A quad‐cistronic fluorescent biosensor system for real‐time detection of subcellular Ca²⁺ signals

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Background and Purpose The calcium ion (Ca 2+ ) is a versatile cellular messenger regulating a variety of biological processes. Compounds modulating subcellular Ca 2+ signals hold substantial pharmacological potential. Advances in fluorescent biosensors have revolutionised Ca 2+ imaging. However, co‐expression of targeted biosensors for simultaneous measurement of Ca 2+ signals in multiple cellular compartments is still complicated by heterogeneous expression levels of the various sensors. Experimental Approach We developed the ribosomal skipping‐based quad‐cistronic fluorescent biosensor system CARMEN, enabling high‐content Ca 2+ imaging across three compartments. CARMEN allows proportional co‐expression of spectrally distinct Ca 2+ biosensors: the near‐infrared Ca 2+ biosensor for the cytosol (NIR‐GECO2G‐NES), the green Ca 2+ biosensor for mitochondria (CEPIA3mt) and the red Ca 2+ biosensor for the endoplasmic reticulum (R‐CEPIA1er), along with a Ca 2+ ‐insensitive blue fluorescent protein targeted to the nucleus (NLS‐mTagBFP2), serving as a normalisation reference. Key Results CARMEN allows spatiotemporal correlation of Ca 2+ signals across the cytosol, endoplasmic reticulum and mitochondria, revealing distinct dynamics. We noted delayed mitochondrial Ca 2+ uptake compared to the other compartments. We validated CARMEN across three cell types and tested two recently identified mitochondrial Ca 2+ uniporter inhibitors (MCUis), MCUi4 and MCUi11, showcasing the potential of CARMEN for its application in pharmacological research. Our results show that while both MCUi4 and MCUi11 inhibited mitochondrial Ca 2+ uptake in HeLa S3 cells, MCUi4 reduced cytosolic Ca 2+ signals and oscillations, whereas MCUi11 had opposing effects. Conclusions and Implications CARMEN is a powerful tool for real‐time, multiplexed analysis of compartment‐specific Ca 2+ signals, with the potential for automation in high‐content drug screening.