Science.gov

Sample records for mitochondrial membrane potential

  1. Wafer-scale Mitochondrial Membrane Potential Assays

    PubMed Central

    Lim, Tae-Sun; Davila, Antonio; Zand, Katayoun; Douglas, Wallace C.; Burke, Peter J.

    2012-01-01

    It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4” standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems. PMID:22627274

  2. Mitochondrial membrane potential is regulated by vimentin intermediate filaments

    PubMed Central

    Chernoivanenko, Ivan S.; Matveeva, Elena A.; Gelfand, Vladimir I.; Goldman, Robert D.; Minin, Alexander A.

    2015-01-01

    This study demonstrates that the association of mitochondria with vimentin intermediate filaments (VIFs) measurably increases their membrane potential. This increase is detected by quantitatively comparing the fluorescence intensity of mitochondria stained with the membrane potential-sensitive dye tetramethylrhodamine-ethyl ester (TMRE) in murine vimentin-null fibroblasts with that in the same cells expressing human vimentin (∼35% rise). When vimentin expression is silenced by small hairpin RNA (shRNA) to reduce vimentin by 90%, the fluorescence intensity of mitochondria decreases by 20%. The increase in membrane potential is caused by specific interactions between a subdomain of the non-α-helical N terminus (residues 40 to 93) of vimentin and mitochondria. In rho 0 cells lacking mitochondrial DNA (mtDNA) and consequently missing several key proteins in the mitochondrial respiratory chain (ρ0 cells), the membrane potential generated by an alternative anaerobic process is insensitive to the interactions between mitochondria and VIF. The results of our studies show that the close association between mitochondria and VIF is important both for determining their position in cells and their physiologic activity.—Chernoivanenko, I. S., Matveeva, E. A., Gelfand, V. I., Goldman, R. D., Minin, A. A. Mitochondrial membrane potential is regulated by vimentin intermediate filaments. PMID:25404709

  3. Proteasome Impairment Induces Recovery of Mitochondrial Membrane Potential and an Alternative Pathway of Mitochondrial Fusion

    PubMed Central

    Shirozu, Ryohei; Yashiroda, Hideki

    2015-01-01

    Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of the short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to the long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment. PMID:26552703

  4. Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin

    PubMed Central

    Wang, Zhi-hao; Luo, Yu; Zhang, Xiangnan; Liu, Xiu-Ping; Feng, Qiong; Wang, Qun; Yue, Zhenyu; Chen, Zhong; Ye, Keqiang; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-01-01

    Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTEN-induced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies. PMID:26943044

  5. TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

    PubMed

    Martínez-Reyes, Inmaculada; Diebold, Lauren P; Kong, Hyewon; Schieber, Michael; Huang, He; Hensley, Christopher T; Mehta, Manan M; Wang, Tianyuan; Santos, Janine H; Woychik, Richard; Dufour, Eric; Spelbrink, Johannes N; Weinberg, Samuel E; Zhao, Yingming; DeBerardinis, Ralph J; Chandel, Navdeep S

    2016-01-21

    Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation. PMID:26725009

  6. Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI.

    PubMed

    Jensen, Kristian H R; Rekling, Jens C

    2010-10-01

    Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine, which is a suspected mitochondrial toxicant. CCCP and DNP have short-term depolarizing effects, and thioridazine has long-term hyperpolarizing effects on the mitochondrial membrane potential of Chinese hamster ovary (CHO) cells. The assay also detected changes of the mitochondrial membrane potential in CHO cells exposed to cobalt (mimicking hypoxia) and in PC12 cells exposed to amyloid β, demonstrating that the assay can be used in cellular models of hypoxia and Alzheimer's disease. The assay needs no washing steps, has a Z' value >0.5, can be used on standard fluorometers, has good post liquid-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction. PMID:20713988

  7. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL.

    PubMed

    Jin, Seok Min; Lazarou, Michael; Wang, Chunxin; Kane, Lesley A; Narendra, Derek P; Youle, Richard J

    2010-11-29

    PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson's disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function. PMID:21115803

  8. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL

    PubMed Central

    Jin, Seok Min; Lazarou, Michael; Wang, Chunxin; Kane, Lesley A.; Narendra, Derek P.

    2010-01-01

    PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson’s disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function. PMID:21115803

  9. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    PubMed

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously. PMID:26369275

  10. Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

    SciTech Connect

    Montaigne, David; Marechal, Xavier; Baccouch, Riadh; Modine, Thomas; Preau, Sebastien; Zannis, Konstantinos; Marchetti, Philippe; Lancel, Steve; Neviere, Remi

    2010-05-01

    The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 muM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt{sub max} of 105 +- 8 mN/s in control hearts vs. 49 +- 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 +- 0.2 in control hearts vs. 2.2 +- 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 +- 1 muM cytochrome c/min/mg in control hearts vs. 14 +- 3 muM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

  11. Biophysical significance of the inner mitochondrial membrane structure on the electrochemical potential of mitochondria.

    PubMed

    Song, Dong Hoon; Park, Jonghyun; Maurer, Laura L; Lu, Wei; Philbert, Martin A; Sastry, Ann Marie

    2013-12-01

    The available literature supports the hypothesis that the morphology of the inner mitochondrial membrane is regulated by different energy states, that the three-dimensional morphology of cristae is dynamic, and that both are related to biochemical function. Examination of the correlation between the inner mitochondrial membrane (IMM) structure and mitochondrial energetic function is critical to an understanding of the links between mesoscale morphology and function in progressive mitochondrial dysfunction such as aging, neurodegeneration, and disease. To investigate this relationship, we develop a model to examine the effects of three-dimensional IMM morphology on the electrochemical potential of mitochondria. The two-dimensional axisymmetric finite element method is used to simulate mitochondrial electric potential and proton concentration distribution. This simulation model demonstrates that the proton motive force (Δp) produced on the membranes of cristae can be higher than that on the inner boundary membrane. The model also shows that high proton concentration in cristae can be induced by the morphology-dependent electric potential gradient along the outer side of the IMM. Furthermore, simulation results show that a high Δp is induced by the large surface-to-volume ratio of an individual crista, whereas a high capacity for ATP synthesis can primarily be achieved by increasing the surface area of an individual crista. The mathematical model presented here provides compelling support for the idea that morphology at the mesoscale is a significant driver of mitochondrial function. PMID:24483502

  12. Biophysical significance of the inner mitochondrial membrane structure on the electrochemical potential of mitochondria

    PubMed Central

    Song, Dong Hoon; Park, Jonghyun; Maurer, Laura L.; Lu, Wei; Philbert, Martin A.; Sastry, Ann Marie

    2014-01-01

    The available literature supports the hypothesis that the morphology of the inner mitochondrial membrane is regulated by different energy states, that the three-dimensional morphology of cristae is dynamic and that both are related to biochemical function. Examination of the correlation between the inner mitochondrial membrane (IMM) structure and mitochondrial energetic function is critical to an understanding of the links between meso-scale morphology and function in progressive mitochondrial dysfunction such as aging, neurodegeneration, and disease. To investigate this relationship, we develop a model to examine the effects of three-dimensional IMM morphology on the electrochemical potential of mitochondria. The 2D axisymmetric finite element method is used to simulate mitochondrial electric potential and proton concentration distribution. This simulation model demonstrates that the proton motive force (PMF) produced on the membranes of cristae can be higher than that on the inner boundary membrane. The model also shows that high proton concentration in cristae can be induced by the morphology-dependent electric potential gradient along the outer side of the IMM. Furthermore, simulation results show that a high PMF is induced by the large surface-to-volume ratio of an individual crista, whereas a high capacity for ATP synthesis can primarily be achieved by increasing the surface area of an individual crista. The mathematical model presented here provides compelling support for the idea that morphology at the meso-scale is a significant driver of mitochondrial function. PMID:24483502

  13. Biophysical significance of the inner mitochondrial membrane structure on the electrochemical potential of mitochondria

    NASA Astrophysics Data System (ADS)

    Song, Dong Hoon; Park, Jonghyun; Maurer, Laura L.; Lu, Wei; Philbert, Martin A.; Sastry, Ann Marie

    2013-12-01

    The available literature supports the hypothesis that the morphology of the inner mitochondrial membrane is regulated by different energy states, that the three-dimensional morphology of cristae is dynamic, and that both are related to biochemical function. Examination of the correlation between the inner mitochondrial membrane (IMM) structure and mitochondrial energetic function is critical to an understanding of the links between mesoscale morphology and function in progressive mitochondrial dysfunction such as aging, neurodegeneration, and disease. To investigate this relationship, we develop a model to examine the effects of three-dimensional IMM morphology on the electrochemical potential of mitochondria. The two-dimensional axisymmetric finite element method is used to simulate mitochondrial electric potential and proton concentration distribution. This simulation model demonstrates that the proton motive force (Δp) produced on the membranes of cristae can be higher than that on the inner boundary membrane. The model also shows that high proton concentration in cristae can be induced by the morphology-dependent electric potential gradient along the outer side of the IMM. Furthermore, simulation results show that a high Δp is induced by the large surface-to-volume ratio of an individual crista, whereas a high capacity for ATP synthesis can primarily be achieved by increasing the surface area of an individual crista. The mathematical model presented here provides compelling support for the idea that morphology at the mesoscale is a significant driver of mitochondrial function.

  14. [HOMOCYSTEINE-INDUCED MEMBRANE CURRENTS, CALCIUM RESPONSES AND CHANGES OF MITOCHONDRIAL POTENTIAL IN RAT CORTICAL NEURONS].

    PubMed

    Abushik, P A; Karelina, T V; Sibarov, D A; Stepanenko, J D; Giniatullin, R; Antonov, S M

    2015-01-01

    Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential. PMID:26547950

  15. Expression of a mitochondrial progesterone receptor in human spermatozoa correlates with a progestin-dependent increase in mitochondrial membrane potential.

    PubMed

    Tantibhedhyangkul, J; Hawkins, K C; Dai, Q; Mu, K; Dunn, C N; Miller, S E; Price, T M

    2014-11-01

    The hyperactivation of human spermatozoa necessary for fertilization requires a substantial increase in cellular energy production. The factors responsible for increasing cellular energy remain poorly defined. This article proposes a role for a novel mitochondrial progesterone receptor (PR-M) in modulation of mitochondrial activity. Basic science studies demonstrate a 38 kDa protein with western blot analysis, consistent with PR-M; whereas imaging studies with confocal and immunoelectron microscopy demonstrate a PR on the mitochondria. Treatment with a PR-specific progestin shows increased mitochondrial membrane potential, not related to induction of an acrosome reaction. The increase in mitochondrial membrane potential was inhibited by a specific PR antagonist, but not affected by an inhibitor to the progesterone-dependent Catsper voltage-activated channel. In conclusion, these studies suggest expression of a novel mitochondrial PR in human spermatozoa with a progestin-dependent increase in mitochondrial activity. This mechanism may serve to enhance cellular energy production as the spermatozoa traverse the female genital tract being exposed to increasing concentrations of progesterone. PMID:25187426

  16. Effect of hydrogen peroxide and hypochlorite on mitochondrial membrane potential in permeabilized rat heart cells

    SciTech Connect

    Konno, N.; Kako, K.J. )

    1991-03-15

    The chemiosmotic theory states that the proton electrochemical potential gradient across the membrane drives mitochondrial energy transduction. Mitochondria can take up Ca accumulated in the cytosol. Therefore, oxidant-induced ATP depletion and Ca overload in the cell may be the result of mitochondrial dysfunction. Consequently, the authors measured membrane potential of mitochondria in situ in isolated rat heart myocytes with {sup 3}H-triphenylmethylphosphonium. This was followed by permeabilization using digitonin and rapid centrifugation using density gradient of bromododecane. They found that the membrane potentials, 118 mV with isolated and 161 mV with in situ mitochondria, were relatively well maintained under oxidant stress. High concentrations of oxidants reduced also the cellular ATP level, whereas the matrix volume was not significantly changed. The H{sub 2}O{sub 2} effect on the mitochondrial membrane potential was more pronounced when the extra-mitochondrial free Ca concentration was increased in permeabilized myocytes. These results support the view that heart mitochondria are equipped with well developed defense mechanisms against oxidants and thus the electrochemical gradient of inner membrane is affected only by a relatively large concentration of H{sub 2}O{sub 2} and HOCl.

  17. Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling.

    PubMed

    Wigdal, Susan S; Kirkland, Rebecca A; Franklin, James L; Haak-Frendscho, Mary

    2002-09-01

    It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons. PMID:12358750

  18. Assessment of mitochondrial membrane potential using an on-chip microelectrode in a microfluidic device

    PubMed Central

    Dávila, Antonio; Wallace, Douglas C.; Burke, Peter

    2010-01-01

    The mitochondrial membrane potential is used to generate and regulate energy in living systems, driving the conversion of ADP to ATP, regulating ion homeostasis, and controlling apoptosis, all central to human health and disease. Therefore, there is a need for tools to study its regulation in a controlled environment for potential clinical and scientific applications. For this aim, an on-chip tetraphenylphosphonium (TPP+) selective microelectrode sensor was constructed in a microfluidic environment. The concentration of isolated mitochondria (Heb7A) used in a membrane potential measurement was 0.3 ng μL−1, four orders of magnitude smaller than the concentration used in conventional assays (3 μg μL−1). In addition, the volume of the chamber (85 μL) is 2 orders of magnitude smaller than traditional experiments. As a demonstration, changes in the membrane potential are clearly measured in response to a barrage of well-known substrates and inhibitors of the electron transport chain. This general approach, which to date has not been demonstrated for study of mitochondrial function and bio-energetics in generally, can be instrumental in advancing the field of mitochondrial research and clinical applications by allowing high throughput studies of the regulation, dynamics, and statistical properties of the mitochondrial membrane potential in response to inhibitors and inducers of apoptosis in a controlled (microfluidic) chemical environment. PMID:20383402

  19. A biphenyl type two-photon fluorescence probe for monitoring the mitochondrial membrane potential.

    PubMed

    Moritomo, Hiroki; Yamada, Kengo; Kojima, Yuki; Suzuki, Yasutaka; Tani, Seiji; Kinoshita, Hazuki; Sasaki, Akira; Mikuni, Shintaro; Kinjo, Masataka; Kawamata, Jun

    2014-01-01

    Here we describe the design and synthesis of a bifunctional two-photon fluorescence probe, N,N'-‍dimethyl-4,4'-(biphenyl-2,1-ethenediyl)dipyridinium hexafluorophosphate (BP6). HeLa, Hek293, and Paramecium caudatum cells were stained with BP6. BP6 accumulated on the mitochondria of all three cell types when the mitochondrial membrane potential was high. As the mitochondrial membrane potential decreased following the addition of carbonyl cyanide m-chlorophenyl hydrazine, BP6 moved from the mitochondria to the nucleus in a reversible manner, depending on the mitochondrial membrane potential status. The maximum value of the two-photon absorption cross-section of BP6 is 250 GM (1 GM=1×10(-50) cm(4) s molecules(-1) photon(-1)). This value is 3 and 30 times larger, respectively, than that of the conventional mitochondria selective probes, rhodamine 123 and green fluorescence protein. These results suggest that BP6 should be useful for monitoring mitochondrial membrane potential by two-photon excitation. PMID:25319070

  20. Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death.

