An expanded palette of bright and photostable organellar Ca ²⁺ sensors

The use of fluorescent sensors for functional imaging has revolutionized the study of organellar Ca ²⁺ signaling. However, understanding the dynamic interplay between intracellular Ca ²⁺ sinks and sources requires bright, photostable and multiplexed measurements in each signaling compartment of interest to dissect the origins and destinations of Ca ²⁺ fluxes. We introduce a new toolkit of chemigenetic indicators based on HaloCaMP, optimized to report Ca ²⁺ dynamics in the endoplasmic reticulum (ER) and mitochondria of mammalian cells and neurons. Both ER-HaloCaMP and Mito-HaloCaMP present high brightness and responsiveness, and the use of different HaloTag ligands enables tunable red and far-red emission when quantifying organelle Ca ²⁺ dynamics, expanding significantly multiplexing capacities of Ca ²⁺ signaling. The improved brightness of ER-HaloCaMP using either red or far-red HaloTag ligands enabled measuring ER Ca ²⁺ fluxes in axons of neurons, in which the ER is formed by a tiny tubule of 30-60 nanometers of diameter that impeded measurements with previous red ER Ca ²⁺ sensors. When measuring ER Ca ²⁺ fluxes in activated neuronal dendritic spines of cultured neurons, ER-HaloCaMP presented increased photostability compared to the gold-standard ER Ca ²⁺ sensor in the field, ER-GCaMP6-210, while presenting the same responsiveness. On the other hand, Mito-HaloCaMP presented higher responsiveness than current red sensors, and enabled the first measurements of mitochondrial Ca ²⁺ signaling in far-red in cell lines and primary neurons. As a proof-of-concept, we used 3-plex multiplexing to quantify interorganellar Ca ²⁺ signaling. We show that effective transfer of Ca ²⁺ from the ER to mitochondria depends on the ER releasing a critical amount of Ca ²⁺ . When this threshold is not met, the mobilized Ca ²⁺ is diverted to the cytosol instead. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca ²⁺ sensors, which will facilitate future studies of intracellular Ca ²⁺ signaling.