Spatially organised mitochondrial calcium uptake through a novel pathway in chick neurones.
AuthorsCoatesworth W, Bolsover S
JournalCell Calcium
PubMed ID16338004
'A brief depolarisation of chick sensory neurones evokes a calcium increase in mitochondria that peaks 1-2s after the depolarisation event and then decays over tens of seconds. Peripheral mitochondria take up more calcium than do central ones, even when the cytosolic calcium increase is spatially homogeneous. The calcium influx into ... More
Tissue transglutaminase is a multifunctional BH3-only protein.
AuthorsRodolfo C, Mormone E, Matarrese P, Ciccosanti F, Farrace MG, Garofano E, Piredda L, Fimia GM, Malorni W, Piacentini M
JournalJ Biol Chem
PubMed ID15485857
'Tissue transglutaminase (TG2) protein accumulates to high levels in cells during early stages of apoptosis both in vivo and in vitro. The analysis of the TG2 primary sequence showed the presence of an eight amino acid domain, sharing 70% identity with the Bcl-2 family BH3 domain. Cell-permeable peptides, mimicking the ... More
Methods for assessing autophagy and autophagic cell death.
AuthorsTasdemir E, Galluzzi L, Maiuri MC, Criollo A, Vitale I, Hangen E, Modjtahedi N, Kroemer G,
JournalMethods Mol Biol
PubMed ID18425442
'Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. ... More
Trafficking of ganglioside GD3 to mitochondria by tumor necrosis factor-alpha.
AuthorsGarcía-Ruiz C, Colell A, Morales A, Calvo M, Enrich C, Fernández-Checa JC
JournalJ Biol Chem
PubMed ID12118012
'The interaction of mitochondria with proapoptotic proteins activates apoptosis pathways. Previous findings have identified ganglioside GD3 (GD3) as an emerging apoptotic lipid intermediate that targets mitochondria in response to death signals. Using immunoelectron and laser scanning confocal microscopy, we characterize the trafficking of GD3 to mitochondria in response to tumor ... More
Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2.
AuthorsUnkila M, McColl KS, Thomenius MJ, Heiskanen K, Distelhorst CW
JournalJ Biol Chem
PubMed ID11489892
'A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release ... More
Towards an understanding of apoptosis detection by SYTO dyes.
AuthorsWlodkowic D, Skommer J, Pelkonen J
JournalCytometry A
PubMed ID17200958
'BACKGROUND: SYTO probes are gaining momentum as reliable and easy to use markers of apoptotic cell death, but the phenomenon underlying reduced SYTO fluorescence in apoptotic cells as compared with normal cells is still not fully elucidated. Herein, we attempt to provide further insights into mechanisms of reduced SYTO16 fluorescence ... More
Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition.
AuthorsAlano CC, Ying W, Swanson RA
JournalJ Biol Chem
PubMed ID14960594
'Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD(+) depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations ... More
Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes.
'We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes ... More
Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra.
AuthorsTursun P, Tashiro M, Konishi M
JournalBiophys J
PubMed ID15626700
'The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, ... More
Assessment of membrane potentials of mitochondrial populations in living cells.
AuthorsZhang H, Huang HM, Carson RC, Mahmood J, Thomas HM, Gibson GE
JournalAnal Biochem
PubMed ID11757503
'Mitochondrial membrane potentials (MMP) reflect the functional status of mitochondria within cells. Fluorescent probes to estimate these potentials within cells have been available for some time, but measurements of populations of mitochondria are not possible by existing methods. Therefore, comparisons between different cell types (e.g., fibroblasts and neuroblastoma), fibroblast cell ... More
Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells.
AuthorsNagy G, Barcza M, Gonchoroff N, Phillips PE, Perl A
JournalJ Immunol
PubMed ID15356113
'Abnormal T cell activation and cell death underlie the pathology of systemic lupus erythematosus. Although mitochondrial hyperpolarization (MHP) represents an early and reversible checkpoint of T cell activation and apoptosis, lupus T cells exhibit persistent MHP. NO has recently been recognized as a key signal of mitochondrial biogenesis and mediator ... More
Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.
AuthorsRauen U, Kerkweg U, Weisheit D, Petrat F, Sustmann R, de Groot H
JournalFree Radic Biol Med
PubMed ID14680689
'We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4 degrees ... More
Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes.
AuthorsDiaz G, Liu S, Isola R, Diana A, Falchi AM
JournalHistochem Cell Biol
PubMed ID14574587
'Mitochondria are the main source of reactive oxygen species (ROS). The aim of this work was to verify the ROS generation in situ in HeLa cells exposed to prooxidants and antioxidants (menadione, tert-butyl hydroperoxide, antimycin A, vitamin E, N-acetyl-L-cysteine, and butylated hydroxytoluene) using the ROS-sensitive probes 6-carboxy-2'',7''-dichlorodihydrofluorescein diacetate di-acetomethyl ester ... More
Nucleic acid binding agents exert local toxic effects on neurites via a non-nuclear mechanism.
AuthorsPin S, Chen H, Lein PJ, Wang MM
JournalJ Neurochem
PubMed ID16441515
'The mechanism by which drugs that target nucleic acids cause neurotoxicity is not well described. We characterized the neurotoxicity of Hoechst 33342 (bis-benzimide), a common cell permeable nuclear dye, in primary neuronal cultures. The mechanism of cell death was not apoptotic, as death is rapid, not accompanied by typical nuclear ... More
Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.
AuthorsScaduto RC, Grotyohann LW
JournalBiophys J
PubMed ID9876159
'We investigated the use of rhodamine 123 (R123), tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) as fluorescent probes to monitor the membrane potential of mitochondria. These indicator dyes are lipophilic cations accumulated by mitochondria in proportion to DeltaPsi. Upon accumulation, all three dyes exhibit a red shift in ... More
'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress.
AuthorsJohnson-Cadwell LI, Jekabsons MB, Wang A, Polster BM, Nicholls DG
JournalJ Neurochem
PubMed ID17437552
'Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that ''mild uncoupling'' could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Deltapsi(m)) were determined in parallel as a function of FCCP ... More
Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress.
AuthorsGyulkhandanyan AV, Pennefather PS
JournalJ Neurochem
PubMed ID15228597
'We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher ... More
Mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate-induced necrosis, apoptosis, and tolerance.
'A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either delta psi(m) or delta psi(p), ... More
Multiparameter detection of apoptosis using red-excitable SYTO probes.
'Functional assays allowing phenotypic characterization of different cell death parameters at a single-cell level are important tools for preclinical anticancer drug screening. Currently, the selection of cytometric assays is limited by the availability of fluorescent probes with overlapping spectral characteristics. Following on our earlier reports on green and orange fluorescent ... More
Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.
AuthorsGalluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnóczky G, Hardwick JM, Hengartner MO, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Ma
JournalCell Death Differ
PubMed ID19373242
'Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, ... More
Molecular cloning and characterization of a human mitochondrial ceramidase.
AuthorsEl Bawab S, Roddy P, Qian T, Bielawska A, Lemasters JJ, Hannun YA
JournalJ Biol Chem
PubMed ID10781606
'We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of ... More
Methods for the assessment of mitochondrial membrane permeabilization in apoptosis.
AuthorsGalluzzi L, Zamzami N, de La Motte Rouge T, Lemaire C, Brenner C, Kroemer G
JournalApoptosis
PubMed ID17294081
'Mitochondrial membrane permeabilization (MMP) is considered as the "point-of-no-return" in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable ... More
The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.
AuthorsLemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B
JournalBiochim Biophys Acta
PubMed ID9714796
'Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative ... More
Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase.
AuthorsAbramov AY, Canevari L, Duchen MR
JournalJ Neurosci
PubMed ID14724257
'Beta-amyloid (betaA) peptide is strongly implicated in the neurodegeneration underlying Alzheimer''s disease, but the mechanisms of neurotoxicity remain controversial. This study establishes a central role for oxidative stress by the activation of NADPH oxidase in astrocytes as the cause of betaA-induced neuronal death. betaA causes a loss of mitochondrial potential ... More
Gliotoxin-mediated apoptosis of activated human hepatic stellate cells.
'BACKGROUND: Activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis, and apoptosis of activated HSCs might be essential to clear HSCs from injured liver. Gliotoxin induces apoptosis of activated human and rat HSCs by an unknown mechanism. AIM: This study investigated the role of reactive oxygen species ... More
Preconditioning protects by inhibiting the mitochondrial permeability transition.
'Mitochondrial permeability transition (mPT) is a crucial event in the progression to cell death in the setting of ischemia-reperfusion. We have used a model system in which mPT can be reliably and reproducibly induced to test the hypothesis that the profound protection associated with the phenomenon of myocardial preconditioning is ... More
Mitochondrial depolarization following hydrogen sulfide exposure in erythrocytes from a sulfide-tolerant marine invertebrate.
AuthorsJulian D, April KL, Patel S, Stein JR, Wohlgemuth SE
JournalJ Exp Biol
PubMed ID16244170
'Sulfide-tolerant marine invertebrates employ a variety of mechanisms to detoxify sulfide once it has entered their bodies, but their integumentary, respiratory epithelium and circulatory cells may still be exposed to toxic sulfide concentrations. To investigate whether sulfide exposure is toxic to mitochondria of a sulfide-tolerant invertebrate, we used the fluorescent ... More
Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1.
AuthorsFrieden M, James D, Castelbou C, Danckaert A, Martinou JC, Demaurex N
JournalJ Biol Chem
PubMed ID15024001
'Mitochondria modulate Ca(2+) signals by taking up, buffering, and releasing Ca(2+) at key locations near Ca(2+) release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission ... More
Neurotoxic nitric oxide rapidly depolarizes and permeabilizes mitochondria by dynamically opening the mitochondrial transition pore.
'Exposure of SH-SY5Y neuroblastoma or rat cortical neurons to diethylenetriamine-NO (DETA-NO) rapidly depolarized mitochondria. In SH-SY5Y DETA-NO activated caspase 3 and produced cell death. Mitochondrial depolarization in SH-SY5Y was visualized both with JC-1 accumulation and as dequenching of calcein fluorescence in mitochondria initially loaded with calcein-AM and tetramethylrhodamine methyl ester ... More
Role of hepatic transporters in the disposition and hepatotoxicity of a HER2 tyrosine kinase inhibitor CP-724,714.
AuthorsFeng B, Xu JJ, Bi YA, Mireles R, Davidson R, Duignan DB, Campbell S, Kostrubsky VE, Dunn MC, Smith AR, Wang HF,
JournalToxicol Sci
PubMed ID19223659
'CP-724,714, a potent and selective orally active HER2 tyrosine kinase inhibitor, was discontinued from clinical development due to unexpected hepatotoxicity in cancer patients. Based on the clinical manifestation of the toxicity, CP-724,714 likely exerted its hepatotoxicity via both hepatocellular injury and hepatobiliary cholestatic mechanisms. The direct cytotoxic effect, hepatobiliary disposition ... More
Modulation of mitochondrial Ca(2+) homeostasis by Bcl-2.
'We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. ... More
F16, a mitochondriotoxic compound, triggers apoptosis or necrosis depending on the genetic background of the target carcinoma cell.
AuthorsFantin VR, Leder P
JournalCancer Res
PubMed ID14729642
'Mutations that lead to the emergence of resistance to apoptosis are commonly observed among tumor cells. Some of the proteins affected are integral parts of the apoptotic cascade such as pro- and antiapoptotic members of the Bcl-2 family. F16 is a small molecule that accumulates in mitochondria of a variety ... More
Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential.
AuthorsXu JJ, Diaz D, O'Brien PJ
JournalChem Biol Interact
PubMed ID15522265
'While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a ... More
Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals.
AuthorsIchas F, Jouaville LS, Mazat JP
JournalCell
PubMed ID9215636
'We report Ca2(+)-induced release of Ca2+ from mitochondria (mCICR) dependent on transitory opening of the permeability transition pore (PTP) operating in a low conductance mode. The Ca2+ fluxes taking place during mCICR are a direct consequence of the mitochondrial depolarization spike (mDPS) caused by PTP opening. Both mDPS and mCICR ... More
Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype.
AuthorsJazayeri M, Andreyev A, Will Y, Ward M, Anderson CM, Clevenger W,
JournalJ Biol Chem
PubMed ID12645575
'We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) ... More
Bax regulates production of superoxide in both apoptotic and nonapoptotic neurons: role of caspases.
AuthorsKirkland RA, Saavedra GM, Cummings BS, Franklin JL,
JournalJ Neurosci
PubMed ID21123558
'A Bax- and, apparently, mitochondria-dependent increase in superoxide (O(2)(·-)) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant ... More
Examining intracellular organelle function using fluorescent probes: from animalcules to quantum dots.
AuthorsZorov DB, Kobrinsky E, Juhaszova M, Sollott SJ
JournalCirc Res
PubMed ID15297386
'Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in ... More
Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study.
'The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine ... More
Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.
AuthorsGeibel S, Zimmermann D, Zifarelli G, Becker A, Koenderink JB, Hu YK, Kaplan JH, Friedrich T, Bamberg E
JournalAnn N Y Acad Sci
PubMed ID12763772
'We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus ... More
Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity.
AuthorsKang HJ, Choi YS, Hong SB, Kim KW, Woo RS, Won SJ, Kim EJ, Jeon HK, Jo SY, Kim TK, Bachoo R, Reynolds IJ, Gwag BJ, Lee HW
JournalJ Neurosci
PubMed ID14960598
'The catalytic subunit of telomerase reverse transcriptase (TERT) protects dividing cells from replicative senescence in vitro. Here, we show that expression of TERT mRNA is induced in the ipsilateral cortical neurons after occlusion of the middle cerebral artery in adult mice. Transgenic mice that overexpress TERT showed significant resistance to ... More
Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide.
AuthorsNieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ
JournalBiochem J
PubMed ID7718000
'We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria ... More
Granzyme B induces BID-mediated cytochrome c release and mitochondrial permeability transition.
AuthorsAlimonti JB, Shi L, Baijal PK, Greenberg AH
JournalJ Biol Chem
PubMed ID11114298
'Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical ... More
"Mitochondrial" photochemical drugs do not release toxic amounts of 1O(2) within the mitochondrial matrix space.
AuthorsPetrat F, Pindiur S, Kirsch M, de Groot H
JournalArch Biochem Biophys
PubMed ID12667484
'Previously, we demonstrated that mitochondrial NAD(P)H is the primary target of singlet oxygen (1O(2)) generated by photoactivation of mitochondria-selective rhodamine derivatives. Hence, local NAD(P)H oxidation/fluorescence decrease may be used to reveal the site of intracellular 1O(2) generation. Therefore, in addition to the previously used tetramethylrhodamine methylester (TMRM), 2(''),4(''),5(''),7('')-tetrabromorhodamine 123 bromide ... More
Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling.
AuthorsScorrano L, Penzo D, Petronilli V, Pagano F, Bernardi P
JournalJ Biol Chem
PubMed ID11134037
'We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent ... More
Rapid Ca2+-dependent increase in oxygen consumption by mitochondria in single mammalian central neurons.
AuthorsHayakawa Y, Nemoto T, Iino M, Kasai H
JournalCell Calcium
PubMed ID15755497
'Oxygen consumption increases within a fraction of a second after the onset of neuronal activity, a phenomenon referred to as the "initial dip" in functional imaging studies of the living brain. The cellular mechanism that underlies this rapid increase in oxygen consumption has remained unclear, however. We have now used ... More
Galectin-1 sensitizes resting human T lymphocytes to Fas (CD95)-mediated cell death via mitochondrial hyperpolarization, budding, and fission.
AuthorsMatarrese P, Tinari A, Mormone E, Bianco GA, Toscano MA, Ascione B, Rabinovich GA, Malorni W
JournalJ Biol Chem
PubMed ID15556941
'Galectins have emerged as a novel family of immunoregulatory proteins implicated in T cell homeostasis. Recent studies showed that galectin-1 (Gal-1) plays a key role in tumor-immune escape by killing antitumor effector T cells. Here we found that Gal-1 sensitizes human resting T cells to Fas (CD95)/caspase-8-mediated cell death. Furthermore, ... More
Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.
AuthorsLoew LM, Tuft RA, Carrington W, Fay FS
JournalBiophys J
PubMed ID8312478
'Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative ... More
Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence.
AuthorsPetronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F
JournalBiophys J
PubMed ID9929477
'The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was ... More
Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death.
AuthorsNakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H, Inohara H, Kubo T, Tsujimoto Y
JournalNature
PubMed ID15800626
'Mitochondria play an important role in energy production, Ca2+ homeostasis and cell death. In recent years, the role of the mitochondria in apoptotic and necrotic cell death has attracted much attention. In apoptosis and necrosis, the mitochondrial permeability transition (mPT), which leads to disruption of the mitochondrial membranes and mitochondrial ... More
Characterization of 3-D collagen hydrogels for functional cell-based biosensing.
AuthorsMao C, Kisaalita WS
JournalBiosens Bioelectron
PubMed ID15018963
'To address the growing demand for functional cell-based assay technologies with accelerated drug discovery applications, we have proposed the use of human neuroblastoma cells (IMR-32) immobilized in three-dimensional (3-D) collagen hydrogel matrices. The gel protects weakly adherent cells from fluid mechanical forces while providing a more physiologically relevant 3-D environment. ... More
Life cell quantification of mitochondrial membrane potential at the single organelle level.
AuthorsDistelmaier F, Koopman WJ, Testa ER, de Jong AS, Swarts HG, Mayatepek E, Smeitink JA, Willems PH,
JournalCytometry A
PubMed ID18163486
'Mitochondrial membrane potential (Deltapsi) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Deltapsi in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is ... More
'Photosensitizers are chromophores that generate reactive oxygen species (ROS) upon light irradiation. They are used for inactivation of specific proteins by chromophore-assisted light inactivation (CALI) and for light-induced cell killing in photodynamic therapy. Here we report a genetically encoded photosensitizer, which we call KillerRed, developed from the hydrozoan chromoprotein anm2CP, ... More
Homogeneous longitudinal profiles and synchronous fluctuations of mitochondrial transmembrane potential.
AuthorsDiaz G, Falchi AM, Gremo F, Isola R, Diana A
JournalFEBS Lett
PubMed ID10869560
'This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mDeltapsi) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mDeltapsi fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same ... More
Effect of transient and permanent permeability transition pore opening on NAD(P)H localization in intact cells.
'To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume, and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, tetramethylrhodamine methyl ester, MitoTracker, and NAD(P)H ... More
Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1).
AuthorsSaengkhae C, Loetchutinat C, Garnier-Suillerot A
JournalBiophys J
PubMed ID12944313
'Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of ... More
Flow cytometry-based apoptosis detection.
AuthorsWlodkowic D, Skommer J, Darzynkiewicz Z,
JournalMethods Mol Biol
PubMed ID19609746
'An apoptosing cell demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the stimuli and the cell type. The gross majority of classical apoptotic hallmarks can be rapidly examined by flow and image cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. ... More
Interactions of chloromethyltetramethylrosamine (Mitotracker Orange) with isolated mitochondria and intact cells.
AuthorsScorrano L, Petronilli V, Colonna R, Di Lisa F, Bernardi P
JournalAnn N Y Acad Sci
PubMed ID10672275
Fluorescent indicators of membrane potential: microspectrofluorometry and imaging.
AuthorsGross D, Loew LM
JournalMethods Cell Biol
PubMed ID2648110
Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence.
AuthorsPlymale DR, Haskins JR, de la Iglesia FA
JournalNat Med
PubMed ID10086396
Confocal microscopy of potentiometric fluorescent dyes.
AuthorsLoew LM
JournalMethods Cell Biol
PubMed ID8246781
Assessment of mitochondrial membrane potential in situ using single potentiometric dyes and a novel fluorescence resonance energy transfer technique.
AuthorsDykens JA, Stout AK
JournalMethods Cell Biol
PubMed ID11381600
Mitochondrial membrane potential and neuronal glutamate excitotoxicity: mortality and millivolts.
AuthorsNicholls DG, Ward MW
JournalTrends Neurosci
PubMed ID10717676
In the past few years it has become apparent that mitochondria have an essential role in the life and death of neuronal and non-neuronal cells. The central mitochondrial bioenergetic parameter is the protonmotive force, Deltap. Much research has focused on the monitoring of the major component of Deltap, the mitochondrial ... More
Assessing mitochondrial potential, calcium, and redox state in isolated mammalian cells using confocal microscopy.
AuthorsDavidson SM, Yellon D, Duchen MR,
JournalMethods Mol Biol
PubMed ID18314743
Mitochondria play a vital role in the regulation of intracellular calcium dynamics. Fluorescent dyes can be used to provide a direct measurement of the redox state, mitochondrial membrane potential, and mitochondrial calcium content. The simplicity of this approach lends itself to high-throughput assays and time-resolved analyses; however, care must be ... More
Analyzing mitochondrial changes during apoptosis.
AuthorsGottlieb RA, Granville DJ
JournalMethods
PubMed ID12054925
Mitochondria play a central role in programmed cell death through the release of cytochrome c and other proapoptotic factors. Fluorescence microscopy is used to visualize cytochrome c translocation and loss of mitochondrial membrane potential. Flow cytometry can also be used to measure mitochondrial membrane potential. Cytochrome c content in cytosol ... More
Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells.
AuthorsHeiskanen KM, Bhat MB, Wang HW, Ma J, Nieminen AL
JournalJ Biol Chem
PubMed ID10026183
Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester ... More
Hereditary early-onset Parkinson's disease caused by mutations in PINK1.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated with PARK6. We have identified ... More
Tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements of mitochondrial membrane potential in cells treated with digitonin.
AuthorsFloryk D, Houstek J
JournalBiosci Rep
PubMed ID10379904
A new method for cytofluorometric analysis of mitochondrial membrane potential deltapsi has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells ... More
Protein kinase C beta and prolyl isomerase 1 regulate mitochondrial effects of the life-span determinant p66Shc.
AuthorsPinton P, Rimessi A, Marchi S, Orsini F, Migliaccio E, Giorgio M, Contursi C, Minucci S, Mantovani F, Wieckowski MR, Del Sal G, Pelicci PG, Rizzuto R
JournalScience
PubMed ID17272725
The 66-kilodalton isoform of the growth factor adapter Shc (p66Shc) translates oxidative damage into cell death by acting as reactive oxygen species (ROS) producer within mitochondria. However, the signaling link between cellular stress and mitochondrial proapoptotic activity of p66Shc was not known. We demonstrate that protein kinase C beta, activated ... More
Mitochondrial permeability transition: a common pathway to necrosis and apoptosis.
AuthorsKim JS, He L, Lemasters JJ
JournalBiochem Biophys Res Commun
PubMed ID12729580
Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these ... More
Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.
Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus ... More
Nongenetic method for purifying stem cell-derived cardiomyocytes.
AuthorsHattori F, Chen H, Yamashita H, Tohyama S, Satoh YS, Yuasa S, Li W, Yamakawa H, Tanaka T, Onitsuka T, Shimoji K, Ohno Y, Egashira T, Kaneda R, Murata M, Hidaka K, Morisaki T, Sasaki E, Suzuki T, Sano M, Makino S, Oikawa S, Fukuda K,
JournalNat Methods
PubMed ID19946277
Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and ... More
Mutual dependence of Foxo3a and PGC-1alpha in the induction of oxidative stress genes.
AuthorsOlmos Y, Valle I, Borniquel S, Tierrez A, Soria E, Lamas S, Monsalve M,
JournalJ Biol Chem
PubMed ID19324885
Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here ... More
A cell-based high-throughput assay system reveals modulation of oxidative and nonoxidative glucose metabolism due to commonly used organic solvents.
AuthorsZimmermann S, Zarse K, Schulz TJ, Siems K, Müller-Kuhrt L, Birringer M, Ristow M,
JournalHorm Metab Res
PubMed ID18197582
A 96-well format screening system was generated to quantify changes in nonoxidative glucose metabolism and oxidative pyruvate metabolism. D-Glucose uptake from the supernatant media was quantified by the glucose oxidase method, and L-lactate production of cells was quantified by the lactate dehydrogenase method applied on supernatant media. Mitochondrial membrane potential ... More
Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone.
AuthorsYadava N, Nicholls DG,
JournalJ Neurosci
PubMed ID17611283
Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 ... More
TOXI-SIM-A simulation tool for the analysis of mitochondrial and plasma membrane potentials.
AuthorsHuber HJ, Plchut M, Weisová P, Düssmann H, Wenus J, Rehm M, Ward MW, Prehn JH,
JournalJ Neurosci Methods
PubMed ID18824028
Changes in the electrochemical gradients across biological membranes are excellent indicators of pathophysiological processes, drug action, or drug toxicity. Our previous studies have utilized the potentiometric probe tetramethylrhodamine methyl ester (TMRM) to characterize changes in mitochondrial function by monitoring alterations in the mitochondrial membrane potential (Deltapsi(m)) over time during glutamate ... More
In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone.
AuthorsDykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y,
JournalToxicol Sci
PubMed ID18344530
Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, ... More
Autophagy in neurite injury and neurodegeneration: in vitro and in vivo models.
Recent advances indicate that maintaining a balanced level of autophagy is critically important for neuronal health and function. Pathologic dysregulation of macroautophagy has been implicated in synaptic dysfunction, cellular stress, and neuronal cell death. Autophagosomes and autolysosomes are induced in acute and chronic neurological disorders including stroke, brain trauma, neurotoxin ... More
Coordinated behavior of mitochondria in both space and time: a reactive oxygen species-activated wave of mitochondrial depolarization.
Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte ... More
Caspase-cleaved tau expression induces mitochondrial dysfunction in immortalized cortical neurons: implications for the pathogenesis of Alzheimer disease.
AuthorsQuintanilla RA, Matthews-Roberson TA, Dolan PJ, Johnson GV,
JournalJ Biol Chem
PubMed ID19389700
In Alzheimer disease (AD) mitochondrial abnormalities occur early in the pathogenic process and likely play a significant role in disease progression. Tau is a microtubule-associated protein that is abnormally processed in AD, and a connection between tau pathology and mitochondrial impairment has been proposed. However, few studies have examined the ... More
SYTO probes in the cytometry of tumor cell death.
AuthorsWlodkowic D, Skommer J, Darzynkiewicz Z,
JournalCytometry A
PubMed ID18260152
Apoptosis is a complex and finely controlled cell death process of great relevance in tissue homeostasis and pathogenesis. The majority of classical apoptotic features can be examined by flow as well as image cytometry. Therefore, cytometry has been used as a technology of choice in studies of tumor cell demise. ... More
Rosiglitazone treatment prevents mitochondrial dysfunction in mutant huntingtin-expressing cells: possible role of peroxisome proliferator-activated receptor-gamma (PPARgamma) in the pathogenesis of Huntington disease.
AuthorsQuintanilla RA, Jin YN, Fuenzalida K, Bronfman M, Johnson GV,
JournalJ Biol Chem
PubMed ID18640979
Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the PPAR family of transcription factors. Synthetic PPARgamma agonists are used as oral anti-hyperglycemic drugs for the treatment of non-insulin-dependent diabetes. However, emerging evidence indicates that PPARgamma activators can also prevent or attenuate neurodegeneration. Given these previous findings, the focus of this ... More
High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening.
AuthorsO'Brien PJ, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C,
JournalArch Toxicol
PubMed ID16598496
To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells ... More
Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo.
AuthorsMortensen M, Ferguson DJ, Edelmann M, Kessler B, Morten KJ, Komatsu M, Simon AK,
JournalProc Natl Acad Sci U S A
PubMed ID20080761
Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by ... More
Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations.
Mutations of mitochondrial DNA are associated with a wide spectrum of disorders, primarily affecting the central nervous system and muscle function. The specific consequences of mitochondrial DNA mutations for neuronal pathophysiology are not understood. In order to explore the impact of mitochondrial mutations on neuronal biochemistry and physiology, we have ... More
PINK1-associated Parkinson's disease is caused by neuronal vulnerability to calcium-induced cell death.
AuthorsGandhi S, Wood-Kaczmar A, Yao Z, Plun-Favreau H, Deas E, Klupsch K, Downward J, Latchman DS, Tabrizi SJ, Wood NW, Duchen MR, Abramov AY,
JournalMol Cell
PubMed ID19285945
Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na(+)/Ca(2+) exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, ... More
Mitochondrial free calcium transients during excitation-contraction coupling in rabbit cardiac myocytes.
AuthorsChacon E, Ohata H, Harper IS, Trollinger DR, Herman B, Lemasters JJ
JournalFEBS Lett
PubMed ID8612759
Mitochondrial free Ca2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Fluo-3 to measure free Ca2+ and tetramethylrhodamine methylester to identify mitochondria, we measured cytosolic and mitochondrial Ca2+ transients during the contractile cycle. In resting cells, cytosolic ... More
Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products.
AuthorsRigoni M, Pizzo P, Schiavo G, Weston AE, Zatti G, Caccin P, Rossetto O, Pozzan T, Montecucco C
JournalJ Biol Chem
PubMed ID17311918
Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated ... More
Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes.
