Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ~1,200 slices of 10 nm thickness from a 28 μm × 15 μm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule (STIM)-gated ORAI channels at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites maintains adequate levels of Ca2+ within the ER lumen during Ca2+ signaling. Disruption of ER Ca2+ homeostasis activates the unfolded protein response (UPR) to restore proteostasis. Here, we report that the UPR transducer inositol-requiring enzyme 1 (IRE1) interacts with STIM1, promotes ER-PM contact sites, and enhances SOCE. IRE1 deficiency reduces T cell activation and human myoblast differentiation. In turn, STIM1 deficiency reduces IRE1 signaling after store depletion. Using a CaMPARI2-based Ca2+ genome-wide screen, we identify CAMKG2 and slc105a as SOCE enhancers during ER stress. Our findings unveil a direct crosstalk between SOCE and UPR via IRE1, acting as key regulator of ER Ca2+ and proteostasis in T cells and muscles. Under ER stress, this IRE1-STIM1 axis boosts SOCE to preserve immune cell functions, a pathway that could be targeted for cancer immunotherapy.

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

T-type Ca(2+) channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca(2+) elevations during anoxia. To test this hypothesis, we measured the Ca(2+) concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca(2+) elevations that were reduced by Cav3.2 inhibition (50 μm Ni(2+)) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca(2+) elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni(2+) and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni(2+) and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca(2+) load actively extruded by plasma membrane Ca(2+) pumps. The cytosolic Ca(2+) elevations were paralleled by mitochondrial Ca(2+) elevations that were also increased by Cav3.2 overexpression and decreased by Ni(2+) but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca(2+) uniporter, the major mitochondrial Ca(2+) uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca(2+) elevations. Selective activation of T-type Ca(2+) channels evoked both cytosolic and mitochondrial Ca(2+) elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca(2+) ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca(2+) overload.

The uncoupling proteins UCP2 and UCP3 have been postulated to catalyze Ca(2+) entry across the inner membrane of mitochondria, but this proposal is disputed, and other, unrelated proteins have since been identified as the mitochondrial Ca(2+) uniporter. To clarify the role of UCPs in mitochondrial Ca(2+) handling, we down-regulated the expression of the only uncoupling protein of HeLa cells, UCP3, and measured Ca(2+) and ATP levels in the cytosol and in organelles with genetically encoded probes. UCP3 silencing did not alter mitochondrial Ca(2+) uptake in permeabilized cells. In intact cells, however, UCP3 depletion increased mitochondrial ATP production and strongly reduced the cytosolic and mitochondrial Ca(2+) elevations evoked by histamine. The reduced Ca(2+) elevations were due to inhibition of store-operated Ca(2+) entry and reduced depletion of endoplasmic reticulum (ER) Ca(2+) stores. UCP3 depletion accelerated the ER Ca(2+) refilling kinetics, indicating that the activity of sarco/endoplasmic reticulum Ca(2+) (SERCA) pumps was increased. Accordingly, SERCA inhibitors reversed the effects of UCP3 depletion on cytosolic, ER, and mitochondrial Ca(2+) responses. Our results indicate that UCP3 is not a mitochondrial Ca(2+) uniporter and that it instead negatively modulates the activity of SERCA by limiting mitochondrial ATP production. The effects of UCP3 on mitochondrial Ca(2+) thus reflect metabolic alterations that impact on cellular Ca(2+) homeostasis. The sensitivity of SERCA to mitochondrial ATP production suggests that mitochondria control the local ATP availability at ER Ca(2+) uptake and release sites.

Nutrient secretagogues activate mitochondria of the pancreatic beta-cell through the provision of substrate, hyperpolarisation of the inner mitochondrial membrane and mitochondrial calcium rises. We report that mitochondrial matrix pH, a parameter not previously studied in the beta-cell, also exerts an important control function in mitochondrial metabolism. During nutrient stimulation matrix pH alkalinises, monitored by the mitochondrial targeted fluorescent pH-sensitive protein mtAlpHi or (31)P-NMR inorganic phosphate chemical shifts following saturation transfer. Compared with other cell types, the resting mitochondrial pH was surprisingly low, rising from pH 7.25 to 7.7 during nutrient stimulation of rat beta-cells. As cytosolic alkalinisation to the nutrient was of much smaller amplitude, the matrix alkalinisation was accompanied by a pronounced increase of the DeltapH across the inner mitochondrial membrane. Furthermore, matrix alkalinisation closely correlates with the cytosolic ATP net increase, which is also associated with elevated ATP synthesis rates in mitochondria. Preventing DeltapH increases in permeabilised cells abrogated substrate-driven ATP synthesis. We propose that the mitochondrial pH and DeltapH are key determinants of mitochondrial energy metabolism and metabolite transport important for cell activation.

Selective Serotonin Reuptake Inhibitor antidepressants, such as fluoxetine (Prozac), have been shown to induce cell death in cancer cells, paving the way for their potential use as cancer therapy. These compounds are able to increase cytosolic calcium concentration ([Ca2+]cyt), but the involved mechanisms and their physiological consequences are still not well understood. Here, we show that fluoxetine induces an increase in [Ca2+]cyt by emptying the endoplasmic reticulum (ER) through the translocon, an ER Ca2+ leakage structure. Our data also show that fluoxetine inhibits oxygen consumption and lowers mitochondrial ATP. This latter is essential for Ca2+ reuptake into the ER, and we postulated therefore that the fluoxetine-induced decrease in mitochondrial ATP production results in the emptying of the ER, leading to capacitative calcium entry. Furthermore, Ca2+ quickly accumulated in the mitochondria, leading to mitochondrial Ca2+ overload and cell death. We found that fluoxetine could induce an early necrosis in human peripheral blood lymphocytes and Jurkat cells, and could also induce late apoptosis, especially in the tumor cell line. These results shed light on fluoxetine-induced cell death and its potential use in cancer treatment.

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψm) whose origin is unclear. Using structurally distinct genetically encoded pH-sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed "mitopHlashes". Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψm drops or respiratory chain inhibition. Opa1 ablation does not alter Δψm fluctuations but drastically decreases the efficiency of mitopHlash/Δψm coupling, which is restored by re-expressing fusion-deficient OPA1K301A and preserved in cells lacking the outer-membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψm uncoupling occurs early during staurosporine-induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψm by an explosive matrix pH flash.

Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G1 transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G1 cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski-/- MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.

The NADPH-oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H+ extrusion through depolarization-activated H+ channels. Whether H+ fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H+ channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H+ current (IH), causing an approximately -40-mV shift in the activation threshold that leads to the appearance of inward IH. To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn2+]. IH from PMA-treated cells activated faster and at lower voltages, enabling sustained H+ influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie was delayed, and sometimes prevented, by cytosolic ATP and GTP-gamma-S. A good correlation was observed between the amplitude of Ie and both inward and outward IH when a stable driving force for e- was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and IH amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.

Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However, the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1), YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP), a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction, in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally, advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly, gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together, our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy.

Tubular aggregate myopathy (TAM) is a progressive skeletal muscle disease associated with gain-of-function mutations in the ER Ca2+ sensor STIM1 that mediates store-operated Ca2+ entry (SOCE) across the Ca2+-release-activated (CRAC) Ca2+ channel ORAI1. A frameshift mutation in STIM1 inactivation domain, STIM1I484R, was identified in a TAM patient and reported to decrease SOCE. Using ion imaging and electrophysiology, we show that the STIM1I484R mutation instead renders STIM1 constitutively active. In ion imaging experiments, STIM1I484R was less efficient than native STIM1 when expressed alone but enhanced SOCE and increased basal Ca2+ and Mn2+ influx when expressed together with ORAI1. In patch-clamp recordings, STIM1I484R generated larger pre-activated CRAC currents lacking slow Ca2+-dependent inhibition (SCDI). STIM1I484R was pre-recruited in plasma membrane clusters when co-expressed with ORAI1, as were mutants truncated at the frameshift residue or lacking EB-1-binding, which recapitulated STIM1I484R gain-of-function. When expressed alone in human primary myoblasts, STIM1I484R was pre-recruited in large clusters and increased basal Ca2+ entry. These observations establish that STIM1I484R confers a gain of CRAC channel function due to the loss of critical inhibitory C-terminal domains that prevent STIM1 binding to ORAI1, enable STIM1 trapping by microtubules, and mediate SCDI, providing a mechanistic explanation for the muscular defects of TAM patients bearing this mutation.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

Accumulation of misfolded proteins in the brain is a common hallmark of most age-related neurodegenerative diseases. Previous studies from our group identified the presence of anti-inflammatory and antioxidant compounds in leaves derived from the Chilean berry Ugni molinae (murtilla), in addition to show a potent anti-aggregation activity in models of Alzheimer´s disease. However, possible beneficial effects of berry extracts of murtilla was not investigated. Here we evaluated the efficacy of fruit extracts from different genotypes of Chilean-native U. molinae on reducing protein aggregation using cellular models of Huntington´s disease and assess the correlation with their chemical composition. Berry extraction was performed by exhaustive maceration with increasing-polarity solvents. An unbiased automatic microscopy platform was used for cytotoxicity and protein aggregation studies in HEK293 cells using polyglutamine-EGFP fusion proteins, followed by secondary validation using biochemical assays. Phenolic-rich extracts from murtilla berries of the 19-1 genotype (ETE 19-1) significantly reduced polyglutamine peptide aggregation levels, correlating with the modulation in the expression levels of autophagy-related proteins. Using LC-MS and molecular network analysis we correlated the presence of flavonoids, phenolic acids, and ellagitannins with the protective effects of ETE 19-1 effects on protein aggregation. Overall, our results indicate the presence of bioactive components in ethanolic extracts from U. molinae berries that reduce the load of protein aggregates in living cells.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH(m) (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.