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

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.