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bioRxiv · 10.1101/2025.01.10.632364

An expanded palette of bright and photostable organellar Ca2+ sensors

Moret, A.·Farrants, H.·Fan, R.·Zingg, K.·Gee, C. E.·Oertner, T. G.·Rangaraju, V.·Schreiter, E. R.·de Juan-Sanz, J.
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Abstract

The use of fluorescent sensors for functional imaging has revolutionized the study of organellar Ca2+ signaling. However, understanding the dynamic interplay between intracellular Ca2+ sinks and sources has been hindered by the lack of bright, photostable, and multiplexed measurements in different organelles, limiting our ability to define how Ca2+ shapes cell physiology across fields of biology. Here we introduce a new toolkit of chemigenetic organellar Ca2+ indicators whose color is tunable by reconstituting their fluorescence with different exogenous rhodamine dye-ligands, which significantly expand the capacity for multiplexing organellar Ca2+ measurements. These sensors, which we named ER-HaloCaMP and Mito-HaloCaMP, are optimized to report Ca2+ dynamics in the endoplasmic reticulum (ER) and mitochondria of mammalian cells and neurons, and show significantly improved brightness, photostability and responsiveness when compared to current best-in-class alternatives. Using either red or far-red dye-ligands, both ER-HaloCaMP and Mito-HaloCaMP enable visualizing ER and mitochondrial Ca2+ dynamics in neuronal axons, a subcellular location that only contains a few ER tubules and small mitochondria, structural limitations that have impaired measurements with previous red sensors. To show the expanded multiplexing capacities of our toolkit, we measured interorganellar Ca2+ fluxes simultaneously in three different subcellular compartments in live cells, revealing that the amplitude of ER Ca2+ release controls the efficacy of ER-mitochondria Ca2+ coupling in a cooperative manner. Organellar HaloCaMPs enable also measuring Ca2+ dynamics in intact brain tissue from flies and rodents, demonstrating their versatility across biological models. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca2+ sensors, which will facilitate future studies of intracellular Ca2+ signaling across fields of biology in health and disease.

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Moret, A., Farrants, H., Fan, R., Zingg, K., Gee, C. E., Oertner, T. G., Rangaraju, V., Schreiter, E. R., de Juan-Sanz, J.. 2025-01-12. An expanded palette of bright and photostable organellar Ca2+ sensors. https://doi.org/10.1101/2025.01.10.632364

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