Using aequorin probes to measure Ca²⁺ in intracellular organelles

Aequorins are excellent tools for measuring intra-organellar Ca²⁺ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca²⁺ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca²⁺ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca²⁺ indicator for monitoring physiological Ca²⁺ signaling ex vivo and in vivo.