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SHR Neuro Krebs Kardio Lipid Stoffw Microb

Lischnig, A; Erdoğan, YC; Gottschalk, B; Gruber, M; Groselj-Strele, A; Burgstaller, S; Graier, WF; Malli, R.
A quad-cistronic fluorescent biosensor system for real-time detection of subcellular Ca²⁺ signals.
Br J Pharmacol. 2025; Doi: 10.1111/bph.70211
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Autor*innen der Med Uni Graz:

Burgstaller Sandra

Erdogan Yusuf Ceyhun

Gottschalk Benjamin

Graier Wolfgang

Groselj-Strele Andrea

Lischnig Anna

Malli Roland

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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.

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