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Find video protocols related to scientific articles indexed in Pubmed.
Molecular identity of the mitochondrial permeability transition pore and its role in ischemia-reperfusion injury.
J. Mol. Cell. Cardiol.
PUBLISHED: 08-27-2014
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The mitochondrial permeability transition is a key event in cell death. Intense research efforts have been focused on elucidating the molecular components of the mitochondrial permeability transition pore (mPTP) to improve the understanding and treatment of various pathologies, including neurodegenerative disorders, cancer and cardiac diseases. Several molecular factors have been proposed as core components of the mPTP; however, further investigation has indicated that these factors are among a wide range of regulators. Thus, the scientific community lacks a clear model of the mPTP. Here, we review the molecular factors involved in the regulation and formation of the mPTP. Furthermore, we propose that the mitochondrial ATP synthase, specifically its c subunit, is the central core component of the mPTP complex. Moreover, we discuss the involvement of the mPTP in ischemia and reperfusion as well as the results of clinical studies targeting the mPTP to ameliorate ischemia-reperfusion injury. This article is part of a Special Issue entitled "Mitochondria".
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Methods to monitor and compare mitochondrial and glycolytic ATP production.
Meth. Enzymol.
PUBLISHED: 05-28-2014
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ATP is commonly considered as the main energy unit of the cell and participates in a variety of cellular processes. Thus, intracellular ATP concentrations rapidly vary in response to a wide variety of stimuli, including nutrients, hormones, cytotoxic agents, and hypoxia. Such alterations not necessarily affect cytosolic and mitochondrial ATP to similar extents. From an oncological perspective, this is particularly relevant in the course of tumor progression as well as in the response of cancer cells to therapy. In normal cells, mitochondrial oxidative phosphorylation (OXPHOS) is the predominant source of ATP. Conversely, many cancer cells exhibit an increased flux through glycolysis irrespective of oxygen tension. Assessing the relative contribution of glycolysis and OXPHOS to intracellular ATP production is fundamental not only for obtaining further insights into the peculiarities and complexities of oncometabolism but also for developing therapeutic and diagnostic tools. Several techniques have been developed to measure intracellular ATP levels including enzymatic methods based on hexokinase, glucose-6-phosphate dehydrogenase, and firefly luciferase. Here, we summarize conventional methods for measuring intracellular ATP levels and we provide a detailed protocol based on cytosol- and mitochondrion-targeted variants of firefly luciferase to determine the relative contribution of glycolysis and OXPHOS to ATP synthesis.
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Methods to monitor ROS production by fluorescence microscopy and fluorometry.
Meth. Enzymol.
PUBLISHED: 05-28-2014
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Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also lead to the premature death of the entire organism. Less severe increases in ROS levels may lead to the nonlethal oxidation of fundamental cellular components, such as proteins, phospholipids, and DNA, hence exerting a mutagenic effect that promotes oncogenesis and tumor progression. Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism.
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STAT3 Activities and Energy Metabolism: Dangerous Liaisons.
Cancers (Basel)
PUBLISHED: 05-13-2014
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STAT3 mediates cytokine and growth factor receptor signalling, becoming transcriptionally active upon tyrosine 705 phosphorylation (Y-P). Constitutively Y-P STAT3 is observed in many tumors that become addicted to its activity, and STAT3 transcriptional activation is required for tumor transformation downstream of several oncogenes. We have recently demonstrated that constitutively active STAT3 drives a metabolic switch towards aerobic glycolysis through the transcriptional induction of Hif-1? and the down-regulation of mitochondrial activity, in both MEF cells expressing constitutively active STAT3 (Stat3C/C) and STAT3-addicted tumor cells. This novel metabolic function is likely involved in mediating pre-oncogenic features in the primary Stat3C/C MEFs such as resistance to apoptosis and senescence and rapid proliferation. Moreover, it strongly contributes to the ability of primary Stat3C/C MEFs to undergo malignant transformation upon spontaneous immortalization, a feature that may explain the well known causative link between STAT3 constitutive activity and tumor transformation under chronic inflammatory conditions. Taken together with the recently uncovered role of STAT3 in regulating energy metabolism from within the mitochondrion when phosphorylated on Ser 727, these data place STAT3 at the center of a hub regulating energy metabolism under different conditions, in most cases promoting cell survival, proliferation and malignant transformation even though with distinct mechanisms.