    PubMed

    Shang, Tiesong; Joseph, Joy; Hillard, Cecilia J; Kalyanaraman, B

    2005-10-14

    We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed. PMID:16085644

  1. Toxicity and Loss of Mitochondrial Membrane Potential Induced by Alkyl Gallates in Trypanosoma cruzi

    PubMed Central

    Andréo, Rogério; Regasini, Luís Octávio; Petrônio, Maicon Segalla; Chiari-Andréo, Bruna Galdorfini; Tansini, Aline; Silva, Dulce Helena Siqueira; Cicarelli, Regina Maria Barretto

    2015-01-01

    American trypanosomiasis or Chagas disease is a debilitating disease representing an important social problem that affects, approximately, 10 million people in the world. The main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease. In this context, the search for trypanocidal substances isolated from plants, synthetic or semi synthetic molecules, is an important strategy. Here, the trypanocidal potential of gallates was assayed in epimastigotes forms of T. cruzi and also, the interference of these substances on the mitochondrial membrane potential of the parasites was assessed, allowing the study of the mechanism of action of the gallates in the T. cruzi organisms. Regarding the preliminary structure-activity relationships, the side chain length of gallates plays crucial role for activity. Nonyl, decyl, undecyl, and dodecyl gallates showed potent antitrypanosomal effect (IC50 from 1.46 to 2.90 μM) in contrast with benznidazole (IC50 = 34.0 μM). Heptyl gallate showed a strong synergistic activity with benznidazole, reducing by 105-fold the IC50 of benznidazole. Loss of mitochondrial membrane potential induced by these esters was revealed. Tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential, at IC50 value.

  2. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    PubMed

    Kozjak-Pavlovic, Vera; Dian-Lothrop, Elke A; Meinecke, Michael; Kepp, Oliver; Ross, Katharina; Rajalingam, Krishnaraj; Harsman, Anke; Hauf, Eva; Brinkmann, Volker; Günther, Dirk; Herrmann, Ines; Hurwitz, Robert; Rassow, Joachim; Wagner, Richard; Rudel, Thomas

    2009-10-01

    The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection. PMID:19851451

  3. Ischemic Preconditioning Preserves Mitochondrial Membrane Potential and Limits Reactive Oxygen Species Production

    PubMed Central

    Quarrie, Ricardo; Lee, Daniel S.; Steinbaugh, Gregory; Cramer, Brandon; Erdahl, Warren; Pfeiffer, Douglas R.; Zweier, Jay L.; Crestanello, Juan A.

    2012-01-01

    Background Mitochondrial superoxide radical (O2•−) production increases after cardiac ischemia-reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O2•− production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O2•− production; mitochondrial depolarization decreases O2•− formation. We examined the relationship between O2•− production and mΔΨ during IR and IPC. Materials/Methods Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end-equilibration (End EQ), end-ischemia (End I) and end-reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O2•− production was measured by electron paramagnetic resonance (EPR) using DMPO spin trap. Cytochrome c levels were measured using high pressure liquid chromatography. Results IPC preserved mΔΨ at End I (−156±5 vs. −131±6 mV, p<0.001) and End RP (−168±2 vs. −155±2 mV, p<0.05). At End RP, IPC attenuated O2•− production (2527±221 vs. 3523±250 AU/mg protein, p<0.05). IPC preserved cytochrome c levels (351±14 vs. 269±16 picomoles/mg protein, p<0.05) at End RP, and decreased mitochondrial cristae disruption (10±4 vs. 33±7%, p<0.05) and amorphous density formation (18±4 vs. 28±1%, p<0.05). Conclusion We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O2•− production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O2•− production, our results indicate that preservation of mΔΨ is associated with decreased O2•− and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential. PMID:22763215

  4. Mitochondrial membrane potential and ATP production in primary disorders of ATP synthase.

    PubMed

    Vojtísková, Alena; Jesina, Pavel; Kalous, Martin; Kaplanová, Vilma; Houstek, Josef; Tesarová, Markéta; Fornůsková, Daniela; Zeman, Jirí; Dubot, Audrey; Godinot, Catherine

    2004-01-01

    Studies of fibroblasts with primary defects in mitochondrial ATP synthase (ATPase) due to heteroplasmic mtDNA mutations in the ATP6 gene, affecting protonophoric function or synthesis of subunit a, show that at high mutation loads, mitochondrial membrane potential DeltaPsi(m) at state 4 is normal, but ADP-induced discharge of DeltaPsi(m) is impaired and ATP synthesis at state 3-ADP is decreased. Increased DeltaPsi(m) and low ATP synthesis is also found when the ATPase content is diminished by altered biogenesis of the enzyme complex. Irrespective of the different pathogenic mechanisms, elevated DeltaPsi(m) in primary ATPase disorders could increase mitochondrial production of reactive oxygen species and decrease energy provision. PMID:20021115

  5. ELECTROCHEMICAL POTENTIAL OF THE INNER MITOCHONDRIAL MEMBRANE AND Ca2+ HOMEOSTASIS OF MYOMETRIUM CELLS.

    PubMed

    Danylovych, Yu V; Karakhim, S A; Danylovych, H V; Kolomiets, O V; Kosterin, S O

    2015-01-01

    We demonstrated using Ca(2+)-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes' inner mitochondrial membrane by aprotonophore carbonyl cyanide m-chlorophenyl hydrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3'-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane's electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hydrazone causes substantial increase in fluorescence of Ca(2+)-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca(2+)-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane

  6. Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons.

    PubMed

    Nicholls, David G

    2006-05-26

    Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV. PMID:16551630

  7. Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.

    PubMed

    Valmas, Nicholas; Zuryn, Steven; Ebert, Paul R

    2008-10-30

    Phosphine is the most widely used fumigant for the protection of stored commodities against insect pests, especially food products such as grain. However, pest insects are developing resistance to phosphine and thereby threatening its future use. As phosphine inhibits cytochrome c oxidase (complex IV) of the mitochondrial respiratory chain and reduces the strength of the mitochondrial membrane potential (DeltaPsi(m)), we reasoned that mitochondrial uncouplers should act synergistically with phosphine. The mitochondrial uncouplers FCCP and PCP caused complete mortality in populations of both wild-type and phosphine-resistant lines of Caenorhabditis elegans simultaneously exposed to uncoupler and phosphine at concentrations that were individually nonlethal. Strong synergism was also observed with a third uncoupler DNP. We have also tested an alternative complex IV inhibitor, azide, with FCCP and found that this also caused a synergistic enhancement of toxicity in C. elegans. To investigate potential causes of the synergism, we measured DeltaPsi(m), ATP content, and oxidative damage (lipid hydroperoxides) in nematodes subjected to phosphine-FCCP treatment and found that neither an observed 50% depletion in ATP nor oxidative stress accounted for the synergistic effect. Instead, a synergistic reduction in DeltaPsi(m) was observed upon phosphine-FCCP co-treatment suggesting that this is directly responsible for the subsequent mortality. These results support the hypothesis that phosphine-induced mortality results from the in vivo disruption of normal mitochondrial activity. Furthermore, we have identified a novel pathway that can be targeted to overcome genetic resistance to phosphine. PMID:18755236

  8. VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

    PubMed

    Lemeshko, Victor V

    2014-05-01

    The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death. PMID:24412217

  9. Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential

    PubMed Central

    Attene-Ramos, Matias S.; Huang, Ruili; Michael, Sam; Witt, Kristine L.; Richard, Ann; Tice, Raymond R.; Simeonov, Anton; Austin, Christopher P.

    2014-01-01

    Background: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases. Objectives: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP). Methods: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells. Results: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP. Conclusions: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity. Citation: Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox

  10. Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.

    PubMed

    Uribe, P; Boguen, R; Treulen, F; Sánchez, R; Villegas, J V

    2015-03-01

    Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility. PMID:25425609

  11. The force exerted by the membrane potential during protein import into the mitochondrial matrix

    NASA Technical Reports Server (NTRS)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2004-01-01

    The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.

  12. Left ventricular noncompaction (LVNC) and low mitochondrial membrane potential are specific for Barth syndrome.

    PubMed

    Karkucinska-Wieckowska, Agnieszka; Trubicka, Joanna; Werner, Bozena; Kokoszynska, Katarzyna; Pajdowska, Magdalena; Pronicki, Maciej; Czarnowska, Elzbieta; Lebiedzinska, Magdalena; Sykut-Cegielska, Jolanta; Ziolkowska, Lidia; Jaron, Weronika; Dobrzanska, Anna; Ciara, Elzbieta; Wieckowski, Mariusz R; Pronicka, Ewa

    2013-11-01

    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 (mtΔΨ 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 mtΔΨ and the inhibition of complex V activity. It can be hypothesised that impaired mtΔΨ 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. PMID:23361305

  13. The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

    NASA Technical Reports Server (NTRS)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2002-01-01

    The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

  14. Dietary Tocotrienol/γ-Cyclodextrin Complex Increases Mitochondrial Membrane Potential and ATP Concentrations in the Brains of Aged Mice

    PubMed Central

    Schloesser, Anke; Esatbeyoglu, Tuba; Piegholdt, Stefanie; Dose, Janina; Ikuta, Naoko; Okamoto, Hinako; Ishida, Yoshiyuki; Terao, Keiji; Matsugo, Seiichi; Rimbach, Gerald

    2015-01-01

    Brain aging is accompanied by a decrease in mitochondrial function. In vitro studies suggest that tocotrienols, including γ- and δ-tocotrienol (T3), may exhibit neuroprotective properties. However, little is known about the effect of dietary T3 on mitochondrial function in vivo. In this study, we monitored the effect of a dietary T3/γ-cyclodextrin complex (T3CD) on mitochondrial membrane potential and ATP levels in the brain of 21-month-old mice. Mice were fed either a control diet or a diet enriched with T3CD providing 100 mg T3 per kg diet for 6 months. Dietary T3CD significantly increased mitochondrial membrane potential and ATP levels compared to those of controls. The increase in MMP and ATP due to dietary T3CD was accompanied by an increase in the protein levels of the mitochondrial transcription factor A (TFAM). Furthermore, dietary T3CD slightly increased the mRNA levels of superoxide dismutase, γ-glutamyl cysteinyl synthetase, and heme oxygenase 1 in the brain. Overall, the present data suggest that T3CD increases TFAM, mitochondrial membrane potential, and ATP synthesis in the brains of aged mice. PMID:26301044

  15. MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells.

    PubMed

    Vowinckel, Jakob; Hartl, Johannes; Butler, Richard; Ralser, Markus

    2015-09-01

    Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available. PMID:26184437

  16. MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells

    PubMed Central

    Vowinckel, Jakob; Hartl, Johannes; Butler, Richard; Ralser, Markus

    2015-01-01

    Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available. PMID:26184437

  17. A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts.

    PubMed

    Wan, B; Doumen, C; Duszynski, J; Salama, G; LaNoue, K F

    1993-08-01

    The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8368347

  18. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.

    PubMed Central

    Di Lisa, F; Blank, P S; Colonna, R; Gambassi, G; Silverman, H S; Stern, M D; Hansford, R G

    1995-01-01

    1. The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation. delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye. 2. De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission. This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells. 3. A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials. The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission. Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges. 4. At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained. The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture. 5. The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ([Mg2+]i) monitored by mag-indo-1 epifluorescence

  19. Protective effect of silymarin on viability, motility and mitochondrial membrane potential of ram sperm treated with sodium arsenite

    PubMed Central

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    Background: Sodium arsenite can impair male reproductive function by inducing oxidative stress. Silymarin is known as a potent antioxidant. Objective: This study was performed to investigate if silymarin can prevent the adverse effect of sodium arsenite on ram sperm viability, motility and mitochondrial membrane potential. Materials and Methods: Epidydimal spermatozoa obtained from ram were divided into five groups: 1) Spermatozoa at 0 hr, 2) spermatozoa at 180 min (control), 3) spermatozoa treated with sodium arsenite (10 μM) for 180 min, 4) spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min and 5) spermatozoa treated with silymarin (20 μM) for 180 min. MTT assay and Rhodamine 123 staining were used to assess sperm viability and mitochondrial membrane potential respectively. Sperm motility was performed according to World Health Organization (WHO) guidelines. Results: Viability (p<0.01), nonprogressive motility (p<0.001) and intact mitochondrial membrane potential (p<0.001) of the spermatozoa were significantly decreased in sodium arsenite treated group compared to control group. In silymarin + sodium arsenite group, silymarin could significantly reverse the adverse effect of sodium arsenite on these sperm parameters compared to sodium arsenite group (p<0.001). In addition, the application of silymarin alone for 180 minutes could significantly increase progressively motile sperm (p<0.001) and decrease non motile sperm (p<0.01) compared to the control. Conclusion: Silymarin could compensate the adverse effect of sodium arsenite on viability, nonprogressive motility and mitochondrial membrane potential of ram sperm. PMID:27525323

  20. Fibrates inhibit the apoptosis of Batten disease lymphoblast cells via autophagy recovery and regulation of mitochondrial membrane potential.

    PubMed

    Hong, Minho; Song, Ki Duk; Lee, Hak-Kyo; Yi, SunShin; Lee, Yong Seok; Heo, Tae-Hwe; Jun, Hyun Sik; Kim, Sung-Jo

    2016-03-01

    Batten disease (BD; also known as juvenile neuronal ceroid lipofuscinosis) is a genetic disorder inherited as an autosomal recessive trait and is characterized by blindness, seizures, cognitive decline, and early death resulting from the inherited mutation of the CLN3 gene. Mitochondrial oxidative stress, endoplasmic reticulum (ER) stress, disrupted autophagy, and enhanced apoptosis have been suggested to play a role in BD pathogenesis. Fibrates, a class of lipid-lowering drugs that induce peroxisome proliferator-activated receptor-α (PPAR-α) activation, are the most commonly used PPAR agonists. Assuming that fibrates have a neuroprotective effect, we studied the effects of fibrates, fenofibrate, bezafibrate, and gemfibrozil on apoptosis, depolarization of mitochondrial membrane, and defective autophagy in BD lymphoblast cells. The viability of fibrate-treated BD lymphoblast cells increased to levels of normal lymphoblast cells. In addition, treatment with fibrates inhibited depolarization of mitochondrial membrane potential in BD lymphoblast cells. Defective autophagy in BD lymphoblast cells was normalized when treated with fibrates as indicated by increased acridine orange staining. The recovery of autophagy in BD lymphoblast cells is most likely attributed to the upregulation of autophagy proteins, lysosomal-associated membrane protein 1 (LAMP1), and LC3 I/II, after treatment with fibrates. This study therefore suggests that fibrates may have a therapeutic potential against BD. PMID:26659390

  1. Profiling of the Tox21 Chemical Collection for Mitochondrial Function: I. Compounds that Decrease Mitochondrial Membrane Potential

    EPA Science Inventory

    Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding how different environmental chemicals and drug-like molecules impact mitochondrial function rep...

  2. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.

    PubMed

    Logan, Angela; Pell, Victoria R; Shaffer, Karl J; Evans, Cameron; Stanley, Nathan J; Robb, Ellen L; Prime, Tracy A; Chouchani, Edward T; Cochemé, Helena M; Fearnley, Ian M; Vidoni, Sara; James, Andrew M; Porteous, Carolyn M; Partridge, Linda; Krieg, Thomas; Smith, Robin A J; Murphy, Michael P

    2016-02-01

    The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo. PMID:26712463

  3. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry

    PubMed Central

    Logan, Angela; Pell, Victoria R.; Shaffer, Karl J.; Evans, Cameron; Stanley, Nathan J.; Robb, Ellen L.; Prime, Tracy A.; Chouchani, Edward T.; Cochemé, Helena M.; Fearnley, Ian M.; Vidoni, Sara; James, Andrew M.; Porteous, Carolyn M.; Partridge, Linda; Krieg, Thomas; Smith, Robin A.J.; Murphy, Michael P.

    2016-01-01

    Summary The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by “click” chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo. PMID:26712463

  4. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

    PubMed Central

    Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C.; Fernyhough, Paul

    2015-01-01

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. PMID:26647379

  5. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

    PubMed

    Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

    2016-01-01

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. PMID:26647379

  6. Mitochondrial Membrane Potential in a Small Subset of Artemisinin-Induced Dormant Plasmodium falciparum Parasites In Vitro.

    PubMed

    Peatey, Christopher L; Chavchich, Marina; Chen, Nanhua; Gresty, Karryn J; Gray, Karen-Ann; Gatton, Michelle L; Waters, Norman C; Cheng, Qin

    2015-08-01

    Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage. PMID:25635122

  7. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes

    PubMed Central

    2013-01-01

    Background The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. Methods THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. Results P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. Conclusions P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species

  8. Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

    PubMed

    Pyatrikas, Darya V; Fedoseeva, Irina V; Varakina, Nina N; Rusaleva, Tatyana M; Stepanov, Alexei V; Fedyaeva, Anna V; Borovskii, Gennadii B; Rikhvanov, Eugene G

    2015-06-01

    Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential. PMID:25991811

  9. Quantitative Analysis of Mitochondrial Morphology and Membrane Potential in Living Cells Using High-Content Imaging, Machine Learning, and Morphological Binning

    PubMed Central

    Leonard, Anthony P.; Cameron, Robert B.; Speiser, Jaime L.; Wolf, Bethany J.; Peterson, Yuri K.; Schnellmann, Rick G.; Beeson, Craig C.; Rohrer, Baerbel

    2014-01-01

    Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological

  10. Silencing of the methionine sulfoxide reductase A gene results in loss of mitochondrial membrane potential and increased ROS production in human lens cells

    PubMed Central

    Marchetti, Maria A.; Lee, Wanda; Cowell, Tracy L.; Wells, Tracy M.; Weissbach, Herbert; Kantorow, Marc

    2010-01-01

    Accumulation of methionine sulfoxide (Met(O)) is a significant feature of human cataract and previous studies have shown that methionine sulfoxide reductase A (MsrA), which acts to repair Met(O), can defend human lens cells against oxidative stress induced cell death. A key feature of oxidative stress is increased reactive oxygen species (ROS) in association with loss of mitochondrial function. Here, we sought to establish a potential role for MsrA in the accumulation of ROS in lens cells and the corresponding mitochondrial membrane potential in these cells. Targeted gene silencing was used to establish populations of lens cells expressing different levels of MsrA, and the mitochondrial membrane potential and ROS levels of these cell populations were monitored. Decreased MsrA levels were found to be associated with loss of cell viability, decreased mitochondrial membrane potential, and increased ROS levels in the absence of oxidative stress. These effects were augmented upon oxidative stress treatment. These results provide evidence that MsrA is a major determinant for accumulation of ROS in lens cells and that increased ROS levels in lens cells are associated with a corresponding decrease in mitochondrial membrane potential that is likely related to the requirement for MsrA in lens cell viability. PMID:16934804

  11. Lipid metabolism in mitochondrial membranes.

    PubMed

    Mayr, Johannes A

    2015-01-01

    Mitochondrial membranes have a unique lipid composition necessary for proper shape and function of the organelle. Mitochondrial lipid metabolism involves biosynthesis of the phospholipids phosphatidylethanolamine, cardiolipin and phosphatidylglycerol, the latter is a precursor of the late endosomal lipid bis(monoacylglycero)phosphate. It also includes mitochondrial fatty acid synthesis necessary for the formation of the lipid cofactor lipoic acid. Furthermore the synthesis of coenzyme Q takes place in mitochondria as well as essential parts of the steroid and vitamin D metabolism. Lipid transport and remodelling, which are necessary for tailoring and maintaining specific membrane properties, are just partially unravelled. Mitochondrial lipids are involved in organelle maintenance, fission and fusion, mitophagy and cytochrome c-mediated apoptosis. Mutations in TAZ, SERAC1 and AGK affect mitochondrial phospholipid metabolism and cause Barth syndrome, MEGDEL and Sengers syndrome, respectively. In these disorders an abnormal mitochondrial energy metabolism was found, which seems to be due to disturbed protein-lipid interactions, affecting especially enzymes of the oxidative phosphorylation. Since a growing number of enzymes and transport processes are recognised as parts of the mitochondrial lipid metabolism, a further increase of lipid-related disorders can be expected. PMID:25082432

  12. SB203580 enhances the RV-induced loss of mitochondrial membrane potential and apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Li, Hai-yang; Zhuang, Cai-ping; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψm) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

  13. Mitochondrial fusion through membrane automata.

    PubMed

    Giannakis, Konstantinos; Andronikos, Theodore

    2015-01-01

    Studies have shown that malfunctions in mitochondrial processes can be blamed for diseases. However, the mechanism behind these operations is yet not sufficiently clear. In this work we present a novel approach to describe a biomolecular model for mitochondrial fusion using notions from the membrane computing. We use a case study defined in BioAmbient calculus and we show how to translate it in terms of a P automata variant. We combine brane calculi with (mem)brane automata to produce a new scheme capable of describing simple, realistic models. We propose the further use of similar methods and the test of other biomolecular models with the same behaviour. PMID:25417022

  14. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

    PubMed Central

    Gerencser, Akos A; Chinopoulos, Christos; Birket, Matthew J; Jastroch, Martin; Vitelli, Cathy; Nicholls, David G; Brand, Martin D

    2012-01-01

    Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV. PMID:22495585

  15. Ram spermatozoa migrating through artificial mucus in vitro have reduced mitochondrial membrane potential but retain their viability.

    PubMed

    Martínez-Rodríguez, Carmen; Alvarez, Mercedes; López-Urueña, Elena; Gomes-Alves, Susana; Anel-López, Luis; Chamorro, Cesar A; Anel, Luis; de Paz, Paulino

    2015-06-01

    Sperm motility in vitro is one of the most common predictors of fertility in male screening. We propose that a mucus-penetration assay can isolate a cellular subpopulation critical to reproductive success. To this end, a device was designed with three modules (sample, test and collection) and its conditions of use evaluated (length of mucus, incubation time, mucus medium, sperm concentration and position in relation to the horizontal). The number of spermatozoa migrating and the viability and acrosomal status of the spermatozoa not migrating were calculated. The second objective was to evaluate the qualitative parameters of the spermatozoa migrating in 1.6% polyacrylamide for 30min. The number of spermatozoa migrating and the sperm motility, viability and the acrosomal and mitochondrial status of three sperm populations (fresh, not migrating and migrating) were determined. A higher number of migrating spermatozoa were observed after 60min of incubation, but this situation adversely affected sperm quality. The methylcellulose-based test showed a significantly lower number of migrating spermatozoa than the polyacrylamide test. The position at an angle of 45° resulted in a higher number of migrating spermatozoa in the polyacrylamide-based test. The sperm counts for three consecutive assays indicated an acceptable repeatability of the method. The viability and acrosomal status of the migrating spermatozoa showed no significant changes with regard to the control when the device was placed at 45°, whereas these parameters showed lower values at 0°. The percentage of high mitochondrial membrane potential spermatozoa was significantly reduced in the population of migrating spermatozoa. PMID:25413445

  16. Formation and Regulation of Mitochondrial Membranes

    PubMed Central

    Schenkel, Laila Cigana

    2014-01-01

    Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases. PMID:24578708

  17. Ferric nitrilotriacetate (Fe-NTA)-induced reactive oxidative species protects human hepatic stellate cells from apoptosis by regulating Bcl-2 family proteins and mitochondrial membrane potential

    PubMed Central

    Liu, Mei; Li, Shu-Jie; Xin, Yong-Ning; Ji, Shu-Sheng; Xie, Rui-Jin; Xuan, Shi-Ying

    2015-01-01

    Reactive oxidative species (ROS)-induced apoptosis of human hepatic stellate (HSC) is one of the treatments for liver fibrosis. However, how ROS (reactive oxygen species) affect HSC apoptosis and liver fibrosis is still unknown. In our study, ROS in human HSC cell line LX-2 was induced by ferric nitrilotriacetate (Fe-NTA) and assessed by superoxide dismutase (SOD) activity and methane dicarboxylic aldehyde (MDA) level. We found that in LX2 cells Fe-NTA induced notable ROS, which played a protective role in HSCs cells apoptosis by inhibiting Caspase-3 activation. Fe-NTA-induced ROS increased mRNA and protein level of anti-apoptosis Bcl-2 and decreased mRNA protein level of pro-apoptosis gene Bax, As a result, maintaining mitochondrial membrane potential of HSCs. Fe-NTA-induced ROS play a protective role in human HSCs by regulating Bcl-2 family proteins and mitochondrial membrane potential. PMID:26770403

  18. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells

    PubMed Central

    Gerencser, Akos A.; Mookerjee, Shona A.; Jastroch, Martin; Brand, Martin D.

    2016-01-01

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions. PMID:27404273

  19. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

    PubMed

    Gerencser, Akos A; Mookerjee, Shona A; Jastroch, Martin; Brand, Martin D

    2016-01-01

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions. PMID:27404273

  20. Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells

    PubMed Central

    Buckler, K J; Vaughan-Jones, R D

    1998-01-01

    Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4-dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mm Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. In perforated-patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to −70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3–4 s. We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage-gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation. PMID:9824720

  1. Effects of mitochondrial uncouplers on intracellular calcium, pH and membrane potential in rat carotid body type I cells.

    PubMed

    Buckler, K J; Vaughan-Jones, R D

    1998-12-15

    1. Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4-dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mM Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. 2. In perforated-patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to -70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. 3. Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. 4. FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3-4 s. 5. We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage-gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation. PMID:9824720

  2. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    SciTech Connect

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  3. Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro.

    PubMed

    Rogers, Susannah K; Shapiro, Lee A; Tobin, Richard P; Tow, Benjamin; Zuzek, Aleksej; Mukherjee, Sanjib; Newell-Rogers, M Karen

    2014-01-01

    Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor-ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action. PMID:24600432

  4. The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons

    PubMed Central

    Koyano, Fumika; Okatsu, Kei; Ishigaki, Shinsuke; Fujioka, Yusuke; Kimura, Mayumi; Sobue, Gen; Tanaka, Keiji; Matsuda, Noriyuki

    2013-01-01

    PINK1 and PARKIN are causal genes for hereditary Parkinsonism. Recent studies have shown that PINK1 and Parkin play a pivotal role in the quality control of mitochondria, and dysfunction of either protein likely results in the accumulation of low-quality mitochondria that triggers early-onset familial Parkinsonism. As neurons are destined to degenerate in PINK1/Parkin-associated Parkinsonism, it is imperative to investigate the function of PINK1 and Parkin in neurons. However, most studies investigating PINK1/Parkin have used non-neuronal cell lines. Here we show that the principal PINK1 and Parkin cellular events that have been documented in non-neuronal lines in response to mitochondrial damage also occur in primary neurons. We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria. Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin–ester formation at Cys431 of Parkin. As a result, mitochondrial substrates in neurons become ubiquitylated. These results underscore the relevance of the PINK1/Parkin-mediated mitochondrial quality control pathway in primary neurons and shed further light on the underlying mechanisms of the PINK1 and Parkin pathogenic mutations that predispose Parkinsonism in vivo. PMID:23751051

  5. Dysfunction of Rice Mitochondrial Membrane Induced by Yb3+.

    PubMed

    Gao, Jia-Ling; Wu, Man; Liu, Wen; Feng, Zhi-Jiang; Zhang, Ye-Zhong; Jiang, Feng-Lei; Liu, Yi; Dai, Jie

    2015-12-01

    Ytterbium (Yb), a widely used rare earth element, is treated as highly toxic to human being and adverseness to plant. Mitochondria play a significant role in plant growth and development, and are proposed as a potential target for ytterbium toxicity. In this paper, the biological effect of Yb(3+) on isolated rice mitochondria was investigated. We found that Yb(3+) with high concentrations (200 ~ 600 μM) not only induced mitochondrial membrane permeability transition (mtMPT), but also disturbed the mitochondrial ultrastructure. Moreover, Yb(3+) caused the respiratory chain damage, ROS formation, membrane potential decrease, and mitochondrial complex II activity reverse. The results above suggested that Yb(3+) with high concentrations could induce mitochondrial membrane dysfunction. These findings will support some valuable information to the safe application of Yb-based agents. PMID:26305923

  6. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) by using fluorescence-activated flow cytometry.

    PubMed

    Guthrie, H David; Welch, Glenn R

    2008-01-01

    A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (Deltapsi(m)), >80-100 mV using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with membranes possessing a high Deltapsi(m), JC-1 forms aggregates (J(agg)) that are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator, menadione reduced sperm motility and reduced Deltapsi(m) in a dose responsive fashion that was closely correlated with the loss of motility. PMID:19082941

  7. Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential.

    PubMed

    Xiao, Dong; Lew, Karen L; Zeng, Yan; Xiao, Hui; Marynowski, Stanley W; Dhir, Rajiv; Singh, Shivendra V

    2006-11-01

    The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC), which is a cancer chemopreventive constituent of cruciferous vegetables, using PC-3 human prostate cancer cells as a model. The PEITC-induced cell death in PC-3 cells was associated with disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria to the cytosol and generation of reactive oxygen species (ROS), which were blocked in the presence of a combined mimetic of superoxide dismutase and catalase (Euk134). Ectopic expression of Bcl-xL, whose protein level is reduced markedly on treatment of PC-3 cells with PEITC, conferred partial protection against PEITC-induced apoptosis only at higher drug concentrations (>10 microM). Administration of 12 micromol PEITC/day (Monday through Friday) by oral gavage significantly retarded growth of PC-3 xenografts in athymic mice. For instance, 31 days after the initiation of PEITC administration, the average tumor volume in control mice (721 +/- 153 mm3) was approximately 2-fold higher compared with mice receiving 12 micromol PEITC/day. The PEITC-mediated inhibition of PC-3 xenograft growth was associated with induction of Bax and Bid proteins. In conclusion, the present study indicates that the PEITC-induced apoptosis in PC-3 cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential and regulated by Bax and Bid. PMID:16774948

  8. Two Trichothecene Mycotoxins from Myrothecium roridum Induce Apoptosis of HepG-2 Cells via Caspase Activation and Disruption of Mitochondrial Membrane Potential.

    PubMed

    Ye, Wei; Chen, Yuchan; Li, Haohua; Zhang, Weimin; Liu, Hongxin; Sun, Zhanghua; Liu, Taomei; Li, Saini

    2016-01-01

    Trichothecene mycotoxins are a type of sesquiterpenoid produced by various kinds of plantpathogenic fungi. In this study, two trichothecene toxins, namely, a novel cytotoxic epiroridin acid and a known trichothecene, mytoxin B, were isolated from the endophytic fungus Myrothecium roridum derived from the medicinal plant Pogostemon cablin. The two trichothecene mytoxins were confirmed to induce the apoptosis of HepG-2 cells by cytomorphology inspection, DNA fragmentation detection, and flow cytometry assay. The cytotoxic mechanisms of the two mycotoxins were investigated by quantitative real time polymerase chain reaction, western blot, and detection of mitochondrial membrane potential. The results showed that the two trichothecene mycotoxins induced the apoptosis of cancer cell HepG-2 via activation of caspase-9 and caspase-3, up-regulation of bax gene expression, down-regulation of bcl-2 gene expression, and disruption of the mitochondrial membrane potential of the HepG-2 cell. This study is the first to report on the cytotoxic mechanism of trichothecene mycotoxins from M. roridum. This study provides new clues for the development of attenuated trichothecene toxins in future treatment of liver cancer. PMID:27322225

  9. Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions.

    PubMed

    Belosludtsev, Konstantin N; Belosludtseva, Natalia V; Agafonov, Alexey V; Penkov, Nikita V; Samartsev, Victor N; Lemasters, John J; Mironova, Galina D

    2015-10-01

    The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10-50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening. PMID:26014488

  10. Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions

    PubMed Central

    Belosludtsev, Konstantin N.; Belosludtseva, Natalia V.; Agafonov, Alexey V.; Penkov, Nikita V.; Samartsev, Victor N.; Lemasters, John J.; Mironova, Galina D.

    2016-01-01

    The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10–50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening. PMID:26014488

  11. Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry.

    PubMed

    Krumschnabel, Gerhard; Eigentler, Andrea; Fasching, Mario; Gnaiger, Erich

    2014-01-01

    The mitochondrial transmembrane potential (Δψmt or mtMP) is directly influenced by oxidative phosphorylation (OXPHOS). The exact nature of the interactions between respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. Here, we describe a combination of high-resolution respirometry and fluorometry based on the OROBOROS Oxygraph-2k and the widely applied mtMP indicator safranin. The analysis of OXPHOS in mouse brain homogenates revealed that, at commonly applied concentrations, safranin inhibits Complex I-driven OXPHOS capacity, primarily targeting the phosphorylation system, but has no effects on LEAK respiration. Conversely, Complex II-driven OXPHOS capacity was inhibited by <20% by safranin concentrations normally used for mtMP monitoring. The mtMP was higher in the LEAK state without adenylates than at identical LEAK respiration after ADP stimulation and Complex V inhibition with oligomycin. The maximal electron transfer system (ETS) capacity was reached in uncoupler titrations before the mtMP fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations, resulting in the progressive reduction of mtMP. In a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to the inhibition of OXPHOS to 50% of its normal capacity, but robustly responded to inhibitors when respiration was limited by substrate depletion. The optimal concentration of uncoupler supporting maximal ETS capacity varied as a function of pharmacological intervention. Taken together, the combined measurement of respiration and mtMP greatly enhances the informative potential of OXPHOS studies. The respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore employed to assess mtMP in any respiratory state, tissue type, and pathophysiological condition. The methodological issues analyzed herein are relevant for

  12. Trypanosoma cruzi mitochondrial swelling and membrane potential collapse as primary evidence of the mode of action of naphthoquinone analogues

    PubMed Central

    2013-01-01

    Background Naphthoquinones (NQs) are privileged structures in medicinal chemistry due to the biological effects associated with the induction of oxidative stress. The present study evaluated the activities of sixteen NQs derivatives on Trypanosoma cruzi. Results Fourteen NQs displayed higher activity against bloodstream trypomastigotes of T. cruzi than benznidazole. Further assays with NQ1, NQ8, NQ9 and NQ12 showed inhibition of the proliferation of axenic epimastigotes and intracelulluar amastigotes interiorized in macrophages and in heart muscle cells. NQ8 was the most active NQ against both proliferative forms of T. cruzi. In epimastigotes the four NQs induced mitochondrial swelling, vacuolization, and flagellar blebbing. The treatment with NQs also induced the appearance of large endoplasmic reticulum profiles surrounding different cellular structures and of myelin-like membranous contours, morphological characteristics of an autophagic process. At IC50 concentration, NQ8 totally disrupted the ΔΨm of about 20% of the parasites, suggesting the induction of a sub-population with metabolically inactive mitochondria. On the other hand, NQ1, NQ9 or NQ12 led only to a discrete decrease of TMRE + labeling at IC50 values. NQ8 led also to an increase in the percentage of parasites labeled with DHE, indicative of ROS production, possibly the cause of the observed mitochondrial swelling. The other three NQs behaved similarly to untreated controls. Conclusions NQ1, NQ8, NQ9 and NQ12 induce an autophagic phenotype in T. cruzi epimastigoted, as already observed with others NQs. The absence of oxidative stress in NQ1-, NQ9- and NQ12-treated parasites could be due to the existence of more than one mechanism of action involved in their trypanocidal activity, leaving ROS generation suppressed by the detoxification system of the parasite. The strong redox effect of NQ8 could be associated to the presence of the acetyl group in its structure facilitating quinone reduction, as

  13. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zhu, Yue-Yong; Huang, Hong-Yan; Wu, Yin-Lian

    2015-10-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‑123 DNA‑binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‑dependent, as well as time‑dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‑G1 (apoptotic) phase of the cell cycle, in a dose‑dependent manner. Staining with Annexin V‑fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  14. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

    PubMed Central

    Strauss, Mike; Hofhaus, Götz; Schröder, Rasmus R; Kühlbrandt, Werner

    2008-01-01

    ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long ∼1-μm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of ∼0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance. PMID:18323778

  15. In boar sperm capacitation L-lactate and succinate, but not pyruvate and citrate, contribute to the mitochondrial membrane potential increase as monitored via safranine O fluorescence.

    PubMed

    Paventi, Gianluca; Lessard, Christian; Bailey, Janice L; Passarella, Salvatore

    2015-07-01

    Having ascertained using JC-1 as a probe that, in distinction with the controls, during capacitation boar sperm maintains high mitochondrial membrane potential (ΔΨ), to gain some insight into the role of mitochondria in capacitation, we monitored ΔΨ generation due to externally added metabolites either in hypotonically-treated spermatozoa (HTS) or in intact cells by using safranine O as a probe. During capacitation, the addition to HTS of L-lactate and succinate but not those of pyruvate, citrate and ascorbate + TMPD resulted in increase of ΔΨ generation. Accordingly, the addition of L-lactate and succinate, but not that of citrate, to intact sperm resulted in ΔΨ generation increased in capacitation. PMID:25956060

  16. Radiosensitization by fullerene-C60 dissolved in squalene on human malignant melanoma through lipid peroxidation and enhanced mitochondrial membrane potential

    NASA Astrophysics Data System (ADS)

    Kato, Shinya; Kimura, Masatsugu; Miwa, Nobuhiko

    2014-04-01

    We examined fullerene-C60 dissolved in squalene (C60/Sqe) for the ability to potentiate the radiosensitization under X-ray irradiation on human malignant melanoma HMV-II cells, which were treated with C60/Sqe and thereafter irradiated with X-ray. The cell proliferation for C60/Sqe was inhibited more markedly than for Sqe alone. Meanwhile, cell proliferation was almost unaltered for C60/squalane (Sqa) or Sqa, a hydrogenated form of Sqe, as compared to no-additive control. Thus radiosensitization of C60/Sqe is attributed to peroxidation of unsaturated bonds of squalene by X-ray-excited C60 in contrast to squalane. The fluorescence images of HMV-II cells stained with Rhodamine123, an indicator for mitochondrial membrane potential, were monitored for 6 h after X-ray irradiation. C60/Sqe obviously exhibited more augmented fluorescence intensity on perinuclear region of HMV-II cells than Sqe alone. TBARS assay showed that the lipid peroxidation level as malondialdehyde-equivalent increased by combination of C60/Sqe and X-ray dose-dependently on X-ray doses. C60/Sqe exhibited lipid peroxidation more markedly by 1.2-fold than Sqe alone. Thus the level of lipid peroxidation of squalene was sufficiently higher in C60/Sqe than in Sqe in the absence of C60 under X-ray irradiation, suggesting the combination of C60/Sqe and X-ray irradiation induced radiosensitization on HMV-II cells by peroxidation of absorbed Sqe in mitochondrial membrane via oxidative stress mediated by fullerene-C60.

  17. Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

    PubMed Central

    Richter, Uwe; Lahtinen, Taina; Marttinen, Paula; Suomi, Fumi

    2015-01-01

    Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes. PMID:26504172

  18. Paraquat Induces Cell Death Through Impairing Mitochondrial Membrane Permeability.

    PubMed

    Huang, Chuen-Lin; Chao, Chih-Chang; Lee, Yi-Chao; Lu, Mei-Kuang; Cheng, Jing-Jy; Yang, Ying-Chen; Wang, Vin-Chi; Chang, Wen-Chang; Huang, Nai-Kuei

    2016-05-01

    Paraquat (PQ) as a Parkinsonian mimetic has been demonstrated to impair dopaminergic (DAergic) neurons and is highly correlated with the etiology of Parkinson's disease (PD) where the death of DAergic neurons has been mainly attributed to impaired mitochondrial functioning. In this study, PQ-induced cytotoxicity focusing on mitochondrial membrane permeability (MMP), which has been implicated to play a part in neurodegeneration, was investigated. Primarily, PQ-induced cytotoxicity and reactive oxygen species (ROS) were inhibited by an inhibitor of NADPH oxidase (NOX), indicating the toxic effect of PQ redox cycling. Further, dibucaine and cyclosporin A which respectively inhibit mitochondrial apoptosis-induced channels (MAC) and mitochondrial permeability transition pores (mPTP) were used and found to prevent PQ-induced mitochondrial dysfunction, such as decreased mitochondrial membrane potential and increased MMP, mitochondrial ROS, and pro-apoptotic factor release. Knockdown of bax and/or bak blocked PQ-induced mitochondrial clusterization of Bax and/or Bak and cytotoxicity, demonstrating the significance of MAC which is composed of Bax and/or Bak. This clusterization coincided with the release of mitochondrial apoptotic factors before there was an increase in inner MMP, indicating that MAC may precede mPTP formation. Besides, NOX inhibitor but not dibucaine attenuated the earlier PQ-induced cytosolic ROS formation or Bax and/or Bak clusterization indicating PQ redox cycling may account for MAC formation. In this model, we have resolved for the first that PQ cytotoxicity through redox cycling may sequentially result in increased outer (MAC) and inner (mPTP) MMP and suggested MMP could be implicated as a therapeutic target in treating neurodegenerative diseases like PD. PMID:25947082

  19. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) using flow cytometry.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (''m), > 80-100 mV using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear ...

  20. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase

    PubMed Central

    Munday, Jane C.; Tagoe, Daniel N. A.; Stelmanis, Valters; Schnaufer, Achim

    2016-01-01

    Background Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. Methodology/Principal Findings Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. Conclusions/Significance Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial

  1. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  2. Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin.

    PubMed

    Ismail, Samhani; Haris, Khalilah; Abdul Ghani, Abdul Rahman Izaini; Abdullah, Jafri Malin; Johan, Muhammad Farid; Mohamed Yusoff, Abdul Aziz

    2013-09-01

    Aloe emodin, one of the active compounds found in Aloe vera leaves, plays an important role in the regulation of cell growth and death. It has been reported to promote the anti-cancer effects in various cancer cells by inducing apoptosis. However, the mechanism of inducing apoptosis by this agent is poorly understood in glioma cells. This research is to investigate the apoptosis and cell cycle arrest inducing by aloe emodin on U87 human malignant glioma cells. Aloe emodin showed a time- and dose-dependent inhibition of U87 cells proliferation and decreased the percentage of viable U87 cells via the induction of apoptosis. Characteristic morphological changes, such as the formation of apoptotic bodies, were observed with confocal microscope by Annexin V-FITC/PI staining, supporting our viability study and flow cytometry analysis results. Our data also demonstrated that aloe emodin arrested the cell cycle in the S phase and promoted the loss of mitochondrial membrane potential in U87 cells that indicated the early event of the mitochondria-induced apoptotic pathway. PMID:23869465

  3. Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells.

    PubMed

    Villaverde, Marcela Solange; Targovnik, Alexandra Marisa; Miranda, María Victoria; Finocchiaro, Liliana María Elena; Glikin, Gerardo Claudio

    2016-08-01

    Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy. PMID:27236354

  4. Selenium (sodium selenite) causes cytotoxicity and apoptotic mediated cell death in PLHC-1 fish cell line through DNA and mitochondrial membrane potential damage.

    PubMed

    Selvaraj, Vellaisamy; Tomblin, Justin; Yeager Armistead, Mindy; Murray, Elizabeth

    2013-01-01

    Elevated concentration of selenium poses a toxic threat to organisms inhabiting aquatic ecosystems influenced by excessive inputs from anthropogenic sources. Selenium is also an essential micronutrient in living things, particularly in fish, and provides antioxidant properties to tissues. Whole fish and hepatocytes in primary culture show selenite toxicity above threshold levels. The present study was designed to investigate the process by which selenite exposure causes cellular toxicity and apoptotic and necrotic cell death in fish hepatoma cell line PLHC-1. PLHC-1 cells were exposed to various selenite concentrations (1, 10, 50 and 100 μM) for 10, 20 and 40 h intervals. The 24h inhibitory concentration 50 (IC₅₀) of selenite in PLHC-1 cell line was found to be 237 μM. Flow cytometery data showed that selenite exposed cells promote apoptotic and necrotic mediated cell death when selenite concentrations were ≥10 μM compared to control. Selenite exposure was associated with a significant increase of caspase-3 activities suggesting the induction of apoptosis. Selenite exposure at high levels (≥10 μM) and longer exposure times (≥20 h) induces mitochondrial membrane potential damage (ΔΨ(m)), DNA damage and elevated production of ROS which could be associated with cell death. PMID:23158585

  5. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

    PubMed Central

    Montes de Oca Balderas, Pavel; Aguilera, Penélope

    2015-01-01

    Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase. PMID:25954808

  6. Inhibition of N-Methyl-D-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential.

    PubMed

    Marshall, John; Wong, Kwoon Y; Rupasinghe, Chamila N; Tiwari, Rakesh; Zhao, Xiwu; Berberoglu, Eren D; Sinkler, Christopher; Liu, Jenney; Lee, Icksoo; Parang, Keykavous; Spaller, Mark R; Hüttemann, Maik; Goebel, Dennis J

    2015-09-01

    It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons. PMID:26100636

  7. NADPH Oxidase-Mediated Superoxide Production by Intermediary Bacterial Metabolites of Dibenzofuran: A Potential Cause for Trans-Mitochondrial Membrane Potential (ΔΨm) Collapse in Human Hepatoma Cells.

    PubMed

    Jaiswal, Prashant Kumar; Gupta, Jyotsana; Shahni, Shweta; Thakur, Indu Shekhar

    2015-09-01

    Dibenzofuran is a direct precursor of extremely toxic compounds such as dioxins. It is widely distributed persistent organic pollutant in environment that potentiate oxidative stress, apoptosis, and necrosis through bioactivation in HepG2 cells. An alkalotolerent Pseudomonas strain ISTDF1 can metabolize dibenzofuran as a sole source of carbon and energy through diverse dioxygenation. However, there is a paucity of information about the potential toxic effects of the intermediary metabolites that are formed during treatment with dibenzofuran. We have assessed and discovered the potential mechanism of toxicity induced by metabolites of dibenzofuran that were formed at 18 and 36 h. Cell viability, CYP1A2 induction, ROS activity, Superoxide production, mitochondrial NADPH oxidase activity, and mitochondrial trans-membrane potential were studied using different assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), confocal laser scanning microscopy, and flow cytometry. Analysis revealed formation of 2-(1-carbonyl methylidine)-2,3-dihydrobenzofuranlidene after 18 h of bacterial treatment due to oxygenation at carbon (C3-C4). This compound induces higher mitochondrial NADPH oxidase-dependent superoxide production that makes it more toxic than the parent compound. It was evident that after 36 h of bacterial treatment, toxicity induced by dibenzofuran and its metabolites was completely removed. This study highlights the fact that despite of efficient biodegradation of toxicants, bioactive toxic intermediates can be formed. Therefore, it is necessary to assess the toxicity of each intermediary for complete mitigation of associated risk. PMID:26032510

  8. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

    PubMed Central

    de Vantéry Arrighi, Corinne; Lucas, Hervé; Chardonnens, Didier; de Agostini, Ariane

    2009-01-01

    Background Externalization of phosphatidylserine (EPS) occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS) on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality. Methods EPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB) technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC). The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP) by Rhodamine 123. Results Mean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h. Conclusion Human ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation. PMID:19133142

  9. Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages.

    PubMed

    Giardina, Tindaro M; Steer, James H; Lo, Susan Z Y; Joyce, David A

    2008-02-01

    Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments. PMID:18082129

  10. Bax inserts into the mitochondrial outer membrane by different mechanisms.

    PubMed

    Cartron, Pierre-François; Bellot, Grégory; Oliver, Lisa; Grandier-Vazeille, Xavier; Manon, Stephen; Vallette, François M

    2008-09-01

    Bax insertion into the mitochondrial outer membrane is essential for the implementation of apoptosis. However, little is known about the first stage of Bax integration into the mitochondrial outer membrane. We have recently shown that TOM22, a mitochondrial outer membrane receptor, is important for insertion, although other reports have suggested that only mitochondrial lipids are involved in this process. Here, we show that monomers, but not dimers, of Bax require the presence of TOM22 and TOM40 to integrate into mitochondria. In addition we show that once inserted into the membrane, Bax can act as a receptor for cytosolic Bax. PMID:18687331

  11. Bcl-2 apoptosis proteins, mitochondrial membrane curvature, and cancer

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Schmidt, Nathan; Sanders, Lori; Mishra, Abhijit; Wong, Gerard; Ivashyna, Olena; Christenson, Eric; Schlesinger, Paul; Akabori, Kiyotaka; Santangelo, Christian

    2012-02-01

    Critical interactions between Bcl-2 family proteins permeabilize the outer mitochondrial membrane, a common decision point early in the intrinsic apoptotic pathway that irreversibly commits the cell to death. However, a unified picture integrating the essential non-passive role of lipid membranes with the contested dynamics of Bcl-2 regulation remains unresolved. Correlating results between synchrotron x-ray diffraction and microscopy in cell-free assays, we report activation of pro-apoptotic Bax induces strong pure negative Gaussian membrane curvature topologically necessary for pore formation and membrane remodeling events. Strikingly, Bcl-xL suppresses not only Bax-induced pore formation, but also membrane remodeling by disparate systems including cell penetrating, antimicrobial or viral fusion peptides, and bacterial toxin, none of which have BH3 allosteric domains to mediate direct binding. We propose a parallel mode of Bcl-2 pore regulation in which Bax and Bcl-xL induce antagonistic and mutually interacting Gaussian membrane curvatures. The universal nature of curvature-mediated interactions allows synergy with direct binding mechanisms, and potentially accounts for the Bcl-2 family modulation of mitochondrial fission/fusion dynamics.

  12. Pyr3, a TRPC3 channel blocker, potentiates dexamethasone sensitivity and apoptosis in acute lymphoblastic leukemia cells by disturbing Ca(2+) signaling, mitochondrial membrane potential changes and reactive oxygen species production.

    PubMed

    Abdoul-Azize, Souleymane; Buquet, Catherine; Vannier, Jean-Pierre; Dubus, Isabelle

    2016-08-01

    Dexamethasone (Dex) is used as a chemotherapeutic drug in the treatment of acute lymphoblastic leukemia (ALL) because of its capacity to induce apoptosis. However, some ALL patients acquire resistance to glucocorticoids (GC). Thus, it is important to explore new agents to overcome GC resistance. The aim of the present work was to assess the ability of Pyr3, a selective inhibitor of transient receptor potential canonical 3 (TRPC3), to sensitize human ALL cells to Dex. We show here, for the first time, that Pyr3 enhances Dex sensitivity through the distraction of Dex-mediated Ca(2+) signaling in ALL cells (in vitro) and primary blasts (ex vivo) associated with mitochondrial-mediated reactive oxygen species production in ALL cells. Pyr3 alone induced Ca(2+) signaling via only endoplasmic reticulum-released Ca(2+) and exerted inhibitory effect on store-operated Ca(2+) entry in dose-dependent manner in ALL cell lines. Pre-incubation of cells with Pyr3 significantly curtailed the thapsigargin- and Dex-evoked Ca(2+) signaling in ALL cell lines. Pyr3 synergistically potentiated Dex lethality, as shown by the induction of cell mortality, G2/M cell cycle arrest and apoptosis in ALL cell lines. Moreover, Pyr3 disrupted Dex-mediated Ca(2+) signaling and increased the sensitivity of Dex-induced cell death in primary blasts from ALL patients. Additional analysis showed that co-treatment with Dex and Pyr3 results in mitochondrial membrane potential depolarization and reactive oxygen species production in ALL cells. Together, Pyr3 exhibited potential therapeutic benefit in combination with Dex to inverse glucocorticoid resistance in human ALL and probably in other lymphoid malignancies. PMID:27179991

  13. Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

    PubMed Central

    Bohnert, Maria; Wenz, Lena-Sophie; Zerbes, Ralf M.; Horvath, Susanne E.; Stroud, David A.; von der Malsburg, Karina; Müller, Judith M.; Oeljeklaus, Silke; Perschil, Inge; Warscheid, Bettina; Chacinska, Agnieszka; Veenhuis, Marten; van der Klei, Ida J.; Daum, Günther; Wiedemann, Nils; Becker, Thomas; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins. PMID:22918945

  14. Sanguinarine inhibits angiotensin II-induced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential

    PubMed Central

    LIU, YUAN; JIAO, RONG; MA, ZHEN-GUO; LIU, WEI; WU, QING-QING; YANG, ZHENG; LI, FANG-FANG; YUAN, YUAN; BIAN, ZHOU-YAN; TANG, QI-ZHU

    2015-01-01

    Cell apoptosis induced by Angiotensin II (Ang II) has a critical role in the development of cardiovascular diseases. The aim of the present study was to investigate whether sanguinarine (SAN), a drug which was proved to have anti-oxidant, anti-proliferative and immune enhancing effects, can abolish cell apoptosis induced by Ang II. In the present study, H9c2 cardiac cells were stimulated with 10 µM Ang II with or without SAN. The level of intracellular reactive oxygen species (ROS) generation was assessed using dichlorodihydrofluorescein diacetate, and changes of the mitochondrial membrane potential (MMP) were assessed using JC-1 staining. Furthermore, mRNA expression of NOX2 was determined by reverse transcription quantitative polymerase chain reaction, and apoptosis was detected by Annexin V/propidium iodide staining and flow cytometry. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) as well as cleaved (c)-caspase 3 and -9 were detected by western blot analysis, and the activity of caspase 3 and -9 was detected using an ELISA. The results of the present study showed that NOX2 expression and ROS generation induced by Ang II were inhibited by SAN, and the Ang 2-induced MMP loss was also ameliorated. Furthermore, Ang II-induced H9c2 cardiac cell apoptosis as well as c-caspase 3 and -9 levels were significantly reduced by SAN. Investigation of the possible pathway involved in the anti-apoptotic effect of SAN showed that the expression of Bcl-2 was decreased, while that of Bax was increased following stimulation with Ang II, which was reversed following treatment with SAN. In addition, Ang II enhanced the activity of caspase 9 and cleaved downstream caspases such as caspase-3, initiating the caspase cascade, while pre-treatment of H9c2 cardiac cells with SAN blocked these effects. In conclusion, the findings of the present study indicated that SAN inhibits the apoptosis of H9c2 cardiac cells induced by Ang II, most likely via restoring

  15. Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane

    PubMed Central

    Sen, Aditya; Cox, Rachel T.

    2016-01-01

    ABSTRACT Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1–Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1–Parkin pathway. PMID:26834020

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

    PubMed

    Zhang, Cuilin; Shi, Zhun; Zhang, Lingzhi; Zhou, Zehua; Zheng, Xiaoyuan; Liu, Guiying; Bu, Guojun; Fraser, Paul E; Xu, Huaxi; Zhang, Yun-Wu

    2016-03-01

    Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology. PMID:26813789

  17. Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1

    PubMed Central

    Povelones, Megan L.; Tiengwe, Calvin; Gluenz, Eva; Gull, Keith; Englund, Paul T.; Jensen, Robert E.

    2016-01-01

    Summary In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knockdown of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential, and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes. PMID:23336702

  18. Deficiency in the inner mitochondrial membrane peptidase 2-like (Immp21) gene increases ischemic brain damage and impairs mitochondrial function

    PubMed Central

    Ma, Yi; Mehta, Suresh L.; Lu, Baisong; Andy Li, P.

    2011-01-01

    Mitochondrial dysfunction plays an important role in mediating ischemic brain damage. Immp2l is an inner mitochondrial membrane peptidase that processes mitochondrial proteins cytochrome c1 (Cyc1). Homozygous mutation of Immp2l (Immp2lTg(Tyr)979Ove or Immp2l−/−) elevates mitochondrial membrane potential, increases superoxide (•O2−) production in the brain and impairs fertility. The objectives of this study are to explore the effects of heterozygous mutation of lmmp2l (Immp2l+/−) on ischemic outcome and to determine the influence of Immp2l deficiency on brain mitochondria after stroke. Male Immp2l+/− and wild-type (WT) mice were subjected to 1-h focal cerebral ischemia. Their brains were harvested after 5 and 24-h of reperfusion. The results showed that infarct volume and DNA oxidative damage significantly increased in the Immp2l+/− mice. There were no obvious cerebral vasculature abnormalities between the two types of mice viewed by Indian ink perfusion. The increased damage in Immp2l+/− mice was associated with early increase in •O2− production. Mitochondrial respiratory rate, total mitochondrial respiratory capacity and mitochondrial respiratory complex activities were decreased at 5-h of recirculation in Immp2l+/− mice compared to WT mice. Our results suggest that lmmp2l deficiency increases ischemic brain damage by enhancing •O2− production and damaging mitochondrial functional performance. PMID:21824519

  19. Bidirectional fluxes of spermine across the mitochondrial membrane.

    PubMed

    Grancara, Silvia; Martinis, Pamela; Manente, Sabrina; García-Argáez, Aida Nelly; Tempera, Giampiero; Bragadin, Marcantonio; Dalla Via, Lisa; Agostinelli, Enzo; Toninello, Antonio

    2014-03-01

    The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (ΔΨ). The presence of phosphate increases spermine uptake by reducing ΔpH and enhancing ΔΨ. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce the ΔΨ value and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop in ΔΨ able to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis. PMID:24043461

  20. The tRNA(Gly) T10003C mutation in mitochondrial haplogroup M11b in a Chinese family with diabetes decreases the steady-state level of tRNA(Gly), increases aberrant reactive oxygen species production, and reduces mitochondrial membrane potential.

    PubMed

    Li, Wei; Wen, Chaowei; Li, Weixing; Wang, Hailing; Guan, Xiaomin; Zhang, Wanlin; Ye, Wei; Lu, Jianxin

    2015-10-01

    Mitochondrial diabetes originates mainly from mutations located in maternally transmitted, mitochondrial tRNA-coding genes. In a genetic screening program of type 2 diabetes conducted with a Chinese Han population, we found one family with suggestive maternally transmitted diabetes. The proband's mitochondrial genome was analyzed using DNA sequencing. Total 42 known nucleoside changes and 1 novel variant were identified, and the entire mitochondrial DNA sequence was assigned to haplogroup M11b. Phylogenetic analysis showed that a homoplasmic mutation, 10003T>C transition, occurred at the highly conserved site in the gene encoding tRNA(Gly). Using a transmitochondrial cybrid cell line harboring this mutation, we observed that the steady-state level of tRNA(Gly) significantly affected and the amount of tRNA(Gly) decreased by 97%, production of reactive oxygen species was enhanced, and mitochondrial membrane potential, mtDNA copy number and cellular oxygen consumption rate were remarkably decreased compared with wild-type cybrid cells. The homoplasmic 10003T>C mutation in the mitochondrial tRNA(Gly) gene suggested to be as a pathogenesis-related mutation which might contribute to the maternal inherited diabetes in the Han Chinese family. PMID:26134044

  1. Mitochondrial Membrane Studies Using Impedance Spectroscopy with Parallel pH Monitoring

    PubMed Central

    Padmaraj, Divya; Pande, Rohit; Miller, John H.; Wosik, Jarek; Zagozdzon-Wosik, Wanda

    2014-01-01

    A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample’s complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles. PMID:25010497

  2. Bcl-2 maintains the mitochondrial membrane potential, but fails to affect production of reactive oxygen species and endoplasmic reticulum stress, in sodium palmitate-induced β-cell death

    PubMed Central

    Welsh, Nils

    2014-01-01

    Background Sodium palmitate causes apoptosis of β-cells, and the anti-apoptotic protein Bcl-2 has been shown to counteract this event. However, the exact mechanisms that underlie palmitate-induced pancreatic β-cell apoptosis and through which pathway Bcl-2 executes the protective effect are still unclear. Methods A stable Bcl-2-overexpressing RINm5F cell clone (BMG) and its negative control (B45) were exposed to palmitate for up to 8 h, and cell viability, mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, and NF-κB activation were studied in time course experiments. Results Palmitate exposure for 8 h resulted in increased cell death rates, and this event was partially counteracted by Bcl-2. Bcl-2 overexpression promoted in parallel also a delayed induction of GADD153/CHOP and a weaker phosphorylation of BimEL in palmitate-exposed cells. At earlier time points (2–4 h) palmitate exposure resulted in increased generation of ROS, a decrease in mitochondrial membrane potential (Δψm), and a modest increase in the phosphorylation of eIF2α and IRE1α. BMG cells produced similar amounts of ROS and displayed the same eIF2α and IRE1α phosphorylation rates as B45 cells. However, the palmitate-induced dissipation of Δψm was partially counteracted by Bcl-2. In addition, basal NF-κB activity was increased in BMG cells. Conclusions Our results indicate that Bcl-2 counteracts palmitate-induced β-cell death by maintaining mitochondrial membrane integrity and augmenting NF-κB activity, but not by affecting ROS production and ER stress. PMID:25266628

  3. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

    PubMed

    Guthrie, H D; Welch, G R

    2006-08-01

    The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen

  4. The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity.

    PubMed

    Jahani-Asl, Arezu; Pilon-Larose, Karine; Xu, William; MacLaurin, Jason G; Park, David S; McBride, Heidi M; Slack, Ruth S

    2011-02-11

    Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases. PMID:21041314

  5. The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

    SciTech Connect

    Serasinghe, Madhavika N.; Yoon, Yisang

    2008-11-15

    Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.

  6. Mitochondrial outer-membrane permeabilization and remodelling in apoptosis.

    PubMed

    Jourdain, Alexis; Martinou, Jean-Claude

    2009-10-01

    Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP. PMID:19439192

  7. Omega-3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle.

    PubMed

    Herbst, E A F; Paglialunga, S; Gerling, C; Whitfield, J; Mukai, K; Chabowski, A; Heigenhauser, G J F; Spriet, L L; Holloway, G P

    2014-03-15

    Studies have shown increased incorporation of omega-3 fatty acids into whole skeletal muscle following supplementation, although little has been done to investigate the potential impact on the fatty acid composition of mitochondrial membranes and the functional consequences on mitochondrial bioenergetics. Therefore, we supplemented young healthy male subjects (n = 18) with fish oils [2 g eicosapentaenoic acid (EPA) and 1 g docosahexanoic acid (DHA) per day] for 12 weeks and skeletal muscle biopsies were taken prior to (Pre) and following (Post) supplementation for the analysis of mitochondrial membrane phospholipid composition and various assessments of mitochondrial bioenergetics. Total EPA and DHA content in mitochondrial membranes increased (P < 0.05) ∼450 and ∼320%, respectively, and displaced some omega-6 species in several phospholipid populations. Mitochondrial respiration, determined in permeabilized muscle fibres, demonstrated no change in maximal substrate-supported respiration, or in the sensitivity (apparent Km) and maximal capacity for pyruvate-supported respiration. In contrast, mitochondrial responses during ADP titrations demonstrated an enhanced ADP sensitivity (decreased apparent Km) that was independent of the creatine kinase shuttle. As the content of ANT1, ANT2, and subunits of the electron transport chain were unaltered by supplementation, these data suggest that prolonged omega-3 intake improves ADP kinetics in human skeletal muscle mitochondria through alterations in membrane structure and/or post-translational modification of ATP synthase and ANT isoforms. Omega-3 supplementation also increased the capacity for mitochondrial reactive oxygen species emission without altering the content of oxidative products, suggesting the absence of oxidative damage. The current data strongly emphasize a role for omega-3s in reorganizing the composition of mitochondrial membranes while promoting improvements in ADP sensitivity. PMID:24396061

  8. Omega-3 supplementation alters mitochondrial membrane composition and respiration kinetics in human skeletal muscle

    PubMed Central

    Herbst, E A F; Paglialunga, S; Gerling, C; Whitfield, J; Mukai, K; Chabowski, A; Heigenhauser, G J F; Spriet, L L; Holloway, G P

    2014-01-01

    Studies have shown increased incorporation of omega-3 fatty acids into whole skeletal muscle following supplementation, although little has been done to investigate the potential impact on the fatty acid composition of mitochondrial membranes and the functional consequences on mitochondrial bioenergetics. Therefore, we supplemented young healthy male subjects (n = 18) with fish oils [2 g eicosapentaenoic acid (EPA) and 1 g docosahexanoic acid (DHA) per day] for 12 weeks and skeletal muscle biopsies were taken prior to (Pre) and following (Post) supplementation for the analysis of mitochondrial membrane phospholipid composition and various assessments of mitochondrial bioenergetics. Total EPA and DHA content in mitochondrial membranes increased (P < 0.05) ∼450 and ∼320%, respectively, and displaced some omega-6 species in several phospholipid populations. Mitochondrial respiration, determined in permeabilized muscle fibres, demonstrated no change in maximal substrate-supported respiration, or in the sensitivity (apparent Km) and maximal capacity for pyruvate-supported respiration. In contrast, mitochondrial responses during ADP titrations demonstrated an enhanced ADP sensitivity (decreased apparent Km) that was independent of the creatine kinase shuttle. As the content of ANT1, ANT2, and subunits of the electron transport chain were unaltered by supplementation, these data suggest that prolonged omega-3 intake improves ADP kinetics in human skeletal muscle mitochondria through alterations in membrane structure and/or post-translational modification of ATP synthase and ANT isoforms. Omega-3 supplementation also increased the capacity for mitochondrial reactive oxygen species emission without altering the content of oxidative products, suggesting the absence of oxidative damage. The current data strongly emphasize a role for omega-3s in reorganizing the composition of mitochondrial membranes while promoting improvements in ADP sensitivity. PMID:24396061

  9. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: ensuing energetic and oxidative stress implications.

    PubMed

    Pardo-Andreu, Gilberto L; Nuñez-Figueredo, Yanier; Tudella, Valeria G; Cuesta-Rubio, Osmany; Rodrigues, Fernando P; Pestana, Cezar R; Uyemura, Sérgio A; Leopoldino, Andreia M; Alberici, Luciane C; Curti, Carlos

    2011-06-15

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 μM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²⁺ efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. PMID:21549140

  10. The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

    PubMed Central

    Keller, Michael; Taskin, Asli A.; Horvath, Susanne E.; Guan, Xue Li; Prinz, Claudia; Opalińska, Magdalena; Zorzin, Carina; van der Laan, Martin; Wenk, Markus R.; Schubert, Rolf; Wiedemann, Nils; Holzer, Martin

    2015-01-01

    Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein. PMID:26347140

  11. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  12. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  13. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

    SciTech Connect

    Pardo-Andreu, Gilberto L.; Tudella, Valeria G.

    2011-06-15

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 {mu}M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca{sup 2+} efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP{sup +} transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: > We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. > GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. > These actions

  14. Sensitization of U937 leukemia cells to doxorubicin by the MG132 proteasome inhibitor induces an increase in apoptosis by suppressing NF-kappa B and mitochondrial membrane potential loss

    PubMed Central

    2014-01-01

    Background The resistance of cancerous cells to chemotherapy remains the main limitation for cancer treatment at present. Doxorubicin (DOX) is a potent antitumor drug that activates the ubiquitin-proteasome system, but unfortunately it also activates the Nuclear factor kappa B (NF-кB) pathway leading to the promotion of tumor cell survival. MG132 is a drug that inhibits I kappa B degradation by the proteasome-avoiding activation of NF-кB. In this work, we studied the sensitizing effect of the MG132 proteasome inhibitor on the antitumor activity of DOX. Methods U937 human leukemia cells were treated with MG132, DOX, or both drugs. We evaluated proliferation, viability, apoptosis, caspase-3, -8, and −9 activity and cleavage, cytochrome c release, mitochondrial membrane potential, the Bcl-2 and Bcl-XL antiapoptotic proteins, senescence, p65 phosphorylation, and pro- and antiapoptotic genes. Results The greatest apoptosis percentage in U937 cells was obtained with a combination of MG132 + DOX. Likewise, employing both drugs, we observed a decrease in tumor cell proliferation and important caspase-3 activation, as well as mitochondrial membrane potential loss. Therefore, MG132 decreases senescence, p65 phosphorylation, and the DOX-induced Bcl-2 antiapoptotic protein. The MG132 + DOX treatment induced upregulation of proapoptotic genes BAX, DIABLO, NOXA, DR4, and FAS. It also induced downregulation of the antiapoptotic genes BCL-XL and SURVIVIN. Conclusion MG132 sensitizes U937 leukemia cells to DOX-induced apoptosis, increasing its anti-leukemic effectiveness. PMID:24495648

  15. Plant mitochondrial dynamics and the role of membrane lipids

    PubMed Central

    Pan, Ronghui; Hu, Jianping

    2015-01-01

    Mitochondria are highly dynamic organelles that are continuously shaped by the antagonistic fission and fusion processes. The major machineries of mitochondrial fission and fusion, as well as mechanisms that regulate the function of key players in these processes have been analyzed in different experimental systems. In plants however, the mitochondrial fusion machinery is still largely unknown, and the regulatory mechanisms of the fission machinery are just beginning to be elucidated. This review focuses on the molecular mechanisms underlying plant mitochondrial dynamics and regulation of some of the key factors, especially the roles of membrane lipids such as cardiolipin. PMID:26317892

  16. Mitofusins and the mitochondrial permeability transition: the potential downside of mitochondrial fusion

    PubMed Central

    Papanicolaou, Kyriakos N.; Phillippo, Matthew M.

    2012-01-01

    Mitofusins (Mfn-1 and Mfn-2) are transmembrane proteins that bind and hydrolyze guanosine 5′-triphosphate to bring about the merging of adjacent mitochondrial membranes. This event is necessary for mitochondrial fusion, a biological process that is critical for organelle function. The broad effects of mitochondrial fusion on cell bioenergetics have been extensively studied, whereas the local effects of mitofusin activity on the structure and integrity of the fusing mitochondrial membranes have received relatively little attention. From the study of fusogenic proteins, theoretical models, and simulations, it has been noted that the fusion of biological membranes is associated with local perturbations on the integrity of the membrane that present in the form of lipidic holes which open on the opposing bilayers. These lipidic holes represent obligate intermediates that make the fusion process thermodynamically more favorable and at the same time induce leakage to the fusing membranes. In this perspectives article we present the relevant evidence selected from a spectrum of membrane fusion/leakage models and attempt to couple this information with observations conducted with cardiac myocytes or mitochondria deficient in Mfn-1 and Mfn-2. More specifically, we argue in favor of a situation whereby mitochondrial fusion in cardiac myocytes is coupled with outer mitochondrial membrane destabilization that is opportunistically employed during the process of mitochondrial permeability transition. We hope that these insights will initiate research on this new hypothesis of mitochondrial permeability transition regulation, a poorly understood mitochondrial function with significant consequences on myocyte survival. PMID:22636681

  17. Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology*

    PubMed Central

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-01-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei. PMID:23221899

  18. Investigation of the effects of 2.1 GHz microwave radiation on mitochondrial membrane potential (ΔΨm), apoptotic activity and cell viability in human breast fibroblast cells.

    PubMed

    Esmekaya, Meric Arda; Seyhan, Nesrin; Kayhan, Handan; Tuysuz, Mehmet Zahid; Kurşun, Ayşe Canseven; Yağcı, Münci

    2013-01-01

    In the present study we aimed to investigate the effects of 2.1 GHz Wideband Code Division Multiple Access (W-CDMA) modulated Microwave (MW) Radiation on cell survival and apoptotic activity of human breast fibroblast cells. The cell cultures were exposed to W-CDMA modulated MW at 2.1 GHz at a SAR level of 0.607 W/kg for 4 and 24 h. The cell viability was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The percentage of apoptotic cells was analyzed by Annexin V-FITC and PI staining. 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used to measure Mitochondrial Membrane Potential (ΔΨm). sFasL and Fas/APO-1 protein levels were determined by ELISA method. 2.1 GHz MW radiation was shown to be able to inhibit cell proliferation and induce apoptosis in human breast fibroblast cells. The cell viability of MW-exposed cells was decreased significantly. The percentages of Annexin V-FITC positive cells were higher in MW groups. ΔΨm was decreased significantly due to MW radiation exposure. However, neither sFas nor FasL level was significantly changed in MW-exposed fibroblast cells. The results of this study showed that 2.1 GHz W-CDMA modulated MW radiation-induced apoptotic cell death via the mitochondrial pathway. PMID:23723005

  19. Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

    PubMed Central

    Koirala, Sajjan; Guo, Qian; Kalia, Raghav; Bui, Huyen T.; Eckert, Debra M.; Frost, Adam; Shaw, Janet M.

    2013-01-01

    Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity. PMID:23530241

  20. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

    PubMed

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan

    2016-02-15

    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. PMID:26783364

  1. Ubiquilins Chaperone and Triage Mitochondrial Membrane Proteins for Degradation.

    PubMed

    Itakura, Eisuke; Zavodszky, Eszter; Shao, Sichen; Wohlever, Matthew L; Keenan, Robert J; Hegde, Ramanujan S

    2016-07-01

    We investigated how mitochondrial membrane proteins remain soluble in the cytosol until their delivery to mitochondria or degradation at the proteasome. We show that Ubiquilin family proteins bind transmembrane domains in the cytosol to prevent aggregation and temporarily allow opportunities for membrane targeting. Over time, Ubiquilins recruit an E3 ligase to ubiquitinate bound clients. The attached ubiquitin engages Ubiquilin's UBA domain, normally bound to an intramolecular UBL domain, and stabilizes the Ubiquilin-client complex. This conformational change precludes additional chances at membrane targeting for the client, while simultaneously freeing Ubiquilin's UBL domain for targeting to the proteasome. Loss of Ubiquilins by genetic ablation or sequestration in polyglutamine aggregates leads to accumulation of non-inserted mitochondrial membrane protein precursors. These findings define Ubiquilins as a family of chaperones for cytosolically exposed transmembrane domains and explain how they use ubiquitin to triage clients for degradation via coordinated intra- and intermolecular interactions. PMID:27345149

  2. Membrane-lipid unsaturation and mitochondrial function in Saacharomyces cerevisiae.

    PubMed Central

    Watson, K; Houghton, R L; Bertoli, E; Griffiths, D E

    1975-01-01

    The lipid composition of yeast cells was manipulated by the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. There was a 2-3-fold decrease in the concentration of cytochromes a+a3 when the unsaturated fatty acid content of the cells was decreased from 60-70% of the total fatty acid to 20-30%. The amounts of cytochromes b and c were also decreased under these conditions, but to a lesser extent. Further lipid depletion, to proportions of less than 20% unsaturated fatty acid, led to a dramatic decrease in the content of all cytochromes, particularly cytochromes a+a3. The ATPase (adenosine triphosphatase), succinate oxidase and NADH oxidase activities of the isolated mitochondria also varied with the degree of unsaturation of the membrane lipids. The lower the percentage of unsaturated fatty acid, the lower was the enzymic activity. Inhibition of mitochondrial ATPase by oligomycin, on the other hand, was not markedly influenced by the membrane-lipid unsaturation. Npn-linear Arrenius plots of mitochondrial membrane-bound enzymes showed transition temperatures that were dependent on the degree of membrane-lipid unsaturation. The greater the degree of lipid unsaturation, the lower was the transition temperature. It was concluded that the degree of unsaturation of the membrane lipids plays an important role in determining the properties of mitochondrial membrane-bound enzymes. PMID:125585

  3. Proteome analysis of mitochondrial outer membrane from Neurospora crassa

    SciTech Connect

    Schmitt, Simone; Prokisch, Holger; Schlunk, Tilman; Camp, David G.; Ahting, Uwe; Waizenegger, Thomas; Scharfe, Curt M.; Meitinger, Thomas; Imhof, Axel; Neupert, Walter; Oefner, Peter J.; Rapaport, Doron

    2006-01-01

    The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of liquid chromatography tandem mass spectrometry of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS peptide fingerprinting. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (Porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

  4. Functions of outer membrane receptors in mitochondrial protein import.

    PubMed

    Endo, Toshiya; Kohda, Daisuke

    2002-09-01

    Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import. PMID:12191763

  5. Phosphatidylserine Decarboxylase 1 (Psd1) Promotes Mitochondrial Fusion by Regulating the Biophysical Properties of the Mitochondrial Membrane and Alternative Topogenesis of Mitochondrial Genome Maintenance Protein 1 (Mgm1)*

    PubMed Central

    Chan, Eliana Y. L.; McQuibban, G. Angus

    2012-01-01

    Non–bilayer-forming lipids such as cardiolipin, phosphatidic acid, and phosphatidylethanolamine (PE) are proposed to generate negative membrane curvature, promoting membrane fusion. However, the mechanism by which lipids regulate mitochondrial fusion remains poorly understood. Here, we show that mitochondrial-localized Psd1, the key yeast enzyme that synthesizes PE, is required for proper mitochondrial morphology and fusion. Yeast cells lacking Psd1 exhibit fragmented and aggregated mitochondria with impaired mitochondrial fusion during mating. More importantly, we demonstrate that a reduction in PE reduces the rate of lipid mixing during fusion of liposomes with lipid compositions reflecting the mitochondrial membrane. This suggests that the mitochondrial fusion defect in the Δpsd1 strain could be due to the altered biophysical properties of the mitochondrial membrane, resulting in reduced fusion kinetics. The Δpsd1 strain also has impaired mitochondrial activity such as oxidative phosphorylation and reduced mitochondrial ATP levels which are due to a reduction in mitochondrial PE. The loss of Psd1 also impairs the biogenesis of s-Mgm1, a protein essential for mitochondrial fusion, further exacerbating the mitochondrial fusion defect of the Δpsd1 strain. Increasing s-Mgm1 levels in Δpsd1 cells markedly reduced mitochondrial aggregation. Our results demonstrate that mitochondrial PE regulates mitochondrial fusion by regulating the biophysical properties of the mitochondrial membrane and by enhancing the biogenesis of s-Mgm1. While several proteins are required to orchestrate the intricate process of membrane fusion, we propose that specific phospholipids of the mitochondrial membrane promote fusion by enhancing lipid mixing kinetics and by regulating the action of profusion proteins. PMID:23045528

  6. Bergamottin isolated from Citrus bergamia exerts in vitro and in vivo antitumor activity in lung adenocarcinoma through the induction of apoptosis, cell cycle arrest, mitochondrial membrane potential loss and inhibition of cell migration and invasion.

    PubMed

    Wu, Hui-Juan; Wu, Hong-Bo; Zhao, Yan-Qiu; Chen, Li-Juan; Zou, Hong-Zhi

    2016-07-01

    The objective of the present study was to investigate the in vitro and in vivo anticancer properties of bergamottin, a natural furanocoumarin, against human non-small cell lung carcinoma (NSCLC) A549 cells. We also studied its effect on cell proliferation, cell cycle arrest, cell invasion, cell migration as well as cell apoptosis. Antiproliferative activity of bergamottin was estimated by the MTT assay. Phase contrast and fluorescence microscopy as well as flow cytometry using Annexin V-FITC assay were used to study induction of apoptosis by bergamottin in these cells. The effects of bergamottin on cell cycle phase distribution as well as on mitochondrial membrane potential were also demonstrated using flow cytometry. In vitro wound healing assay was used to study the effect of bergamottin on cell migration. The effects of bergamottin on tumor progression were also observed using a nude mouse model. The mice were divided into 4 groups and treated with bergamottin injected intraperitoneally. Bergamottin induced dose-dependent as well as time-dependent cytotoxic effects as well as inhibition of colony formation in the A549 cancer cells. Bergamottin also suppressed cancer cell invasion as well as cancer cell migration. Phase contrast microscopy and fluorescence microscopy revealed that bergamottin induced cell shrinkage, chromatin condensation and the cells became rounded and detached from each other. Bergamottin also induced a potent cell cycle arrest at the G2/M phase of the cell cycle. Experiments in mice showed that 25, 50 and 100 mg/kg bergamottin injection reduced the tumor weight from 1.61 g in the phosphate-buffered saline (PBS)-treated group (control) to 1.21, 0.42 and 0.15 g in the bergamottin-treated groups, respectively. The results of the present study revealed that bergamottin was able to inhibit lung cancer cell growth both in a cell model and a xenograft mouse model by inducing apoptosis, mitochondrial membrane potential loss, G2/M cell cycle

  7. Torilis japonica extract-generated intracellular ROS induces apoptosis by reducing the mitochondrial membrane potential via regulation of the AMPK-p38 MAPK signaling pathway in HCT116 colon cancer.

    PubMed

    Kim, Guen Tae; Lee, Se Hee; Kim, Young Min

    2016-09-01

    Torilis japonica extract (TJE) has been reported to possess diverse medicinal properties including anti‑inflammatory and antibacterial activities. However, the precise mechanism of its anticancer effect is not understood. Thus, we evaluated the apoptotic effects of TJE and examined its underlying molecular mechanisms in HCT116 colorectal cancer cells. Our results show that TJE induces apoptosis through the generation of intracellular reactive oxygen species (ROS), and that it regulates the mitochondrial outer membrane potential via the AMPK/p38 MAPK signaling pathway. Importantly, ~50% of cancer cells have p53 mutations. Thus, the ability to induce apoptosis in a p53-independent manner would be of great value in cancer treatment. Our results show that not only does TJE regulate the AMPK/p38 signaling pathway, but it induces apoptosis in cells in which p53 has been knocked down using siRNA. Moreover, as in in vitro studies, TJE induced apoptosis and regulated apoptosis related-proteins in an HCT 116 xenograft model. Taken together, our results demonstrate that TJE, a natural compound that may provide a substitute for chemotherapeutic drugs, has potential as an anticancer agent. PMID:27314881

  8. Selective sorting and destruction of mitochondrial membrane proteins in aged yeast.

    PubMed

    Hughes, Adam L; Hughes, Casey E; Henderson, Kiersten A; Yazvenko, Nina; Gottschling, Daniel E

    2016-01-01

    Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress. PMID:27097106

  9. Equilibrium Potentials of Membrane Electrodes

    PubMed Central

    Wang, Jui H.; Copeland, Eva

    1973-01-01

    A simple thermodynamic theory of the equilibrium potentials of membrane electrodes is formulated and applied to the glass electrode for measurement of pH. The new formulation assumes the selective adsorption or binding of specific ions on the surface of the membrane which may or may not be permeable to the ion, and includes the conventional derivation based on reversible ion transport across membranes as a special case. To test the theory, a platinum wire was coated with a mixture of stearic acid and methyl-tri-n-octyl-ammonium stearate. When this coated electrode was immersed in aqueous phosphate solution, its potential was found to be a linear function of pH from pH 2 to 12 with a slope equal to the theoretical value of 59.0 mV per pH unit at 24°. PMID:4516194

  10. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  11. Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

    PubMed Central

    Thieffry, M; Chich, J F; Goldschmidt, D; Henry, J P

    1988-01-01

    Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes. Images PMID:2457497

  12. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    PubMed

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers. PMID:17437483

  13. Calcium Flux across Plant Mitochondrial Membranes: Possible Molecular Players

    PubMed Central

    Carraretto, Luca; Checchetto, Vanessa; De Bortoli, Sara; Formentin, Elide; Costa, Alex; Szabó, Ildikó; Teardo, Enrico

    2016-01-01

    Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca2+ transients which are further transduced by Ca2+ sensor proteins into a transcriptional and metabolic response. Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle. PMID:27065186

  14. Calcium Flux across Plant Mitochondrial Membranes: Possible Molecular Players.

    PubMed

    Carraretto, Luca; Checchetto, Vanessa; De Bortoli, Sara; Formentin, Elide; Costa, Alex; Szabó, Ildikó; Teardo, Enrico

    2016-01-01

    Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca(2+) transients which are further transduced by Ca(2+) sensor proteins into a transcriptional and metabolic response. Most of the research on Ca(2+) signaling in plants has been focused on the transport mechanisms for Ca(2+) across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca(2+) signals, but how intracellular organelles such as mitochondria are involved in the process of Ca(2+) signaling is just emerging. The combination of the molecular players and the elicitors of Ca(2+) signaling in mitochondria together with newly generated detection systems for measuring organellar Ca(2+) concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca(2+) across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca(2+) homeostasis for ensuring optimal bioenergetic performance of this organelle. PMID:27065186

  15. Arsenic trioxide (As(2)O(3)) induces apoptosis and necrosis mediated cell death through mitochondrial membrane potential damage and elevated production of reactive oxygen species in PLHC-1 fish cell line.

    PubMed

    Selvaraj, Vellaisamy; Armistead, Mindy Yeager; Cohenford, Menashi; Murray, Elizabeth

    2013-01-01

    Several environmental pollutants, including metals can induce toxicological effect on aquatic animal species. Most studies to understand the toxicity of arsenic compounds were performed in mammalian cells; however, the study of the arsenic toxicity to the aquatic animals' species, including fish, is limited. So the objective of this study was first to investigate the effects of As(2)O(3) induced toxicity particularly on apoptosis and necrosis mediated cell death in fish cell PLHC-1 as compared to the mechanism of toxicity from known mammalian cell lines, secondly to relate in vitro effects in fish to those demonstrated by in vivo systems. To conduct this study, PLHC-1 cells were exposed to various concentrations of As(2)O(3) (0-100 μM) for 10, 20 and 40 h. The results indicate that As(2)O(3) exposure promoted apoptotic and necrotic mediated cell death in a concentration and time dependent manner. Cell death (apoptotic and necrotic) induced by As(2)O(3) was further confirmed by changes in various phases of cell cycle, DNA fragmentation (necro- comet and apo-comet) in the comet assay, alteration in mitochondrial membrane potential and formation of increased reactive oxygen species (ROS). Apoptotic mediated cell death was confirmed further by observing the increased caspase-3 activity and elevated expression of p53, cytochrome c and Bax proteins levels in the same experimental conditions. PLHC-1 cells were shown to be a good model for evaluating biochemical/cytotoxic effects following exposure to various reference chemicals and environmental contaminants. In vitro data obtained from this study provides a comprehensive approach for the elucidating the actual molecular mechanism for As(2)O(3) induced toxicity particularly apoptosis and necrosis mediated cell death in PLHC-1 cell line. PMID:23121984

  16. Neuroprotective and neurorestorative potential of propargylamine derivatives in ageing: focus on mitochondrial targets.

    PubMed

    Bar-Am, Orit; Amit, Tamar; Youdim, Moussa B; Weinreb, Orly

    2016-02-01

    The mitochondrial theory of ageing proposes that accumulation of damage to mitochondrial function and DNA mutation lead to ageing of humans and animals. It has been suggested that mitochondria play dynamic roles in regulating synaptogenesis and morphological/functional responses of synaptic activity, and thus, deteriorating of mitochondrial function (e.g., deficits of the mitochondrial respiratory enzymes, reduced calcium influx, increased accumulation of mitochondrial DNA defects/apoptotic proteins and impairment of mitochondrial membrane potential) can lead to severe neuronal energy deficit, and in the long run, to modifications in neuronal synapses and neurodegeneration in the ageing brain. Hence, considering the mechanisms by which mitochondrial impairment can lead to neuronal death, the development of neuroprotective molecules that target various mitochondrial pathogenic processes can be effective in the treatment of ageing and age-related neurodegenerative diseases. This review addresses several aspects of the neuroprotective effects of propargylamine derivatives (e.g., the monoamine oxidase-B inhibitors, selegiline and rasagiline and the multifunctional drugs, ladostigil, M30 and VAR10303) in ageing with a special focus on mitochondrial molecular protective mechanisms. PMID:25859841

  17. Role of the Transmembrane Potential in the Membrane Proton Leak

    PubMed Central

    Rupprecht, Anne; Sokolenko, Elena A.; Beck, Valeri; Ninnemann, Olaf; Jaburek, Martin; Trimbuch, Thorsten; Klishin, Sergey S.; Jezek, Petr; Skulachev, Vladimir P.; Pohl, Elena E.

    2010-01-01

    Abstract The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨm) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨm (similar to ΔΨm in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨm. The application of ΔΨm up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨm and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA− remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨm more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨm may be key in mitochondrial respiration and metabolism. PMID:20409469

  18. Membrane potential generated by ion adsorption.

    PubMed

    Tamagawa, Hirohisa; Morita, Sachi

    2014-01-01

    It has been widely acknowledged that the Goldman-Hodgkin-Katz (GHK) equation fully explains membrane potential behavior. The fundamental facet of the GHK equation lies in its consideration of permeability of membrane to ions, when the membrane serves as a separator for separating two electrolytic solutions. The GHK equation describes that: variation of membrane permeability to ion in accordance with ion species results in the variation of the membrane potential. However, nonzero potential was observed even across the impermeable membrane (or separator) separating two electrolytic solutions. It gave rise to a question concerning the validity of the GHK equation for explaining the membrane potential generation. In this work, an alternative theory was proposed. It is the adsorption theory. The adsorption theory attributes the membrane potential generation to the ion adsorption onto the membrane (or separator) surface not to the ion passage through the membrane (or separator). The computationally obtained potential behavior based on the adsorption theory was in good agreement with the experimentally observed potential whether the membrane (or separator) was permeable to ions or not. It was strongly speculated that the membrane potential origin could lie primarily in the ion adsorption on the membrane (or separator) rather than the membrane permeability to ions. It might be necessary to reconsider the origin of membrane potential which has been so far believed explicable by the GHK equation. PMID:24957176

  19. Membrane Potential Generated by Ion Adsorption

    PubMed Central

    Tamagawa, Hirohisa; Morita, Sachi

    2014-01-01

    It has been widely acknowledged that the Goldman-Hodgkin-Katz (GHK) equation fully explains membrane potential behavior. The fundamental facet of the GHK equation lies in its consideration of permeability of membrane to ions, when the membrane serves as a separator for separating two electrolytic solutions. The GHK equation describes that: variation of membrane permeability to ion in accordance with ion species results in the variation of the membrane potential. However, nonzero potential was observed even across the impermeable membrane (or separator) separating two electrolytic solutions. It gave rise to a question concerning the validity of the GHK equation for explaining the membrane potential generation. In this work, an alternative theory was proposed. It is the adsorption theory. The adsorption theory attributes the membrane potential generation to the ion adsorption onto the membrane (or separator) surface not to the ion passage through the membrane (or separator). The computationally obtained potential behavior based on the adsorption theory was in good agreement with the experimentally observed potential whether the membrane (or separator) was permeable to ions or not. It was strongly speculated that the membrane potential origin could lie primarily in the ion adsorption on the membrane (or separator) rather than the membrane permeability to ions. It might be necessary to reconsider the origin of membrane potential which has been so far believed explicable by the GHK equation. PMID:24957176

  20. Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane.

