Abstract Endoplasmic/sarcoplasmic reticulum (ER/SR) sits at the heart of the calcium (Ca 2+ ) signaling machinery, yet current genetically encoded Ca 2+ indicators (GECIs) lack the ability to detect elementary Ca 2+ release events from ER/SR, particularly in muscle cells. Here we report a set of organellar GECIs, termed NEMOer, to efficiently capture ER Ca 2+ dynamics with increased sensitivity and responsiveness. Compared to G-CEPIA1er, NEMOer indicators exhibit dynamic ranges that are an order of magnitude larger, which enables up to 5-fold more sensitive detection of Ca 2+ oscillation in both non-excitable and excitable cells. The ratiometric version further allows super-resolution monitoring of local ER Ca 2+ homeostasis and dynamics. Notably, the NEMOer-f variant enabled the inaugural detection of Ca 2+ blinks, elementary Ca 2+ releasing signals from the SR of cardiomyocytes, as well as in vivo spontaneous SR Ca 2+ releases in zebrafish. In summary, the highly dynamic NEMOer sensors expand the repertoire of organellar Ca 2+ sensors that allow real-time monitoring of intricate Ca 2+ dynamics and homeostasis in live cells with high spatiotemporal resolution.

Highly Dynamic and Sensitive NEMOer Calcium Indicators for Imaging ER Calcium Signals in Excitable Cells