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Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V.
Biochim. Biophys. Acta
PUBLISHED: 04-11-2014
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The mitochondrial oxidative phosphorylation (OXPHOS) system consists of four electron transport chain (ETC) complexes (CI-CIV) and the FoF1-ATP synthase (CV), which sustain ATP generation via chemiosmotic coupling. The latter requires an inward-directed proton-motive force (PMF) across the mitochondrial inner membrane (MIM) consisting of a proton (?pH) and electrical charge (??) gradient. CI actively participates in sustaining these gradients via trans-MIM proton pumping. Enigmatically, at the cellular level genetic or inhibitor-induced CI dysfunction has been associated with ?? depolarization or hyperpolarization. The cellular mechanism of the latter is still incompletely understood. Here we demonstrate that chronic (24h) CI inhibition in HEK293 cells induces a proton-based ?? hyperpolarization in HEK293 cells without triggering reverse-mode action of CV or the adenine nucleotide translocase (ANT). Hyperpolarization was associated with low levels of CII-driven O2 consumption and prevented by co-inhibition of CII, CIII or CIV activity. In contrast, chronic CIII inhibition triggered CV reverse-mode action and induced ?? depolarization. CI- and CIII-inhibition similarly reduced free matrix ATP levels and increased the cell's dependence on extracellular glucose to maintain cytosolic free ATP. Our findings support a model in which ?? hyperpolarization in CI-inhibited cells results from low activity of CII, CIII and CIV, combined with reduced forward action of CV and ANT.
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Isolation of plasma membrane-associated membranes from rat liver.
Nat Protoc
PUBLISHED: 01-16-2014
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Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ?8-10 h, and it can be easily modified and adapted to other tissues and cell types.
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Mitochondrial disruption occurs downstream from ?-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: differential activation of stress and survival pathways.
Int. J. Biochem. Cell Biol.
PUBLISHED: 03-18-2013
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?-Adrenergic receptor stimulation plays an important role in cardiomyocyte stress responses, which may result in apoptosis and cardiovascular degeneration. We previously demonstrated that toxicity of the ?-adrenergic agonist isoproterenol on H9c2 cardiomyoblasts depends on the stage of cell differentiation. We now investigate ?-adrenergic receptor downstream signaling pathways and stress responses that explain the impact of muscle cell differentiation on hyper-?-adrenergic stimulation-induced cytotoxicity. When incubated with isoproterenol, differentiated H9c2 muscle cells have increased cytosolic calcium, cyclic-adenosine monophosphate content and oxidative stress, as well as mitochondrial depolarization, increased superoxide anion, loss of subunits from the mitochondrial respiratory chain, decreased Bcl-xL content, increased p53 and phosphorylated-p66Shc as well as activated caspase-3. Undifferentiated H9c2 cells incubated with isoproterenol showed increased Bcl-xL protein and increased superoxide dismutase 2 which may act as protective mechanisms. We conclude that the differentiation of H9c2 is associated with differential regulation of stress responses, which impact the toxicity of several agents, namely those acting through ?-adrenergic receptors and resulting in mitochondrial disruption in differentiated cells only.
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Oxidative stress in cardiovascular diseases and obesity: role of p66Shc and protein kinase C.
Oxid Med Cell Longev
PUBLISHED: 01-25-2013
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Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66(Shc) protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases.
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Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition.
Cell Cycle
PUBLISHED: 01-23-2013
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The term "mitochondrial permeability transition" (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called "permeability transition pore complex" (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.
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Left ventricular noncompaction (LVNC) and low mitochondrial membrane potential are specific for Barth syndrome.
J. Inherit. Metab. Dis.
PUBLISHED: 01-02-2013
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Barth syndrome (BTHS) is an X-linked mitochondrial defect characterised by dilated cardiomyopathy, neutropaenia and 3-methylglutaconic aciduria (3-MGCA). We report on two affected brothers with c.646G > A (p.G216R) TAZ gene mutations. The pathogenicity of the mutation, as indicated by the structure-based functional analyses, was further confirmed by abnormal monolysocardiolipin/cardiolipin ratio in dry blood spots of the patients as well as the occurrence of this mutation in another reported BTHS proband. In both brothers, 2D-echocardiography revealed some features of left ventricular noncompaction (LVNC) despite marked differences in the course of the disease; the eldest child presented with isolated cardiomyopathy from late infancy, whereas the youngest showed severe lactic acidosis without 3-MGCA during the neonatal period. An examination of the patients fibroblast cultures revealed that extremely low mitochondrial membrane potentials (mt?? about 50 % of the control value) dominated other unspecific mitochondrial changes detected (respiratory chain dysfunction, abnormal ROS production and depressed antioxidant defense). 1) Our studies confirm generalised mitochondrial dysfunction in the skeletal muscle and the fibroblasts of BTHS patients, especially a severe impairment in the mt?? and the inhibition of complex V activity. It can be hypothesised that impaired mt?? and mitochondrial ATP synthase activity may contribute to episodes of cardiac arrhythmia that occurred unexpectedly in BTHS patients. 2) Severe lactic acidosis without 3-methylglutaconic aciduria in male neonates as well as an asymptomatic mild left ventricular noncompaction may characterise the ranges of natural history of Barth syndrome.
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Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.
PLoS ONE
PUBLISHED: 01-01-2013
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Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+) at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel ?4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the ?4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.
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p66Shc Aging Protein in Control of Fibroblasts Cell Fate.
Int J Mol Sci
PUBLISHED: 07-06-2011
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Reactive oxygen species (ROS) are wieldy accepted as one of the main factors of the aging process. These highly reactive compounds modify nucleic acids, proteins and lipids and affect the functionality of mitochondria in the first case and ultimately of the cell. Any agent or genetic modification that affects ROS production and detoxification can be expected to influence longevity. On the other hand, genetic manipulations leading to increased longevity can be expected to involve cellular changes that affect ROS metabolism. The 66-kDa isoform of the growth factor adaptor Shc (p66Shc) has been recognized as a relevant factor to the oxygen radical theory of aging. The most recent data indicate that p66Shc protein regulates life span in mammals and its phosphorylation on serine 36 is important for the initiation of cell death upon oxidative stress. Moreover, there is strong evidence that apart from aging, p66Shc may be implicated in many oxidative stress-associated pathologies, such as diabetes, mitochondrial and neurodegenerative disorders and tumorigenesis. This article summarizes recent knowledge about the role of p66Shc in aging and senescence and how this protein can influence ROS production and detoxification, focusing on studies performed on skin and skin fibroblasts.
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Calcium signaling around Mitochondria Associated Membranes (MAMs).
Cell Commun. Signal
PUBLISHED: 06-24-2011
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Calcium (Ca2+) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca2+ concentration is dependent either on Ca2+ influx from the extracellular space through the plasma membrane, or on Ca2+ release from intracellular Ca2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca2+ release and Ca2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.
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Mitochondrial calcium homeostasis as potential target for mitochondrial medicine.
Mitochondrion
PUBLISHED: 06-10-2011
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Mitochondria are crucial in different intracellular pathways of signal transduction. Mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions, ranging from energy production to cell death. The fine modulation of mitochondrial calcium (Ca(2+)) homeostasis plays a fundamental role in many of the processes involving this organelle. When mitochondrial Ca(2+) homeostasis is compromised, different pathological conditions can occur, depending on the cell type involved. Recent data have shed light on the molecular identity of the main proteins involved in the handling of mitochondrial Ca(2+) traffic, opening fascinating and ambitious new avenues for mitochondria-based pharmacological strategies.
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Mitochondria-ros crosstalk in the control of cell death and aging.
J Signal Transduct
PUBLISHED: 06-09-2011
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Reactive oxygen species (ROS) are highly reactive molecules, mainly generated inside mitochondria that can oxidize DNA, proteins, and lipids. At physiological levels, ROS function as "redox messengers" in intracellular signalling and regulation, whereas excess ROS induce cell death by promoting the intrinsic apoptotic pathway. Recent work has pointed to a further role of ROS in activation of autophagy and their importance in the regulation of aging. This review will focus on mitochondria as producers and targets of ROS and will summarize different proteins that modulate the redox state of the cell. Moreover, the involvement of ROS and mitochondria in different molecular pathways controlling lifespan will be reported, pointing out the role of ROS as a "balance of power," directing the cell towards life or death.
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Increased reactive oxygen species (ROS) production and low catalase level in fibroblasts of a girl with MEGDEL association (Leigh syndrome, deafness, 3-methylglutaconic aciduria).
Folia Neuropathol
PUBLISHED: 04-02-2011
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Association of 3-methylglutaconic aciduria (3-MGCA) with sensorineural deafness and Leigh-like encephalopathy (MEGDEL) was described as a very rare mitochondrial disorder without a known molecular background. We present clinical and biochemical characteristics of a 4.5-year-old girl with a similar association. The clinical course of the disease was as follows: in the neonatal period transient adaptation troubles; at 4-5 mo failure to thrive and hypotonia; at 13 mo: extrapyramidal symptoms, sensorineural deafness, Leigh syndrome on MRI, pigmentary degeneration of retina, episodes of respiratory alkalosis, increased lactate in plasma, urine and brain, and increased excretion of 3-MGCA. Mitochondrial encephalopathy was suspected and muscle biopsy performed. Only mild lipid accumulation was found by muscle histopathology and histochemistry. Unspecific decrease of respiratory chain complexes was revealed by muscle homogenate spectrophotometry. The in-gel activity assay in the patients muscle confirmed a combined defect of OXPHOS, particularly indicating suppression of mitochondrial ATP synthase (complex V) activity. Measurements of functional mitochondrial bioenergetic parameters in the patients fibroblasts revealed a decrease in the mitochondrial membrane potential and activity of the mitochondrial respiratory chain. At the same time, a significant increase of ROS production (cytosolic and mitochondrial superoxide and H2O2) with signs of protein damage (protein carbonylation), and decreased antioxidant defence (SOD1 and SOD2) were observed. Additionally, the catalase amount was surprisingly low in comparison with healthy control and other reference 3-MGCA cases (Barth syndrome). Conclusion: (1) the natural history of the disease in the presented patient confirms the existence of previously reported clinical phenotype of MEGDEL (2) antioxidant defence impairment due to abnormal catalase metabolism/transport may characterize an unknown basic defect which led to the development of MEGDEL association.
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Inhibition by purine nucleotides of the release of reactive oxygen species from muscle mitochondria: indication for a function of uncoupling proteins as superoxide anion transporters.
Biochem. Biophys. Res. Commun.
PUBLISHED: 03-17-2011
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Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H?O?) and superoxide anion radical (O?·?), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of O?·? rather than that of H?O? was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of O?·? from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of O?·?, thus enabling its removal from the matrix compartment.
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Regulation and protection of mitochondrial physiology by sirtuins.
Mitochondrion
PUBLISHED: 02-16-2011
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The link between sirtuin activity and mitochondrial biology has recently emerged as an important field. This conserved family of NAD(+)-dependent deacetylase proteins has been described to be particularly involved in metabolism and longevity. Recent studies on protein acetylation have uncovered a high number of acetylated mitochondrial proteins indicating that acetylation/deacetylation processes may be important not only for the regulation of mitochondrial homeostasis but also for metabolic dysfunction in the context of various diseases such as metabolic syndrome/diabetes and cancer. The functional involvement of sirtuins as sensors of the redox/nutritional state of mitochondria and their role in mitochondrial protection against stress are hereby described, suggesting that pharmacological manipulation of sirtuins is a viable strategy against several pathologies.
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Mitochondria associated membranes (MAMs) as critical hubs for apoptosis.
Commun Integr Biol
PUBLISHED: 01-30-2011
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Apoptosis is a process of major biomedical interest, since its deregulation is involved in the pathogenesis of a broad variety of disorders (neoplasia, autoimmune disorders, viral and neurodegenerative diseases, to name a few). It is now firmly established that variations in cellular calcium (Ca(2+)) concentration are pivotal in the control of a variety of cellular functions. Strong evidence has been accumulated supporting a central role of Ca(2+) in the regulation of cell death. In particular, in the context of the biochemical mechanisms of apoptosis, increasing evidence support a role for endoplasmic reticulum (ER)-mitochondria Ca(2+) cross talk as a crucial regulator of several pathways of apoptosis. Recent data highlight as also the promyelocytic leukemia protein (PML), by modulating the ER machinery at the contact sites between ER and mitochondria (the mitochondria associated membranes, MAMs), regulates cell survival through the ER-cytosol/mitochondria Ca(2+) signaling.
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Mitochondrial tolerance to drugs and toxic agents in ageing and disease.
Curr Drug Targets
PUBLISHED: 01-29-2011
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Better understanding of the effect of ageing on mitochondrial metabolism and of the mechanisms of action of various drugs is required to allow optimization of the treatment of many diseases with minimized risk of dangerous impairment of mitochondrial function. Numerous reports show that efficacy of medical treatment depends on the age of treated subjects. This applies particularly to the effect of drugs on various senescence-prone cellular pathways. In this review, we demonstrate how ageing affects various mitochondria-associated pathways and their response to a variety of factors. These factors include registered drugs and other chemicals, and account for diverse consequences which vary depending on the physiological condition. Pharmacological treatments aimed at improving mitochondrial function should thus have in mind the subject age.
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Effect of selenite on basic mitochondrial function in human osteosarcoma cells with chronic mitochondrial stress.
Mitochondrion
PUBLISHED: 01-19-2011
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Mitochondrial chronic stress that originates from defective mitochondria is implicated in a growing list of human diseases. To enhance understanding of pathophysiology of chronic mitochondrial dysfunction we investigated human osteosarcoma cells with 2 types of chronic stress: corresponding to the mutation in ATP synthase subunit 6 encoded by mtDNA (NARP syndrome-mild stress) and to a total lack of mtDNA (Rho0 cells-heavy stress). We previously found that selenium influenced mitochondrial stress response and lowered ROS production. Therefore, in this study effect of selenite on other mitochondrial parameters was investigated. We showed that presence of selenium improved survival of starved cells, modified organization of mitochondrial network in NARP cybrids and decreased cytosolic calcium level in NARP and Rho0 cells. Selenium did not affect mitochondrial membrane potential, ATP level, activity of ATP synthase and activity of complex II of the respiratory chain.
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A STAT3-mediated metabolic switch is involved in tumour transformation and STAT3 addiction.
Aging (Albany NY)
PUBLISHED: 11-19-2010
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The pro-oncogenic transcription factor STAT3 is constitutively activated in a wide variety of tumours that often become addicted to its activity, but no unifying view of a core function determining this widespread STAT3-dependence has yet emerged. We show here that constitutively active STAT3 acts as a master regulator of cell metabolism, inducing aerobic glycolysis and down-regulating mitochondrial activity both in primary fibroblasts and in STAT3-dependent tumour cell lines. As a result, cells are protected from apoptosis and senescence while becoming highly sensitive to glucose deprivation. We show that enhanced glycolysis is dependent on HIF-1? up-regulation, while reduced mitochondrial activity is HIF-1?-independent and likely caused by STAT3-mediated down-regulation of mitochondrial proteins. The induction of aerobic glycolysis is an important component of STAT3 pro-oncogenic activities, since inhibition of STAT3 tyrosine phosphorylation in the tumour cell lines down-regulates glycolysis prior to leading to growth arrest and cell death, both in vitro and in vivo. We propose that this novel, central metabolic role is at the core of the addiction for STAT3 shown by so many biologically different tumours.
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PML regulates apoptosis at endoplasmic reticulum by modulating calcium release.
Science
PUBLISHED: 10-28-2010
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The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.
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[Role of the p66Shc protein in physiological state and in pathologies].
Postepy Biochem.
PUBLISHED: 09-29-2010
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The p66Shc adaptor protein has been in the spotlight of many researchgroups around the world for over adecade. Experiments conducted inrecent years unraveled its structure and enabled the recognition of basic cellular functions. Despite an undoubtedly tremendous progress in the characterization of p66Shc, mechanisms through which this protein potentially impacts the metabolism of mitochondria, and thus the cellular energetics are still waiting to be elucidated. Particularly interesting and profoundly studied is the concept that p66Shc may be a key component of the cell response to oxidative stress which may effectively contribute to the lifespan of the organism. p66Shc phosphorylation at serine 36 triggers a cascade of events leading to an increase in reactive oxygen species (ROS) production. The widely accepted free radical theory of ageing, proposed by Harman inthe - 1950s, assumes that an uncontrolled increase of ROS may lead to oxidation of fundamental cellular components such as proteins and phospholipids even sometimes premature dand cause DNA damage. Accumulation of such lesions in cells may unfavorably affect the functions of tissues and organs, leading to pathologies oreath of the organism. Although well experimentally established, knowledge regarding the involvement of the p66Shc protein in the production of ROS and its impact on the lifespan of organisms remains insufficient and requires a lot of additional research. Further investigation will permit a better understanding ofthe mechanisms governing the processes o f aging andthe emergence of variouspathologies associated with oxidative stress. This work is an attempt to systematize the existing knowledge about the p66Shc protein structure and functions. Another objective was to draw attention to the most interesting aspects and results of in vivo and in vitro studies in different models in the context of oxidative stress-associated pathologies and in aging.
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Methyl-beta-cyclodextrin induces mitochondrial cholesterol depletion and alters the mitochondrial structure and bioenergetics.
FEBS Lett.
PUBLISHED: 07-12-2010
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There is growing evidence of mitochondrial membrane raft-like microdomains that are involved in the apoptotic pathway. The aim of this study was to investigate the effect of methyl-beta-cyclodextrin (M?CD), being a well-known lipid microdomain disrupting agent and cholesterol chelator, on the structure and bioenergetics of rat liver mitochondria (RLM). We observed that M?CD decreases the function of RLM, induces changes in the mitochondrial configuration state and decreases the calcium chloride-induced swelling. These data suggest that disruption of mitochondrial raft-like microdomains by cholesterol efflux on one hand impairs mitochondrial bioenergetics, but on the other hand it protects the mitochondria from swelling.
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Cardiotoxicity of the anticancer therapeutic agent bortezomib.
Am. J. Pathol.
PUBLISHED: 06-04-2010
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Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.
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Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders.
Biochim. Biophys. Acta
PUBLISHED: 03-02-2010
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p66Shc, the growth factor adaptor protein, can have a substantial impact on mitochondrial metabolism through regulation of cellular response to oxidative stress. We investigated relationships between the extent of p66Shc phosphorylation at Ser36, mitochondrial dysfunctions and an antioxidant defense reactions in fibroblasts derived from five patients with various mitochondrial disorders (two with mitochondrial DNA mutations and three with methylglutaconic aciduria and genetic defects localized, most probably, in nuclear genes). We found that in all these fibroblasts, the extent of p66Shc phosphorylation at Ser36 was significantly increased. This correlated with a substantially decreased level of mitochondrial superoxide dismutase (SOD2) in these cells. This suggest that SOD2 is under control of the Ser36 phosphorylation status of p66Shc protein. As a consequence, an intracellular oxidative stress and accumulation of damages caused by oxygen free radicals are observed in the cells.
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Mitochondrial fatty acid oxidation and oxidative stress: lack of reverse electron transfer-associated production of reactive oxygen species.
Biochim. Biophys. Acta
PUBLISHED: 01-12-2010
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Reverse electron transfer (RET) from succinate to NAD+ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH2, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD+ nor intramitochondrial reduction of acetoacetate to beta-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.
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Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.
Nat Protoc
PUBLISHED: 10-08-2009
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Many cellular processes require the proper cooperation between mitochondria and the endoplasmic reticulum (ER). Several recent works show that their functional interactions rely on dynamic structural contacts between both organelles. Such contacts, called mitochondria-associated membranes (MAMs), are crucial for the synthesis and intracellular transport of phospholipids, as well as for intracellular Ca(2+) signaling and for the determination of mitochondrial structure. Although several techniques are available to isolate mitochondria, only few are specifically tuned to the isolation of MAM, containing unique regions of ER membranes attached to the outer mitochondrial membrane and mitochondria without contamination from other organelles (i.e., pure mitochondria). Here we provide optimized protocols to isolate these fractions from tissues and cells. These procedures require 4-5 h and can be easily modified and adapted to different tissues and cell types.
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Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?
Int. J. Biochem. Cell Biol.
PUBLISHED: 05-17-2009
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A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.
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Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles.
Int. J. Biochem. Cell Biol.
PUBLISHED: 02-22-2009
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Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca(2+) homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.
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Guanosine diphosphate exerts a lower effect on superoxide release from mitochondrial matrix in the brains of uncoupling protein-2 knockout mice: new evidence for a putative novel function of uncoupling proteins as superoxide anion transporters.
Biochem. Biophys. Res. Commun.
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In this report, we show new experimental evidence that, in mouse brain mitochondria, uncoupling protein-2 (UCP2) can be involved in superoxide (O(2)(·-)) removal from the mitochondrial matrix. We found that the effect of guanosine 5-diphosphate (GDP) on the rate of reactive oxygen species (ROS) release from brain mitochondria of UCP2 knockout mice was less pronounced compared to the wild type animals. This putative novel UCP2 activity, evaluated by the use of UCP2-knockout transgenic animals, along with the known antioxidant defence systems, may provide additional protection from ROS in brain mitochondria.
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Disrupted ATP synthase activity and mitochondrial hyperpolarisation-dependent oxidative stress is associated with p66Shc phosphorylation in fibroblasts of NARP patients.
Int. J. Biochem. Cell Biol.
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p66Shc is an adaptor protein involved in cell proliferation and differentiation that undergoes phosphorylation at Ser36 in response to oxidative stimuli, consequently inducing a burst of reactive oxygen species (ROS), mitochondrial disruption and apoptosis. Its role during several pathologies suggests that p66Shc mitochondrial signalling can perpetuate a primary mitochondrial defect, thus contributing to the pathophysiology of that condition. Here, we show that in the fibroblasts of neuropathy, ataxia and retinitis pigmentosa (NARP) patients, the p66Shc phosphorylation pathway is significantly induced in response to intracellular oxidative stress related to disrupted ATP synthase activity and mitochondrial membrane hyperpolarisation. We postulate that the increased phosphorylation of p66Shc at Ser36 is partially responsible for further increasing ROS production, resulting in oxidative damage of proteins. Oxidative stress and p66Shc phosphorylation at Ser36 may be mitigated by antioxidant administration or the use of a p66Shc phosphorylation inhibitor. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
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Cardiac mitochondrial dysfunction during hyperglycemia--the role of oxidative stress and p66Shc signaling.
Int. J. Biochem. Cell Biol.