AuthorsPinton P, Leo S, Wieckowski MR, Di Benedetto G, Rizzuto R
JournalJ Cell Biol
PubMed ID15096525
The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating ... More
Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes.
AuthorsJouaville LS, Ichas F, Holmuhamedov EL, Camacho P, Lechleiter JD
JournalNature
PubMed ID7566122
In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is ... More
Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.
AuthorsBelousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S
JournalNat Methods
PubMed ID16554833
We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level ... More
Human hepatitis B virus-X protein alters mitochondrial function and physiology in human liver cells.
AuthorsLee YI, Hwang JM, Im JH, Lee YI, Kim NS, Kim DG, Yu DY, Moon HB, Park SK
JournalJ Biol Chem
PubMed ID14724286
The hepatitis B virus-X protein (HBx) regulates fundamental aspects of mitochondrial physiology. We show that HBx down-regulates mitochondrial enzymes involved in electron transport in oxidative phosphorylation (complexes I, III, IV, and V) and sensitizes the mitochondrial membrane potential in a hepatoma cell line. HBx also increases the level of mitochondrial ... More
Measurement of intracellular calcium.
AuthorsTakahashi A, Camacho P, Lechleiter JD, Herman B
JournalPhysiol Rev
PubMed ID10508230
To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and ... More
High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader.
AuthorsWong A, Cortopassi GA
JournalBiochem Biophys Res Commun
PubMed ID12419317
Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP ... More
Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role?
AuthorsKuznetsov AV, Troppmair J, Sucher R, Hermann M, Saks V, Margreiter R
JournalBiochim Biophys Acta
PubMed ID16712778
Heterogeneity of mitochondria has been reported for a number of various cell types. Distinct mitochondrial subpopulations may be present in the cell and may be differently involved in physiological and pathological processes. However, the origin and physiological roles of mitochondrial heterogeneity are still unknown. In mice skeletal muscle, a much ... More
Effect of oxidative stress on dynamics of mitochondrial reticulum.
AuthorsPletjushkina OY, Lyamzaev KG, Popova EN, Nepryakhina OK, Ivanova OY, Domnina LV, Chernyak BV, Skulachev VP
JournalBiochim Biophys Acta
PubMed ID16829229
Fission of the mitochondrial reticulum (the thread-grain transition) and following gathering of mitochondria in the perinuclear area are induced by oxidative stress. It is shown that inhibitors of the respiratory chain (piericidin and myxothiazol) cause fission of mitochondria in HeLa cells and fibroblasts, whereas a mitochondria-targeted antioxidant (MitoQ) inhibits this ... More
Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2.
AuthorsGingrich JR, Pelkey KA, Fam SR, Huang Y, Petralia RS, Wenthold RJ, Salter MW
JournalProc Natl Acad Sci U S A
PubMed ID15069201
Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained ... More
Mitochondrial pH monitored by a new engineered green fluorescent protein mutant.
AuthorsAbad MF, Di Benedetto G, Magalhães PJ, Filippin L, Pozzan T
JournalJ Biol Chem
PubMed ID14701849
We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. ... More
A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells.
AuthorsDurieux C, Brunet AC, Geeraert V, Coppey J, Coppey-Moisan M
JournalBiol Cell
PubMed ID10629939
The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ... More
Quantitation of mitochondrial alterations associated with apoptosis.
AuthorsCastedo M, Ferri K, Roumier T, Métivier D, Zamzami N, Kroemer G
JournalJ Immunol Methods
PubMed ID12072177
Mitochondria undergo two major changes during early apoptosis. On the one hand, the outer mitochondrial membrane becomes permeable to proteins, resulting in the release of soluble intermembrane proteins (SIMPs) from the mitochondrion. On the other hand, the inner mitochondrial membrane transmembrane potential (DeltaPsi(m)) is reduced. These changes occur in most, ... More
Spontaneous changes in mitochondrial membrane potential in cultured neurons.
AuthorsBuckman JF, Reynolds IJ
JournalJ Neurosci
PubMed ID11438581
Using the mitochondrial membrane potential (DeltaPsi(m))-sensitive fluorescent dyes 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM), we have observed spontaneous changes in the DeltaPsi(m) of cultured forebrain neurons. These fluctuations in DeltaPsi(m) appear to represent partial, transient depolarizations of individual mitochondria. The frequency of these DeltaPsi(m) fluctuations can be significantly ... More