There were errors published in J. Cell Sci. 124, 2143-2152.
In the section given below, PtdIns(3,4,5)P3 was on four occasions incorrectly printed instead of the correct Ins(1,4,5)P3.
We apologise for this mistake.
Increased mitochondrial Ca2+ drives the adaptive metabolic boost observed during early phases of ER stress
Increases in mitochondrial respiration and ATP production are often consequences of increases in mitochondrial Ca2+ (Green and Wang, 2010). In order to determine whether early phases of ER stress induced by tunicamycin increased mitochondrial Ca2+, we treated cells expressing cytosolic or mitochondrial aequorins with histamine [which evokes Ins(1,4,5)P3-dependent Ca2+ release] and compared their mitochondrial Ca2+ uptake. We observed that histamine led to a mitochondrial Ca2+ uptake that was significantly higher in tunicamycin-pretreated cells (P<0.05; 4 hours) than in untreated cells (Fig. 6A). Cytosolic Ca2+ increased similarly in tunicamycin-treated and untreated cells (Fig. 6B). These results indicate that the differences in mitochondrial Ca2+ levels are not due to altered Ca2+ release mediated by the Ins(1,4,5)P3 receptor but to an enhanced mitochondrial Ca2+ uptake, presumably due to the increased apposition of ER and mitochondrial Ca2+ channels. By using a different dye, Fura-2, we monitored the peak cytosolic Ca2+ levels after thapsigargin addition, reflecting the kinetics of Ca2+ release after sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition. After 4 hours of tunicamycin treatment, the thapsigargin-induced Ca2+ peak was increased, and it was further elevated by inhibition of mitochondrial Ca2+ uptake using Ru360 (Fig. 6C). These results suggest that, besides the Ins(1,4,5)P3-receptor-mediated direct Ca2+ transfer from the ER to neighboring mitochondria, an additional phenomenon associated with the early phases of ER stress involves Ca2+ leak from the ER, also resulting in mitochondrial Ca2+ uptake. Indeed, no mitochondrial Ca2+ uptake following the thapsigargin-induced Ca2+ leak was observed in Mfn2-knockout cells (Fig. 6D), which is reflected by the lack of effect of Ru360. This result further indicates that juxtaposition of mitochondria with the ER is necessary for the mitochondrial Ca2+ uptake evoked by Ca2+ leak during early phases of ER stress.
Finally, to test whether mitochondrial Ca2+ levels control the metabolic mitochondrial boost, we measured oxygen consumption rates resulting from OXPHOS in the presence of the Ins(1,4,5)P3 receptor inhibitor xestospongin B or the mitochondrial Ca2+ uptake inhibitor RuRed. We observed that both xestospongin B and RuRed decreased the rate of oxygen consumption after tunicamycin treatment (Fig. 7A,B), which confirms that increased mitochondrial Ca2+ uptake, resulting from ER–mitochondrial coupling, is necessary for the metabolic response observed during early phases of ER stress. Therefore, in order to evaluate whether the early metabolic boost forms part of an adaptive response triggered by ER stress, we inhibited mitochondrial Ca2+ uptake and measured cell viability [through propidium iodide (PI) incorporation] and Δψm. We observed that the inhibition of mitochondrial Ca2+ uptake during the early phase of ER stress increased cell death (PI-positive cells) and also decreased Δψm at 48 hours (Fig. 7C).
In total, the results presented in this study suggest strongly that Ca2+ transfer resulting from enhanced ER–mitochondrial coupling leads to a localized increase in mitochondrial metabolism, thus providing energetic substrates key for a cellular adaptive response in face of ER stress. Further experiments will determine whether this bioenergetic response is necessary for improving the energetic state of the ER, and therefore its folding capacity, or, as it is restricted to perinuclear zones, for the activation of a specific nuclear transcriptional program that participates in the cellular adaptation to stress.