wu, Yukun A Hao, Michael Pan, Mariya Chavarha, Michael Westberg, Francois St-Pierre, Joseph C Wu, Michael Lin
2020 bioRxiv   pre-print
A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP2. Among red
more » ... luorescent proteins tested, mCyRFP2 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP2 provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-mCyRFP2 to visualize membrane voltage changes throughout the cell cycle of motile cells.
doi:10.1101/2020.10.09.328922 fatcat:56mqrkyzbrhvxihauuuysogdfa