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. 2025 Oct 3.
doi: 10.1111/bph.70211. Online ahead of print.

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

Anna Lischnig  1 , Yusuf C Erdoğan  1 , Benjamin Gottschalk  1 , Michael Gruber  2 , Andrea Groselj-Strele  2 , Sandra Burgstaller  3 , Wolfgang F Graier  1   4 , Roland Malli  3   4
Affiliations

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

Anna Lischnig et al. Br J Pharmacol. .

Abstract

Background and purpose: The calcium ion (Ca2+) is a versatile cellular messenger regulating a variety of biological processes. Compounds modulating subcellular Ca2+ signals hold substantial pharmacological potential. Advances in fluorescent biosensors have revolutionised Ca2+ imaging. However, co-expression of targeted biosensors for simultaneous measurement of Ca2+ 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 Ca2+ imaging across three compartments. CARMEN allows proportional co-expression of spectrally distinct Ca2+ biosensors: the near-infrared Ca2+ biosensor for the cytosol (NIR-GECO2G-NES), the green Ca2+ biosensor for mitochondria (CEPIA3mt) and the red Ca2+ biosensor for the endoplasmic reticulum (R-CEPIA1er), along with a Ca2+-insensitive blue fluorescent protein targeted to the nucleus (NLS-mTagBFP2), serving as a normalisation reference.

Key results: CARMEN allows spatiotemporal correlation of Ca2+ signals across the cytosol, endoplasmic reticulum and mitochondria, revealing distinct dynamics. We noted delayed mitochondrial Ca2+ uptake compared to the other compartments. We validated CARMEN across three cell types and tested two recently identified mitochondrial Ca2+ 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 Ca2+ uptake in HeLa S3 cells, MCUi4 reduced cytosolic Ca2+ signals and oscillations, whereas MCUi11 had opposing effects.

Conclusions and implications: CARMEN is a powerful tool for real-time, multiplexed analysis of compartment-specific Ca2+ signals, with the potential for automation in high-content drug screening.

Keywords: Ca2+ multiplexing; MCU inhibitors; fluorescence microscopy; genetically encoded Ca2+ biosensors; spatiotemporal Ca2+ imaging.

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