Direct measurements of luminal Ca with endo-lysosomal GFP-aequorin reveal functional IP receptors.

Endo-lysosomes are considered acidic Ca stores but direct measurements of luminal Ca within them are limited. Here we report that the Ca -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca dynamics in this compartment.

In vitro and in vivo calibration of low affinity genetic Ca indicators.

Calcium is a universal intracellular messenger and proper Caconcentrations ([Ca]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca levels result in malfunction and disease. Selective intraorganellar Cameasurements are best achieved by using targeted genetically encoded Ca indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions.

Use of aequorin-based indicators for monitoring Ca in acidic organelles.

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca upon cell activation. Hence, reliable recording of Ca dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca indicators (GECIs) are valuable tools to monitor Ca in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs.