The fungicide cymoxanil impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition.

Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide.

OPA1 deficiency impairs oxidative metabolism in cycling cells, underlining a translational approach for degenerative diseases.

Dominant optic atrophy is an optic neuropathy with varying clinical symptoms and progression. A severe disorder is associated with certain OPA1 mutations and includes additional symptoms for >20% of patients. This underscores the consequences of OPA1 mutations in different cellular populations, not only retinal ganglionic cells. We assessed the effects of OPA1 loss of function on oxidative metabolism and antioxidant defences using an RNA-silencing strategy in a human epithelial cell line. We observed a decrease in the mitochondrial respiratory chain complexes, associated with a reduction in aconitase activity related to an increase in reactive oxygen species (ROS) production. In response, the NRF2 (also known as NFE2L2) transcription factor was translocated into the nucleus and upregulated SOD1 and GSTP1. This study highlights the effects of OPA1 deficiency on oxidative metabolism in replicative cells, as already shown in neurons. It underlines a translational process to use cycling cells to circumvent and describe oxidative metabolism. Moreover, it paves the way to predict the evolution of dominant optic atrophy using mathematical models that consider mitochondrial ROS production and their detoxifying pathways.

Measurement of fish freshness: Flow cytometry analysis of isolated muscle mitochondria.

Mitochondria are real sensors of the physiological status of tissues. After the death of an animal, they maintain physiological activity for several days. This activity is highly dependent on the availability of nutrients in the tissue. In this study, flow cytometry was used to measure the membrane potential of mitochondria isolated from European seabass (Dicentrarchus labrax) red muscle stored in ice for seven days in order to characterize fish freshness. Two probes, TMRM and Rhodamine 123, were used to measure mitochondrial potential. During the first few days (D0 to D3), isolated mitochondria maintained high potential, and then lost their potential (from D3 to D5), but were always re-polarizable after addition of substrates (glutamate, malate and succinate). From D7, the mitochondria were more strongly depolarized and were difficult to repolarize by the substrates. Using flow cytometry, we demonstrated that mitochondria were an excellent marker to confirm seabass freshness.

Cell fusion enhances energy metabolism of mesenchymal tumor hybrid cells to sustain their proliferation and invasion.

BACKGROUND: Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. RESULTS: Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-ß-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. CONCLUSION: Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress.

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

TMEM70 forms oligomeric scaffolds within mitochondrial cristae promoting in situ assembly of mammalian ATP synthase proton channel.

Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzyme's biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (Fo). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a direct correlation between TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation, two-dimensional blue-native/SDS-PAGE, and pulse-chase experiments reveal that TMEM70 forms large oligomers that interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.

The Warburg Effect in Yeast: Repression of Mitochondrial Metabolism Is Not a Prerequisite to Promote Cell Proliferation.

O. Warburg conducted one of the first studies on tumor energy metabolism. His early discoveries pointed out that cancer cells display a decreased respiration and an increased glycolysis proportional to the increase in their growth rate, suggesting that they mainly depend on fermentative metabolism for ATP generation. Warburg's results and hypothesis generated controversies that are persistent to this day. It is thus of great importance to understand the mechanisms by which cancer cells can reversibly regulate the two pathways of their energy metabolism as well as the functioning of this metabolism in cell proliferation. Here, we made use of yeast as a model to study the Warburg effect and its eventual function in allowing an increased ATP synthesis to support cell proliferation. The role of oxidative phosphorylation repression in this effect was investigated. We show that yeast is a good model to study the Warburg effect, where all parameters and their modulation in the presence of glucose can be reconstituted. Moreover, we show that in this model, mitochondria are not dysfunctional, but that there are fewer mitochondria respiratory chain units per cell. Identification of the molecular mechanisms involved in this process allowed us to dissociate the parameters involved in the Warburg effect and show that oxidative phosphorylation repression is not mandatory to promote cell growth. Last but not least, we were able to show that neither cellular ATP synthesis flux nor glucose consumption flux controls cellular growth rate.

Ascorbate maintains a low plasma oxygen level.

In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 µM in fresh plasma vs < 2 µM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 µM), compared to control (Ascorbateplasma > 15 µM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy.

"Labile" heme critically regulates mitochondrial biogenesis through the transcriptional co-activator Hap4p in Saccharomyces cerevisiae.

