Assessments of individual fiber glycogen and mitochondrial volume percentages reveal a graded reduction in muscle oxidative power during prolonged exhaustive exercise.

During submaximal exercise, there is a heterogeneous recruitment of skeletal muscle fibers, with an ensuing heterogeneous depletion of muscle glycogen both within and between fiber types. Here, we show that the mean (95% CI) mitochondrial volume as a percentage of fiber volume of non-glycogen-depleted fibers was 2 (-10:6), 5 (-21:11), and 12 (-21:-2)% lower than all the sampled fibers after continuing exercise for 1, 2 h, and until task failure, respectively. Therefore, a glycogen-dependent fatigue of individual fibers during submaximal exercise may reduce the muscular oxidative power. These findings suggest a relationship between glycogen and mitochondrial content in individual muscle fibers, which is important for understanding fatigue during prolonged exercise.

Presynaptic Mitochondrial Volume and Packing Density Scale with Presynaptic Power Demand.

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. Neurons have presumably optimized the volume of their bioenergetic machinery to ensure these power demands are met, but the relationship between presynaptic power demands and the volume available to the bioenergetic machinery has never been quantified. Here, we estimated the power demands of six motor nerve terminals in female Drosophila larvae through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min-1·µl-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·µl-1 at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·µl-1 of cytosol at rest, rising to 4.36 during activity. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.SIGNIFICANCE STATEMENT The remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here, we exploit the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the first in vivo estimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.

OPENER Is a Nuclear Envelope and Mitochondria Localized Protein Required for Cell Cycle Progression in Arabidopsis.

Currently one-third of the proteins encoded by the Arabidopsis (Arabidopsis thaliana) genome are of unknown function. Some of these unknown proteins are likely to be involved in uncharacterized vital biological processes. Evolutionarily conserved single copy genes in flowering plants have been shown to be enriched in essential housekeeping functions. This together with publicly available gene expression data allows for a focused search for uncharacterized essential genes. Here we identify an essential single copy gene called OPENER (OPNR) in Arabidopsis. We show that OPNR is predominantly expressed in actively dividing cells and performs essential functions in seed development and root meristem maintenance. Cell cycle tracking using 5-ethynyl-2'-deoxyuridine staining and fluorescent cell cycle markers together with the increased size of nucleolus and nucleus in opnr mutants indicate that OPNR is required for cell cycle progression through the S or G2 phases. Intriguingly, OPNR localizes to the nuclear envelope and mitochondria. Furthermore, the nuclear envelope localization of OPNR is dependent on its interaction with nuclear inner membrane Sad1/UNC-84 (SUN) domain proteins SUN1 and SUN2. Taken together our results open a line of investigation into an evolutionarily conserved essential cellular process occurring in both the nuclear envelopes and mitochondria of dividing cells.

Endurance Training Increases the Running Performance of Untrained Men without Changing the Mitochondrial Volume Density in the Gastrocnemius Muscle.

The activity and quantity of mitochondrial proteins and the mitochondrial volume density (MitoVD) are higher in trained muscles; however, the underlying mechanisms remain unclear. Our goal was to determine if 20 weeks' endurance training simultaneously increases running performance, the amount and activity of mitochondrial proteins, and MitoVD in the gastrocnemius muscle in humans. Eight healthy, untrained young men completed a 20-week moderate-intensity running training program. The training increased the mean speed of a 1500 m run by 14.0% (p = 0.008) and the running speed at 85% of maximal heart rate by 9.6% (p = 0.008). In the gastrocnemius muscle, training significantly increased mitochondrial dynamics markers, i.e., peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) by 23%, mitochondrial transcription factor A (TFAM) by 29%, optic artrophy-1 (OPA1) by 31% and mitochondrial fission factor (MFF) by 44%, and voltage-dependent anion channel 1 (VDAC1) by 30%. Furthermore, training increased the amount and maximal activity of citrate synthase (CS) by 10% and 65%, respectively, and the amount and maximal activity of cytochrome c oxidase (COX) by 57% and 42%, respectively, but had no effect on the total MitoVD in the gastrocnemius muscle. We concluded that not MitoVD per se, but mitochondrial COX activity (reflecting oxidative phosphorylation activity), should be regarded as a biomarker of muscle adaptation to endurance training in beginner runners.

(-)-Epicatechin induces mitochondrial biogenesis and markers of muscle regeneration in adults with Becker muscular dystrophy.

INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.

Mitochondria Turnover and Lysosomal Function in Hematopoietic Stem Cell Metabolism.

Hematopoietic stem cells (HSCs) reside in a hypoxic microenvironment that enables glycolysis-fueled metabolism and reduces oxidative stress. Nonetheless, metabolic regulation in organelles such as the mitochondria and lysosomes as well as autophagic processes have been implicated as essential for the determination of HSC cell fate. This review encompasses the current understanding of anaerobic metabolism in HSCs as well as the emerging roles of mitochondrial metabolism and lysosomal regulation for hematopoietic homeostasis.

Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.SIGNIFICANCE STATEMENT Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle.

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

Fusion and fission: interlinked processes critical for mitochondrial health.

Mitochondria are dynamic organelles that continually undergo fusion and fission. These opposing processes work in concert to maintain the shape, size, and number of mitochondria and their physiological function. Some of the major molecules mediating mitochondrial fusion and fission in mammals have been discovered, but the underlying molecular mechanisms are only partially unraveled. In particular, the cast of characters involved in mitochondrial fission needs to be clarified. By enabling content mixing between mitochondria, fusion and fission serve to maintain a homogeneous and healthy mitochondrial population. Mitochondrial dynamics has been linked to multiple mitochondrial functions, including mitochondrial DNA stability, respiratory capacity, apoptosis, response to cellular stress, and mitophagy. Because of these important functions, mitochondrial fusion and fission are essential in mammals, and even mild defects in mitochondrial dynamics are associated with disease. A better understanding of these processes likely will ultimately lead to improvements in human health.

Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

A Reproducible, Objective Method Using MitoTracker® Fluorescent Dyes to Assess Mitochondrial Mass in T Cells by Flow Cytometry.

MitoTracker ® dyes are fluorescent compounds that allow cellular mitochondrial content to be measured semi-quantitatively by flow cytometry and have been used extensively in immunology publications. However, the parameters commonly reported, mean or median fluorescence intensity and percentage of cells that are MitoTracker® "high", can be influenced by variability in cytometer setup, dye stability, and operator subjectivity, making it difficult to compare data between experiments. Here, we describe a method to identify MitoTracker® "high" populations in an objective manner. When analyzing data, we first removed outliers using a pre-specified threshold, determined the fluorescence intensity of the brightest and dimmest events to obtain the fluorescence range and then gated cells within the top 90% of this range. This strategy substantially reduced variability between technical replicates and produced consistent results when data were analyzed by different operators. Consistent with previous reports and other analysis strategies, this analysis method demonstrated that within an individual, CD4+ T cells exhibit significantly higher mitochondrial mass than CD8+ T cells. Objective gating increases the reliability and utility of data generated using MitoTracker® dyes. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes ß-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

Hypermetabolic macrophages in rheumatoid arthritis and coronary artery disease due to glycogen synthase kinase 3b inactivation.

OBJECTIVES: Accelerated atherosclerotic disease typically complicates rheumatoid arthritis (RA), leading to premature cardiovascular death. Inflammatory macrophages are key effector cells in both rheumatoid synovitis and the plaques of coronary artery disease (CAD). Whether both diseases share macrophage-dependent pathogenic mechanisms is unknown. METHODS: Patients with RA or CAD (at least one myocardial infarction) and healthy age-matched controls were recruited into the study. Peripheral blood CD14+ monocytes were differentiated into macrophages. Metabolic profiles were assessed by Seahorse Analyzer, intracellular ATP concentrations were quantified and mitochondrial protein localisation was determined by confocal image analysis. RESULTS: In macrophages from patients with RA or CAD, mitochondria consumed more oxygen, generated more ATP and built tight interorganelle connections with the endoplasmic reticulum, forming mitochondria-associated membranes (MAM). Calcium transfer through MAM sites sustained mitochondrial hyperactivity and was dependent on inactivation of glycogen synthase kinase 3b (GSK3b), a serine/threonine kinase functioning as a metabolic switch. In patient-derived macrophages, inactivated pGSK3b-Ser9 co-precipitated with the mitochondrial fraction. Immunostaining of atherosclerotic plaques and synovial lesions confirmed that most macrophages had inactivated GSK3b. MAM formation and GSK3b inactivation sustained production of the collagenase cathepsin K, a macrophage effector function closely correlated with clinical disease activity in RA and CAD. CONCLUSIONS: Re-organisation of the macrophage metabolism in patients with RA and CAD drives unopposed oxygen consumption and ultimately, excessive production of tissue-destructive enzymes. The underlying molecular defect relates to the deactivation of GSK3b, which controls mitochondrial fuel influx and as such represents a potential therapeutic target for anti-inflammatory therapy.

Sexual dimorphism of substrate utilization: Differences in skeletal muscle mitochondrial volume density and function.

NEW FINDINGS: What is the central question of this study? Females rely to a greater extent than males on fat oxidation during exercise. Whether any difference in skeletal muscle mitochondrial phenotype and oxidative capacity contributes to this sexual dimorphism remains incompletely explored. What is the main finding and its importance? Female prioritization of fat during exercise occurs in parallel to augmented mitochondrial volume density and intrinsic fatty acid and lactate oxidation in skeletal muscle fibres compared with males, independently of aerobic exercise capacity. The enlarged metabolic machinery in skeletal muscle of females is associated with lower body size and leg mass. ABSTRACT: Fat oxidation during exercise is greater in females than in males. We sought to determine whether sex differences in substrate metabolism are paralleled by distinct skeletal muscle mitochondrial volume density and oxidative capacity. Whole-body substrate (fat and carbohydrate) utilization during submaximal treadmill running was assessed, and skeletal muscle biopsies were taken to determine mitochondrial volume density and function in healthy young females (n = 12) and males (n = 12) matched by aerobic exercise capacity and exercise performance. Females presented a lower respiratory exchange ratio (0.87 ± 0.04 versus 0.91 ± 0.04, P = 0.023) and whole-body carbohydrate oxidation (27.8 ± 8.3 versus 35.8 ± 6.5 mg kg-1  min-1 , P = 0.027), whereas fat oxidation was higher (8.7 ± 2.8 versus 5.9 ± 2.6 mg kg-1  min-1 , P = 0.034) during submaximal exercise compared with males. In skeletal muscle biopsies, females demonstrated augmented mitochondrial volume density (7.51 ± 1.77 versus 5.90 ± 1.72%, P = 0.035) and oxidative capacity for fatty acid [36.6 ± 12.8 versus 24.5 ± 7.3 pmol O2  s-1  (mg wet weight)-1 , P = 0.009] and lactate [71.1 ± 24.4 versus 53.2 ± 14.6 pmol O2  s-1  (mg wet weight)-1 , P = 0.040]. No sex differences in respiratory exchange ratio, whole-body fat oxidation and skeletal muscle variables were detected when adjusted for anthropometric variables including body mass or leg mass, which were lower in females. In conclusion, female prioritization of fat over carbohydrate oxidation during exercise is underpinned by augmented body size-related mitochondrial volume density, fatty acid and lactate oxidative capacity in skeletal muscle fibres.

MicroRNA-7 Regulates the Function of Mitochondrial Permeability Transition Pore by Targeting VDAC1 Expression.

