A StayGold-based calcium ion indicator

Genetically encoded calcium ion (Ca2+) indicators (GECIs) enable visualization of Ca2+ dynamics in living systems but often suffer from limited photostability during prolonged imaging. The recent discovery of StayGold, a green fluorescent protein (FP) with exceptional brightness and photostability, opened the possibility of addressing this longstanding challenge. Here, we sought to establish whether a monomeric variant of StayGold (mStayGold) could be converted into a single FP-based GECI. Through extensive protein engineering, we generated a functional mStayGold-based GECI, HiCaRI (Highly intensiometric Ca2+ Responsive Indicator) by fusing Calmodulin (CaM) and the ckkap binding peptide from K-GECO1 into mStayGold(J). HiCaRI exhibits a large Ca2+-dependent inverse fluorescence response ({triangleup}F/Fmin = -15) while retaining high brightness and improved photostability relative to previously reported GFP-based GECIs. Although the current variant represents a first-generation prototype with shortcomings in terms of Ca2+ affinity and photostability (relative to StayGold and mStayGold(J)), this work demonstrates the feasibility of constructing single FP-based GECIs from a highly photostable fluorescent protein.