The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP ₃ receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.

Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca(2+) homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca(2+) released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca(2+) entering across plasma membrane channels, because the Ca(2+) ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca(2+) entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/rotenone reduced CCE by approximately 35%. These observations indicate that close contact to Ca(2+) influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca(2+) propagation within interconnected mitochondria.

To study the role of calreticulin in Ca²⁺ homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca²⁺ signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with “cameleon” indicators. Induction of calreticulin increased the free Ca²⁺ concentration within the ER lumen, [Ca²⁺]_ER, from 306 ± 31 to 595 ± 53 μm, and doubled the rate of ER refilling. [Ca²⁺]_ER remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca²⁺ATPases. Under these conditions, store-operated Ca²⁺ influx appeared inhibited but could be reactivated by decreasing [Ca²⁺]_ER with the low affinity Ca²⁺ chelatorN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca²⁺]_ER decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca²⁺ response and, surprisingly, with a shorter mitochondrial Ca²⁺ response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca²⁺responses were also observed in cells expressing anN-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca²⁺ fluxes across the ER but decreases mitochondrial Ca²⁺ and membrane potential. The increased Ca²⁺ turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

Mitochondria have been found to sequester and release Ca²⁺ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca²⁺ that sustain activity of capacitative Ca²⁺ entry (CCE). Procedures that prevent mitochondrial Ca²⁺ uptake inhibit local Ca²⁺ buffering and CCE, but it is not clear whether Ca²⁺ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca²⁺ efflux on the ability of mitochondria to buffer subplasmalemmal Ca²⁺, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca²⁺ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca²⁺. However, subsequent removal of extracellular Ca²⁺ produced a reversible decrease in [Ca²⁺]_mito, indicating that Ca²⁺ was continuously taken up and released by mitochondria, although [Ca²⁺]_mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger with CGP 37157 increased [Ca²⁺]_mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca²⁺, resulting in an increased activity of BK_Ca channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca²⁺ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca²⁺ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca²⁺, to facilitate CCE, and to relay Ca²⁺ from the plasma membrane to the ER.

Stromal interaction molecule (STIM) proteins are putative ER Ca²⁺ sensors that recruit and activate store-operated Ca²⁺ (SOC) channels at the plasma membrane, a process triggered by the Ca²⁺ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca²⁺ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca²⁺ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca²⁺ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca²⁺ concentration above physiological values, but no cytosolic Ca²⁺ signals were detected during store refilling even at very high Ca²⁺ concentrations. [Ca²⁺]_ER measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca²⁺]_ER levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca²⁺ entry, because Ca²⁺ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.

Endoplasmic reticulum (ER) Ca2+ refilling is an active process to ensure an appropriate ER Ca2+ content under basal conditions and to maintain or restore ER Ca2+ concentration during/after cell stimulation. The mechanisms to achieve successful ER Ca2+ refilling are multiple and built on a concerted action of processes that provide a suitable reservoir for Ca2+ sequestration into the ER. Despite mitochondria having been found to play an essential role in the maintenance of capacitative Ca2+ entry by buffering subplasmalemmal Ca2+, their contribution to ER Ca2+ refilling was not subjected to detailed analysis so far. Thus, this study was designed to elucidate the involvement of mitochondria in Ca2+ store refilling during and after cell stimulation. ER Ca2+ refilling was found to be accomplished even during continuous inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release by an agonist. Basically, ER Ca2+ refilling depended on the presence of extracellular Ca2+ as the source and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity. Interestingly, in the presence of an IP3-generating agonist, ER Ca2+ refilling was prevented by the inhibition of trans-mitochondrial Ca2+ flux by CGP 37157 (7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one) that precludes the mitochondrial Na+/Ca2+ exchanger as well as by mitochondrial depolarization using a mixture of oligomycin and antimycin A. In contrast, after the removal of the agonist, ER refilling was found to be largely independent of trans-mitochondrial Ca2+ flux. Under these conditions, ER Ca2+ refilling took place even without an associated Ca2+ elevation in the deeper cytosol, thus, indicating that superficial ER domains mimic mitochondrial Ca2+ buffering and efficiently sequester subplasmalemmal Ca2+ and consequently facilitate capacitative Ca2+ entry. Hence, these data point to different contribution of mitochondria in the process of ER Ca2+ refilling based on the presence or absence of IP3, which represents the turning point for the dependence or autonomy of ER Ca2+ refilling from trans-mitochondrial Ca2+ flux.