Mitochondrial dysfunction is one of the major contributors to neurodegenerative disorders including Parkinson disease. The mitochondrial permeability transition pore is a protein complex located on the mitochondrial membrane. Under cellular stress, the pore opens, increasing the release of pro-apoptotic proteins, and ultimately resulting in cell death. MicroRNA-7 (miR-7) is a small non-coding RNA that has been found to exhibit a protective role in the cellular models of Parkinson disease. In the present study, miR-7 was predicted to regulate the function of mitochondria, according to gene ontology analysis of proteins that are down-regulated by miR-7. Indeed, miR-7 overexpression inhibited mitochondrial fragmentation, mitochondrial depolarization, cytochrome c release, reactive oxygen species generation, and release of mitochondrial calcium in response to 1-methyl-4-phenylpyridinium (MPP(+)) in human neuroblastoma SH-SY5Y cells. In addition, several of these findings were confirmed in mouse primary neurons. Among the mitochondrial proteins identified by gene ontology analysis, the expression of voltage-dependent anion channel 1 (VDAC1), a constituent of the mitochondrial permeability transition pore, was down-regulated by miR-7 through targeting 3'-untranslated region of VDAC1 mRNA. Similar to miR-7 overexpression, knockdown of VDAC1 also led to a decrease in intracellular reactive oxygen species generation and subsequent cellular protection against MPP(+). Notably, overexpression of VDAC1 without the 3'-UTR significantly abolished the protective effects of miR-7 against MPP(+)-induced cytotoxicity and mitochondrial dysfunction, suggesting that the protective effect of miR-7 is partly exerted through promoting mitochondrial function by targeting VDAC1 expression. These findings point to a novel mechanism by which miR-7 accomplishes neuroprotection by improving mitochondrial health.

Mitochondrial defects and neuromuscular degeneration caused by altered expression of Drosophila Gdap1: implications for the Charcot-Marie-Tooth neuropathy.

One of the genes involved in Charcot-Marie-Tooth (CMT) disease, an inherited peripheral neuropathy, is GDAP1. In this work, we show that there is a true ortholog of this gene in Drosophila, which we have named Gdap1. By up- and down-regulation of Gdap1 in a tissue-specific manner, we show that altering its levels of expression produces changes in mitochondrial size, morphology and distribution, and neuronal and muscular degeneration. Interestingly, muscular degeneration is tissue-autonomous and not dependent on innervation. Metabolic analyses of our experimental genotypes suggest that alterations in oxidative stress are not a primary cause of the neuromuscular degeneration but a long-term consequence of the underlying mitochondrial dysfunction. Our results contribute to a better understanding of the role of mitochondria in CMT disease and pave the way to generate clinically relevant disease models to study the relationship between mitochondrial dynamics and peripheral neurodegeneration.

Mitochondria-type GPAT is required for mitochondrial fusion.

Glycerol-3-phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER-GPAT and mitochondrial (Mt)-GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt-GPAT is essential for mitochondrial fusion. Mutation of Mt-GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt-GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP-1 and by overexpression of mitochondrial fusion protein FZO-1/mitofusin, suggesting that the fusion/fission balance is affected by Mt-GPAT depletion. Mitochondrial fragmentation was also observed in Mt-GPAT-depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt-GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt-GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein.

Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress.

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

Age decreases mitochondrial motility and increases mitochondrial size in vascular smooth muscle.

KEY POINTS: Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria. ABSTRACT: Mitochondrial function, motility and architecture are each central to cell function. Age-associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill-defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic 'flickers' of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker-assisted localization microscopy; FaLM). In freshly-isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X-Y areas in both young adult (3 months; 0.05-6.58 µm(2) ) and aged rats (18 months; 0.05-13.4 µm(2) ). In cells from young animals, most mitochondria were small (mode area 0.051 µm(2) ) compared to aged animals (0.710 µm(2) ). Cells from older animals contained a subpopulation of highly-elongated mitochondria (5.3% were >2 µm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 µm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 µm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian-like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 µm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly-elongated. These age-associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation.