An expanded palette of bright and photostable organellar Ca2+ sensors

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 requires bright, photostable and multiplexed measurements in each signaling compartment of interest to dissect the origins and destinations of Ca2+ fluxes. We introduce a new toolkit of chemigenetic indicators based on HaloCaMP, optimized to report Ca2+ 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 Ca2+ dynamics, expanding significantly multiplexing capacities of Ca2+ signaling. The improved brightness of ER-HaloCaMP using either red or far-red HaloTag ligands enabled measuring ER Ca2+ 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 Ca2+ sensors. When measuring ER Ca2+ fluxes in activated neuronal dendritic spines of cultured neurons, ER-HaloCaMP presented increased photostability compared to the gold-standard ER Ca2+ 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 Ca2+ signaling in far-red in cell lines and primary neurons. As a proof-of-concept, we used 3-plex multiplexing to quantify interorganellar Ca2+ signaling. We show that effective transfer of Ca2+ from the ER to mitochondria depends on the ER releasing a critical amount of Ca2+. When this threshold is not met, the mobilized Ca2+ is diverted to the cytosol instead. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca2+ sensors, which will facilitate future studies of intracellular Ca2+ signaling.