Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles.

Simultaneous control of different functions by calcium signals is possible because of subcellular compartmentalization. Targeted chemiluminescent Ca2+ probes, such as aequorins (AEQs) are optimal for detecting signals originating in different subcellular domains, but imaging is difficult because of low photon yield causing poor spatiotemporal resolution. To overcome this problem, we have co-expressed two spectrally distinct AEQs in different subcellular locations within the same cells. Seven chimeric proteins containing either green- or red-emitting AEQs, with different targeting sequences and Ca2+ affinities, have been designed and tested. We show here evidence for physical and functional independence of the different probes. Cytosolic Ca2+ signals were mirrored in the nucleus, but amplified inside mitochondria and endoplasmic reticulum, and had different time courses in the various locations. Our results demonstrate that these novel tools permit simultaneous and independent monitoring of [Ca2+] in different subcellular domains of the same cell.