Heme (iron protoporphyrin IX) is a well-known prosthetic group for enzymes involved in metabolic pathways such as oxygen transport and electron transfer through the mitochondrial respiratory chain. However, heme has also been shown to be an important regulatory molecule (as "labile" heme) for diverse processes such as translation, kinase activity, and transcription in mammals, yeast, and bacteria. Taking advantage of a yeast strain deficient for heme production that enabled controlled modulation and monitoring of labile heme levels, here we investigated the role of labile heme in the regulation of mitochondrial biogenesis. This process is regulated by the HAP complex in yeast. Using several biochemical assays along with EM and epifluorescence microscopy, to the best of our knowledge, we show for the first time that cellular labile heme is critical for the post-translational regulation of HAP complex activity, most likely through the stability of the transcriptional co-activator Hap4p. Consequently, we found that labile heme regulates mitochondrial biogenesis and cell growth. The findings of our work highlight a new mechanism in the regulation of mitochondrial biogenesis by cellular metabolites.

Modelling mitochondrial ROS production by the respiratory chain.

ROS (superoxide and oxygen peroxide in this paper) play a dual role as signalling molecules and strong oxidizing agents leading to oxidative stress. Their production mainly occurs in mitochondria although they may have other locations (such as NADPH oxidase in particular cell types). Mitochondrial ROS production depends in an interweaving way upon many factors such as the membrane potential, the cell type and the respiratory substrates. Moreover, it is experimentally difficult to quantitatively assess the contribution of each potential site in the respiratory chain. To overcome these difficulties, mathematical models have been developed with different degrees of complexity in order to analyse different physiological questions ranging from a simple reproduction/simulation of experimental results to a detailed model of the possible mechanisms leading to ROS production. Here, we analyse experimental results concerning ROS production including results still under discussion. We then critically review the three models of ROS production in the whole respiratory chain available in the literature and propose some direction for future modelling work.

Shigella-mediated oxygen depletion is essential for intestinal mucosa colonization.

Pathogenic enterobacteria face various oxygen (O2) levels during intestinal colonization from the O2-deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O2 in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O2 is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.

Mitochondrial Biogenesis and Mitochondrial Reactive Oxygen Species (ROS): A Complex Relationship Regulated by the cAMP/PKA Signaling Pathway.

Mitochondrial biogenesis is a complex process. It requires the contribution of both the nuclear and the mitochondrial genomes and therefore cross talk between the nucleus and mitochondria. Cellular energy demand can vary by great length and it is now well known that one way to adjust adenosine triphosphate (ATP) synthesis to energy demand is through modulation of mitochondrial content in eukaryotes. The knowledge of actors and signals regulating mitochondrial biogenesis is thus of high importance. Here, we review the regulation of mitochondrial biogenesis both in yeast and in mammalian cells through mitochondrial reactive oxygen species.

Advanced glycation end-products disrupt human endothelial cells redox homeostasis: new insights into reactive oxygen species production.

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H2DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H2DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.

Microwell array integrating nanoelectrodes for coupled opto-electrochemical monitorings of single mitochondria.

Chips composed of microwell arrays integrating nanoelectrodes (OptoElecWell) were developed to achieve dual optical and electrochemical detections on isolated biological entities. Each array consists in 106 microwells of 6 µm diameter × 5.2 µm height each, with a transparent bottom surface for optical observations, a platinum nano-ring electrode at its half-height for in situ electrochemistry, and a top open surface to inject solutions. Then, populations of individual mitochondria isolated from yeasts (Saccharomyces cerevisiae) were let to sediment on the array and be trapped within microwells. The trapping efficiency reached 20% but owing to the large number of microwells on the platform, hundreds of them could be filled simultaneously by single mitochondria. This allowed to follow up their individual energetic status based on fluorescence microscopy of their endogenous NADH. Simultaneously, the array of interconnected Pt nanoelectrodes in the microwells was used to monitor in situ variations of dioxygen consumed by all mitochondria captured in the device. Mitochondrial bioenergetics were modulated sequentially using respiratory chain-ATP synthase substrates (ethanol and ADP) and inhibitor (antimycin A). Overall, we show how two complementary analytical approaches, fluorescence and electrochemical detections, can be coupled for a multi-parametric monitoring of mitochondrial activities, with a resolution ranging from a small population (whole device) to the single mitochondrion level (unique well).

Neutrophil Metabolic Shift during their Lifecycle: Impact on their Survival and Activation.