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Diabetes mellitus is a chronic disease caused by a deficiency in the production of insulin and/or by the effects of insulin resistance. Insulin deficiency leads to hyperglycemia which is the major initiator of diabetic cardiovascular complications escalating with time and driven by many complex biochemical and molecular processes. Four hypotheses, which propose mechanisms of diabetes-associated pathophysiology, are currently considered. Cardiovascular impairment may be caused by an increase in polyol pathway flux, by intracellular advanced glycation end-products formation or increased flux through the hexosamine pathway. The latter of these mechanisms involves activation of the protein kinase C. Cellular and mitochondrial metabolism alterations observed in the course of diabetes are partially associated with an excessive production of reactive oxygen species (ROS). Among many processes and factors involved in ROS production, the 66 kDa isoform of the growth factor adaptor shc (p66Shc protein) is of particular interest. This protein plays a key role in the control of mitochondria-dependent oxidative balance thus it involvement in diabetic complications and other oxidative stress based pathologies is recently intensively studied. In this review we summarize the current understanding of hyperglycemia induced cardiac mitochondrial dysfunction with an emphasis on the oxidative stress and p66Shc protein. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.
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ATP synthesis and storage.
Purinergic Signal.
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Since 1929, when it was discovered that ATP is a substrate for muscle contraction, the knowledge about this purine nucleotide has been greatly expanded. Many aspects of cell metabolism revolve around ATP production and consumption. It is important to understand the concepts of glucose and oxygen consumption in aerobic and anaerobic life and to link bioenergetics with the vast amount of reactions occurring within cells. ATP is universally seen as the energy exchange factor that connects anabolism and catabolism but also fuels processes such as motile contraction, phosphorylations, and active transport. It is also a signalling molecule in the purinergic signalling mechanisms. In this review, we will discuss all the main mechanisms of ATP production linked to ADP phosphorylation as well the regulation of these mechanisms during stress conditions and in connection with calcium signalling events. Recent advances regarding ATP storage and its special significance for purinergic signalling will also be reviewed.
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Mitochondrial Ca(2+) and apoptosis.
Cell Calcium
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Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca(2+)) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca(2+) homeostasis and in turn modulate the effectiveness of Ca(2+)-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca(2+) overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.
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Mitochondria-associated membranes (MAMs) as hotspot Ca(2+) signaling units.
Adv. Exp. Med. Biol.
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The tight interplay between endoplasmic reticulum (ER) and mitochondria is a key determinant of cell function and survival through the control of intracellular calcium (Ca(2+)) signaling. The specific sites of physical association between ER and mitochondria are known as mitochondria-associated membranes (MAMs). It has recently become clear that MAMs are crucial for highly efficient transmission of Ca(2+) from the ER to mitochondria, thus controlling fundamental processes involved in energy production and also determining cell fate by triggering or preventing apoptosis. In this contribution, we summarize the main features of the Ca(2+)-signaling toolkit, covering also the latest breakthroughs in the field, such as the identification of novel candidate proteins implicated in mitochondrial Ca(2+) transport and the recent direct characterization of the high-Ca(2+) microdomains between ER and mitochondria. We review the main functions of these two organelles, with special emphasis on Ca(2+) handling and on the structural and molecular foundations of the signaling contacts between them. Additionally, we provide important examples of the physiopathological role of this cross-talk, briefly describing the key role played by MAMs proteins in many diseases, and shedding light on the essential role of mitochondria-ER interactions in the maintenance of cellular homeostasis and the determination of cell fate.
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Effect of mtDNA point mutations on cellular bioenergetics.
Biochim. Biophys. Acta
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This overview discusses the results of research on the effects of most frequent mtDNA point mutations on cellular bioenergetics. Thirteen proteins coded by mtDNA are crucial for oxidative phosphorylation, 11 of them constitute key components of the respiratory chain complexes I, III and IV and 2 of mitochondrial ATP synthase. Moreover, pathogenic point mutations in mitochondrial tRNAs and rRNAs generate abnormal synthesis of the mtDNA coded proteins. Thus, pathogenic point mutations in mtDNA usually disturb the level of key parameter of the oxidative phosphorylation, i.e. the electric potential on the inner mitochondrial membrane (??), and in a consequence calcium signalling and mitochondrial dynamics in the cell. Mitochondrial generation of reactive oxygen species is also modified in the mutated cells. The results obtained with cultured cells and describing biochemical consequences of mtDNA point mutations are full of contradictions. Still they help elucidate the biochemical basis of pathologies and provide a valuable tool for finding remedies in the future. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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