Mitochondria form an apical calcium signaling domain in renal cortical collecting duct cells

Abstract

In several epithelia, Mitochondria sequester intracellular Ca2+ to create Ca2+ signaling domains. We asked whether mitochondria could form barriers to Ca2+ diffusion in renal cortical collecting duct (CCD) cells. We began by staining each of two cell lines (from Xenopus and mouse) with mitotracker red. Mitochondria in both cell lines were localized in bands with a prominent band near the apical surface and a weaker band at the basal pole. To determine the proximity of the apical band to the membrane, we fluorescently tagged PIP2 to label the apical membrane and found that the apical mitochondrial band was localized just beneath the membrane. Many apical membrane proteins are regulated by endoplasmic reticular Ca2+ release. If mitochondria do sequester calcium to create an apical membrane Ca2+ signaling domain, the ER must be able to cross the mitochondrial barrier at certain points so that release of Ca2+ from the ER can reach its desired targets. Cells co‐stained with mitotracker red and ERtracker blue showed that ER crossed the mitochondrial barrier in many places. Next we sought to determine whether mitochondrial bands can prevent movement of intracellular Ca2+ from one pole to another. Live cells stained with the mitochondrial Ca2+ dye Rhodamine 2‐AM showed Ca2+ uptake in the apical but not basal band after treatment with the Ca2+ ionophore ionomycin. Cells transfected with a membrane‐tethered intracellular Ca2+ sensor showed increased Ca2+ at the apical but not basal pole following apical ionomycin. These data suggest that mitochondrial bands in the CCD create an apical signaling domain for Ca2+ regulation of membrane proteins. This work was supported by NIH R37‐DK037963 and T32‐ DK07656 and AHA 13POST16820072.