Mitochondria take up calcium during cell activation thus shaping Ca2+ signaling and exocytosis. In turn, Ca2+ uptake by mitochondria increases respiration and ATP synthesis. Targeted aequorins are excellent Ca2+ probes for subcellular analysis, but single-cell imaging has proven difficult. Here we combine virus-based expression of targeted aequorins with photon-counting imaging to resolve dynamics of the cytosolic, mitochondrial, and nuclear Ca2+ signals at the single-cell level in anterior pituitary cells. These cells exhibit spontaneous electric activity and cytosolic Ca2+oscillations that are responsible for basal secretion of pituitary hormones and are modulated by hypophysiotrophic factors. Aequorin reported spontaneous [Ca2+] oscillations in all the three compartments, bulk cytosol, nucleus, and mitochondria. Interestingly, a fraction of mitochondria underwent much larger [Ca2+] oscillations, which were driven by local high [Ca2+] domains generated by the spontaneous electric activity. These oscillations were large enough to stimulate respiration, providing the basis for local tune-up of mitochondrial function by the Ca2+ signal.
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