There were errors published in J. Cell Sci. 124, 2143Sci. 124, -2152 In the section given below, PtdIns(3,4,5)P 3 was on four occasions incorrectly printed instead of the correct Ins(1,4,5)P 3 .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 Ca 2+ (Green and Wang, 2010). In order to determine whether early phases of ER stress induced by tunicamycin increased mitochondrial Ca 2+ , we treated cells expressing cytosolic or mitochondrial aequorins with histamine [which evokes Ins(1,4,5)P 3 -dependent Ca2+ release] and compared their mitochondrial Ca 2+ uptake. We observed that histamine led to a mitochondrial Ca 2+ uptake that was significantly higher in tunicamycinpretreated cells (P<0.05; 4 hours) than in untreated cells (Fig. 6A). Cytosolic Ca 2+ increased similarly in tunicamycin-treated and untreated cells (Fig. 6B). These results indicate that the differences in mitochondrial Ca 2+ levels are not due to altered Ca 2+ release mediated by the Ins(1,4,5)P 3 receptor but to an enhanced mitochondrial Ca 2+ uptake, presumably due to the increased apposition of ER and mitochondrial Ca 2+ channels. By using a different dye, Fura-2, we monitored the peak cytosolic Ca 2+ levels after thapsigargin addition, reflecting the kinetics of Ca 2+ release after sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibition. After 4 hours of tunicamycin treatment, the thapsigargin-induced Ca 2+ peak was increased, and it was further elevated by inhibition of mitochondrial Ca 2+ uptake using Ru360 (Fig. 6C). These results suggest that, besides the Ins(1,4,5)P 3 -receptor-mediated direct Ca 2+ transfer from the ER to neighboring mitochondria, an additional phenomenon associated with the early phases of ER stress involves Ca 2+ leak from the ER, also resulting in mitochondrial Ca 2+ uptake. Indeed, no mitochondrial Ca 2+ uptake following the thapsigargin-induced Ca 2+ 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 Ca 2+ uptake evoked by Ca 2+ leak during early phases of ER stress.Finally, to test whether mitochondrial Ca 2+ levels control the metabolic mitochondrial boost, we measured oxygen consumption rates resulting from OXPHOS in the presence of the Ins(1,4,5)P 3 receptor inhibitor xestospongin B or the mitochondrial Ca 2+ 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 Ca 2+ 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 triggere...

There were errors published in J. Cell Sci. 124, 2143Sci. 124, -2152 In the section given below, PtdIns(3,4,5)P 3 was on four occasions incorrectly printed instead of the correct Ins(1,4,5)P 3 .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 Ca 2+ (Green and Wang, 2010). In order to determine whether early phases of ER stress induced by tunicamycin increased mitochondrial Ca 2+ , we treated cells expressing cytosolic or mitochondrial aequorins with histamine [which evokes Ins(1,4,5)P 3 -dependent Ca2+ release] and compared their mitochondrial Ca 2+ uptake. We observed that histamine led to a mitochondrial Ca 2+ uptake that was significantly higher in tunicamycinpretreated cells (P<0.05; 4 hours) than in untreated cells (Fig. 6A). Cytosolic Ca 2+ increased similarly in tunicamycin-treated and untreated cells (Fig. 6B). These results indicate that the differences in mitochondrial Ca 2+ levels are not due to altered Ca 2+ release mediated by the Ins(1,4,5)P 3 receptor but to an enhanced mitochondrial Ca 2+ uptake, presumably due to the increased apposition of ER and mitochondrial Ca 2+ channels. By using a different dye, Fura-2, we monitored the peak cytosolic Ca 2+ levels after thapsigargin addition, reflecting the kinetics of Ca 2+ release after sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibition. After 4 hours of tunicamycin treatment, the thapsigargin-induced Ca 2+ peak was increased, and it was further elevated by inhibition of mitochondrial Ca 2+ uptake using Ru360 (Fig. 6C). These results suggest that, besides the Ins(1,4,5)P 3 -receptor-mediated direct Ca 2+ transfer from the ER to neighboring mitochondria, an additional phenomenon associated with the early phases of ER stress involves Ca 2+ leak from the ER, also resulting in mitochondrial Ca 2+ uptake. Indeed, no mitochondrial Ca 2+ uptake following the thapsigargin-induced Ca 2+ 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 Ca 2+ uptake evoked by Ca 2+ leak during early phases of ER stress.Finally, to test whether mitochondrial Ca 2+ levels control the metabolic mitochondrial boost, we measured oxygen consumption rates resulting from OXPHOS in the presence of the Ins(1,4,5)P 3 receptor inhibitor xestospongin B or the mitochondrial Ca 2+ 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 Ca 2+ 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 trigger...

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin-proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.