Monitoring ER Ca by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca.

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.

Using Fluorescent GAP Indicators to Monitor ER Ca.

The endoplasmic reticulum (ER) is the main reservoir of Ca of the cell. Accurate and quantitative measuring of Ca dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca indicators have been developed, including a family of fluorescent Ca indicators, dubbed GFP-Aequorin Proteins (GAPs).

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.