Polymorphonuclear neutrophils (PMNs) are innate immune cells, which represent 50% to 70% of the total circulating leukocytes. How PMNs adapt to various microenvironments encountered during their life cycle, from the bone marrow, to the blood plasma fraction, and to inflamed or infected tissues remains largely unexplored. Metabolic shifts have been reported in other immune cells such as macrophages or lymphocytes, in response to local changes in their microenvironment, and in association with a modulation of their pro-inflammatory or anti-inflammatory functions. The potential contribution of metabolic shifts in the modulation of neutrophil activation or survival is anticipated even though it is not yet fully described. If neutrophils are considered to be mainly glycolytic, the relative importance of alternative metabolic pathways, such as the pentose phosphate pathway, glutaminolysis, or the mitochondrial oxidative metabolism, has not been fully considered during activation. This statement may be explained by the lack of knowledge regarding the local availability of key metabolites such as glucose, glutamine, and substrates, such as oxygen from the bone marrow to inflamed tissues. As highlighted in this review, the link between specific metabolic pathways and neutrophil activation has been outlined in many reports. However, the impact of neutrophil activation on metabolic shifts' induction has not yet been explored. Beyond its importance in neutrophil survival capacity in response to available metabolites, metabolic shifts may also contribute to neutrophil population heterogeneity reported in cancer (tumor-associated neutrophil) or auto-immune diseases (Low/High Density Neutrophils). This represents an active field of research. In conclusion, the characterization of neutrophil metabolic shifts is an emerging field that may provide important knowledge on neutrophil physiology and activation modulation. The related question of microenvironmental changes occurring during inflammation, to which neutrophils will respond to, will have to be addressed to fully appreciate the importance of neutrophil metabolic shifts in inflammatory diseases.

Prestressed cells are prone to cytoskeleton failures under localized shear strain: an experimental demonstration on muscle precursor cells.

We report on a wavelet based space-scale decomposition method for analyzing the response of living muscle precursor cells (C2C12 myoblasts and myotubes) upon sharp indentation with an AFM cantilever and quantifying their aptitude to sustain such a local shear strain. Beyond global mechanical parameters which are currently used as markers of cell contractility, we emphasize the necessity of characterizing more closely the local fluctuations of the shear relaxation modulus as they carry important clues about the mechanisms of cytoskeleton strain release. Rupture events encountered during fixed velocity shear strain are interpreted as local disruptions of the actin cytoskeleton structures, the strongest (brittle) ones being produced by the tighter and stiffer stress fibers or actin agglomerates. These local strain induced failures are important characteristics of the resilience of these cells, and their aptitude to maintain their shape via a quick recovery from local strains. This study focuses on the perinuclear region because it can be considered as a master mechanical organizing center of these muscle precursor cells. Using this wavelet-based method, we combine the global and local approaches for a comparative analysis of the mechanical parameters of normal myoblasts, myotubes and myoblasts treated with actomyosin cytoskeleton disruptive agents (ATP depletion, blebbistatin).

The role of glycolysis-derived hexose phosphates in the induction of the Crabtree effect.

Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect.

Glycated human albumin alters mitochondrial respiration in preadipocyte 3T3-L1 cells.

Diabetes and obesity are strongly associated with increased levels of circulating advanced glycation end products (AGEs) and reactive oxygen species (ROS). These two molecular phenomena affect the physiology of adipose tissue, a biological driver of the metabolic syndrome, leading to an inflammatory profile and insulin resistance, which could contribute to obesity/diabetes-associated complications, such as cardiovascular diseases. Herein, we investigated the impact of AGEs on mitochondrial bioenergetics in murine preadipocyte cells (3T3-L1) and cellular redox homeostasis. We show that incubation of preadipocytes with AGEs stimulates mitochondrial activity and respiration while inducing oxidative stress. This AGE-induced intracellular ROS production was blocked by diphenylene iodonium, an NAD(P)H oxidase inhibitor. In parallel, antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase) were found to be activated upon AGE treatment. Our results suggest that AGE-induced oxidative stress is generated by NAD(P)H oxidase and leads to a cellular proliferation arrest associated with enhanced mitochondrial metabolism and biogenesis, and with increased levels of ROS-detoxifying enzymes, as well. These new data show how AGEs may be involved in hyperglycemia-induced oxidative damage in preadipocytes and their potential links to diabetes progression. © 2017 BioFactors, 43(4):577-592, 2017.