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.
KeywordsCalcium signalling Microdomains Aequorin Chemiluminescence GFP RFP
green fluorescent protein
red fluorescent protein
chimeric GFP-AEQ protein
chimeric mRFP-AEQ protein
cytosolic Ca2+ concentration
nuclear Ca2+ concentration
Ca2+ concentration inside ER
mitochondrial Ca2+ concentration
mitochondrial membrane potential
We thank Mr. Jesús Fernández for technical assistance and the Spanish Ministerio de Educación y Ciencia (MEC; BFU2004-02765/BFI, and BFU2005-02078) and Junta de Castilla y León (VA-088/A06) for financial support. IMM and BD held predoctoral fellowships from MEC and Junta de Comunidades de Castilla-La Mancha, respectively.