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.
Manjarrés
Isabel M
IM
Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid and Consejo Superior de Investigaciones Científicas, c/Sanz y Forés s/n, 47003 Valladolid, Spain.
Chamero
Pablo
P
Domingo
Beatriz
B
Molina
Francisca
F
Llopis
Juan
J
Alonso
María Teresa
MT
García-Sancho
Javier
J
D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002451:Cell Compartmentation / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D000097573:Red Fluorescent Protein
D002118:Calcium; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485192:green fluorescent protein, Aequorea victoria