    PubMed Central

    Fraser, F; Corstorphine, C G; Zammit, V A

    1997-01-01

    The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the

  1. Effects of thyroid hormones on inner mitochondrial membrane fluidity.

    PubMed

    Chimenti, R; Covello, C; De Cicco, T; Bruno, R; Martino, G

    2001-01-01

    Authors studied the effects of thyroid hormones and their diasteroisomers and 3,5-diiodothyronine (LT2) on the fluidity properties of inner mitochondrial membrane (IMM) by specifical fluorescent probe for the internal zone of biological membranes, the 1,6-diphenyl-1,3,5-hexatriene (DPH). The studied parameters are Arrhenius and Perrin plots. The DPH shows a decreased fluorescence quenching in the presence of both T3 and T4. The maximum effect is observed with 2 nM LT2. LT2 is more effective than LT3 in the central zone. The data confirm the selective action of LT3 and LT4 on IMM fluidity. PMID:11822198

  2. Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase

    PubMed Central

    Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M.; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

    2012-01-01

    Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes. PMID:22778261

  3. Polyphenol-rich extract of Salvia chinensis exhibits anticancer activity in different cancer cell lines, and induces cell cycle arrest at the G0/G1-phase, apoptosis and loss of mitochondrial membrane potential in pancreatic cancer cells

    PubMed Central

    ZHAO, QUAN; HUO, XUE-CHEN; SUN, FU-DONG; DONG, RUI-QIAN

    2015-01-01

    Pancreatic cancer (PC) is one of the most aggressive types of human malignancy, which has an overall 5-year survival rate of <2%. PC is the fourth most common cause of cancer-associated mortality in the western world. At present, there is almost no effective treatment available for the treatment of PC. The aim of the present study was to evaluate the anticancer potential of a polyphenol enriched extract obtained from Salvia chinensis, a Chinese medicinal plant. An MTT assay was used to evaluate the cell viability of five cancer cell lines and one normal cell line. In addition, the effects of the extract on apoptotic induction, cell cycle phase distribution, DNA damage and loss of mitochondrial membrane potential (ΛΨm) were evaluated in MiapaCa-2 human PC cells. The effects of the extract on cell cycle phase distribution and ΛΨm were assessed by flow cytometry, using propidium iodide and rhodamine-123 DNA-binding fluorescent dyes, respectively. Fluorescence microscopy, using 4′,6-diamidino-2-phenylindole as a staining agent, was performed in order to detect the morphological changes of the MiapaCa-2 cancer cells and the presence of apoptotic bodies following treatment with the extract. The results of the present study demonstrated that the polyphenol-rich extract from S. chinensis induced potent cytotoxicity in the MCF-7 human breast cancer cells, A549 human lung cancer cells, HCT-116 and COLO 205 human colon cancer cells, and MiapaCa-2 human PC cells. The COLO 205 and MCF-7 cancer cell lines were the most susceptible to treatment with the extract, which exhibited increased rate of growth inhibition. Fluorescence microscopy revealed characteristic morphological features of apoptosis and detected the appearance of apoptotic bodies following treatment with the extract in the PC cells. Flow cytometric analysis demonstrated that the extract induced G0/G1 cell cycle arrest in a dose-dependent manner. In addition, treatment with the extract induced a significant and

  4. Membrane potential and cancer progression

    PubMed Central

    Yang, Ming; Brackenbury, William J.

    2013-01-01

    Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of different ion channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, hyperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be a valuable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis. PMID:23882223

  5. Interaction of fullerene nanoparticles with biomembranes: from the partition in lipid membranes to effects on mitochondrial bioenergetics.

    PubMed

    Santos, Sandra M; Dinis, Augusto M; Peixoto, Francisco; Ferreira, Lino; Jurado, Amália S; Videira, Romeu A

    2014-03-01

    Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics. PMID:24361870

  6. Effect of sphingosine on Ca2+ entry and mitochondrial potential of Jurkat T cells--interaction with Bcl2.

    PubMed

    Dangel, Georg Richard; Lang, Florian; Lepple-Wienhues, Albrecht

    2005-01-01

    Triggers of Jurkat T cell apoptosis include sphingosine and ceramide. Sphingosine and ceramide further inhibit capacitative Ca2+ entry (ICRAC), an effect leading to inactivation but not death of Jurkat T cells. Mitochondria are key organelles in the machinery leading to apoptosis and on the other hand have been shown to participate in the regulation of Ca2+ entry. The present experiments were performed to explore whether treatment of Jurkat T cells with sphingosine leads to apoptosis and reduced Ca2+ entry and whether those effects are sensitive to expression of the antiapoptotic protein Bcl2, localized in the outer mitochondrial membrane. Exposure of Jurkat T cells to 10 microM spingosine was according to DiOC6 fluorescence followed by mitochondrial depolarization and according to Fura-red/Fluo-3 fluorescence followed by decreased capacitative Ca2+ entry. Mitochondrial depolarization was significantly delayed in cells overexpressing wild type Bcl2 or Bcl2 targeted to the mitochondrial membrane, whereas no significant influence on mitochondrial depolarization was observed in cells expressing Bcl2 lacking the membrane targeting motif or Bcl2 targeted to the endoplasmatic reticulum. In contrast to mitochondrial potential, the blunting of capacitative Ca2+ entry following sphingosine treatment was not sensitive to mitochondrial Bcl2 expression. In conclusion sphingosine exposure leads to both, mitochondrial depolarization and inhibition of capacitative Ca2+ entry. Mitochondrial Bcl2 reverses the effect on mitochondria but not on Ca2+ entry and thus leads to dissociation of those two sequelae of sphingosine treatment. PMID:16121028

  7. Liquid membrane potential in nonisothermal systems.

    PubMed Central

    Scibona, G; Fabiani, C; Scuppa, B; Danesi, P R

    1976-01-01

    Electrical membrane potential equations for liquid ion exchange membranes, characterized by the presence of uncharged associated species and by exclusion of co-ions (no electrolyte uptake) have been derived. The irreversible thermodynamic theories already developed for solid membranes with fixed charged site density have been extended to include the different physicochemical aspects of the liquid membranes. To this purpose the dissipation function has been written with reference to the fluxes of all the species present in the membrane. It has been found that the mobile charged site, the counterions, and the uncharged associated species contribute to the electrical membrane potential through their phenomenological coefficients. The electrical membrane potential equations have been integrated in isothermal and nonisothermal conditions for monoionic and biionic systems. The theoretical predictions have been experimentally tested by studying the electrical potential of liquid membranes formed with solutions of tetraheptylammonium salts in omicron-dichlorobenzene. PMID:1276391

  8. Regulation of mitochondrial inner membrane fusion: divergent evolution with similar solutions?

    PubMed

    Wagener, Johannes

    2016-05-01

    Continuous mitochondrial fusion and fission define the dynamic shape of mitochondria. One essential player of mitochondrial fusion is the conserved inner membrane dynamin-like GTPase Mgm1/OPA1. Limited proteolysis of this protein has been proposed as a mechanism to separate and subsequently eliminate dysfunctional parts from the mitochondrial network. Here, I briefly summarize our current knowledge about the underlying proteolytic processing steps in mammals, baker's yeast, Schizosaccharomyces pombe, Drosophila melanogaster and Aspergillus fumigatus. The apparent great diversity in Mgm1/OPA1 processing among the analyzed species indicates a surprising mechanistic heterogeneity in the regulation of mitochondrial inner membrane fusion. PMID:26613727

  9. Melatonin: A Potential Anti-Oxidant Therapeutic Agent for Mitochondrial Dysfunctions and Related Disorders.

    PubMed

    Ganie, Showkat Ahmad; Dar, Tanveer Ali; Bhat, Aashiq Hussain; Dar, Khalid B; Anees, Suhail; Zargar, Mohammad Afzal; Masood, Akbar

    2016-02-01

    Mitochondria play a central role in cellular physiology. Besides their classic function of energy metabolism, mitochondria are involved in multiple cell functions, including energy distribution through the cell, energy/heat modulation, regulation of reactive oxygen species (ROS), calcium homeostasis, and control of apoptosis. Simultaneously, mitochondria are the main producer and target of ROS with the result that multiple mitochondrial diseases are related to ROS-induced mitochondrial injuries. Increased free radical generation, enhanced mitochondrial inducible nitric oxide synthase (iNOS) activity, enhanced nitric oxide (NO) production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pores have all been suggested as factors responsible for impaired mitochondrial function. Because of these, neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and aging, are caused by ROS-induced mitochondrial dysfunctions. Melatonin, the major hormone of the pineal gland, also acts as an anti-oxidant and as a regulator of mitochondrial bioenergetic function. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other anti-oxidants, and thus has emerged as a major potential therapeutic tool for treating neurodegenerative disorders. Multiple in vitro and in vivo experiments have shown the protective role of melatonin for preventing oxidative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. With these functions in mind, this article reviews the protective role of melatonin with mechanistic insights against mitochondrial diseases and suggests new avenues for safe and effective treatment modalities against these devastating neurodegenerative diseases. Future insights are also discussed. PMID:26087000

  10. Mitochondrial membrane lipids in life and death and their molecular modulation by diet: tuning the furnace.

    PubMed

    Monteiro, João P; Morais, Catarina M; Oliveira, Paulo J; Jurado, Amália S

    2014-01-01

    The traditional view of mitochondria as cell powerhouses is a matter of common knowledge, but the overall view of these extraordinary organelles has been revolutionized in the last years. In fact, a large number of important and diverse processes take place at the mitochondrial level, which clearly surpass the energy production scope, intruding the critical fragile balance between cell life and death. The entangled biochemistry of mitochondrial membranes has been found to be dependent on specific lipid requirements, with cardiolipin holding a great part of the raised functional interest. Mitochondria contain a complex membrane system, based on a variety of lipids and exquisite asymmetries. Mitochondria lipid membrane composition depends on a tight interplay with the endoplasmic reticulum, from which some of the lipids present in the mitochondrial membranes have to be imported, at least in the form of precursors. Here, we review some external interventions resulting in alterations of mitochondrial lipid content, namely dietary interventions and genetic manipulation. Such manipulations of mitochondrial membrane lipid composition should result in physiological impact, given the importance of lipid-protein interactions within the mitochondrial membrane boundaries. We provide arguments for future experiments using the most modern chemical and biophysical approaches as well as computer simulation studies applied to appropriate biological membrane model systems, in order to identify the effects exerted by diet-induced lipid changes on membrane physical properties. PMID:24953065

  11. Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

    PubMed Central

    Gerhold, Joachim M.; Cansiz-Arda, Şirin; Lõhmus, Madis; Engberg, Oskar; Reyes, Aurelio; van Rennes, Helga; Sanz, Alberto; Holt, Ian J.; Cooper, Helen M.; Spelbrink, Johannes N.

    2015-01-01

    The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol. PMID:26478270

  12. Dietary lipid quality and mitochondrial membrane composition in trout: responses of membrane enzymes and oxidative capacities.

    PubMed

    Martin, N; Bureau, D P; Marty, Y; Kraffe, E; Guderley, H

    2013-04-01

    To examine whether membrane fatty acid (FA) composition has a greater impact upon specific components of oxidative phosphorylation or on overall properties of muscle mitochondria, rainbow trout (Oncorhynchus mykiss) were fed two diets differing only in FA composition. Diet 1 was enriched in 18:1n-9 and 18:2n-6 while Diet 2 was enriched in 22:6n-3. The FA composition of mitochondrial phospholipids was strongly affected by diet. 22:6n-3 levels were twice as high (49%) in mitochondrial phospholipids of fish fed Diet 2 than in those fed Diet 1. 18:2n-6 content of the phospholipids also followed the diets, whereas 18:1n-9 changed little. All n-6 FA, most notably 22:5n-6, were significantly higher in fish fed Diet 1. Nonetheless, total saturated FA, total monounsaturated FA and total polyunsaturated FA in mitochondrial phospholipids varied little. Despite a marked impact of diet on specific FA levels in mitochondrial phospholipids, only non-phosphorylating (state 4) rates were higher in fish fed Diet 2. Phosphorylating rates (state 3), oxygen consumption due to flux through the electron transport chain complexes as well as the corresponding spectrophotometric activities did not differ with diet. Body mass affected state 4 rates and cytochrome c oxidase and F 0 F 1 ATPase activities while complex I showed a diet-specific effect of body mass. Only the minor FA that were affected by body mass were correlated with functional properties. The regulated incorporation of dietary FA into phospholipids seems to allow fish to maintain critical membrane functions even when the lipid quality of their diets varies considerably, as is likely in their natural environment. PMID:23052948

  13. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    PubMed

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. PMID:26722004

  14. Selective sorting and destruction of mitochondrial membrane proteins in aged yeast

    PubMed Central

    Hughes, Adam L; Hughes, Casey E; Henderson, Kiersten A; Yazvenko, Nina; Gottschling, Daniel E

    2016-01-01

    Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress. DOI: http://dx.doi.org/10.7554/eLife.13943.001 PMID:27097106

  15. Interaction of MDM33 with mitochondrial inner membrane homeostasis pathways in yeast

    PubMed Central

    Klecker, Till; Wemmer, Megan; Haag, Mathias; Weig, Alfons; Böckler, Stefan; Langer, Thomas; Nunnari, Jodi; Westermann, Benedikt

    2015-01-01

    Membrane homeostasis affects mitochondrial dynamics, morphology, and function. Here we report genetic and proteomic data that reveal multiple interactions of Mdm33, a protein essential for normal mitochondrial structure, with components of phospholipid metabolism and mitochondrial inner membrane homeostasis. We screened for suppressors of MDM33 overexpression-induced growth arrest and isolated binding partners by immunoprecipitation of cross-linked cell extracts. These approaches revealed genetic and proteomic interactions of Mdm33 with prohibitins, Phb1 and Phb2, which are key components of mitochondrial inner membrane homeostasis. Lipid profiling by mass spectrometry of mitochondria isolated from Mdm33-overexpressing cells revealed that high levels of Mdm33 affect the levels of phosphatidylethanolamine and cardiolipin, the two key inner membrane phospholipids. Furthermore, we show that cells lacking Mdm33 show strongly decreased mitochondrial fission activity indicating that Mdm33 is critical for mitochondrial membrane dynamics. Our data suggest that MDM33 functionally interacts with components important for inner membrane homeostasis and thereby supports mitochondrial division. PMID:26669658

  16. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

    SciTech Connect

    Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi

    2010-06-25

    Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.

  17. Label-Free Imaging of Membrane Potential Using Membrane Electromotility

    PubMed Central

    Oh, Seungeun; Fang-Yen, Christopher; Choi, Wonshik; Yaqoob, Zahid; Fu, Dan; Park, YongKeun; Dassari, Ramachandra R.; Feld, Michael S.

    2012-01-01

    Electrical activity may cause observable changes in a cell's structure in the absence of exogenous reporter molecules. In this work, we report a low-coherence interferometric microscopy technique that can detect an optical signal correlated with the membrane potential changes in individual mammalian cells without exogenous labels. By measuring milliradian-scale phase shifts in the transmitted light, we can detect changes in the cells' membrane potential. We find that the observed optical signals are due to membrane electromotility, which causes the cells to deform in response to the membrane potential changes. We demonstrate wide-field imaging of the propagation of electrical stimuli in gap-junction-coupled cell networks. Membrane electromotility-induced cell deformation may be useful as a reporter of electrical activity. PMID:22828327

  18. The Taz1p transacylase is imported and sorted into the outer mitochondrial membrane via a membrane anchor domain.

    PubMed

    Herndon, Jenny D; Claypool, Steven M; Koehler, Carla M

    2013-12-01

    Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome. PMID:24078306

  19. The Liver Connexin32 Interactome Is a Novel Plasma Membrane-Mitochondrial Signaling Nexus

    PubMed Central

    2013-01-01

    Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses, given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to both the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority represent plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and the mitochondrial protein, sideroflexin-1, at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatic, biochemical, and confocal analyses support a role for connexin32 in transiently tethering mitochondria to connexin32-enriched plasma membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome. PMID:23590695

  20. Authentic In Vitro Replication of Two Tombusviruses in Isolated Mitochondrial and Endoplasmic Reticulum Membranes

    PubMed Central

    Xu, Kai; Huang, Tyng-Shyan

    2012-01-01

    Replication of plus-stranded RNA viruses takes place on membranous structures derived from various organelles in infected cells. Previous works with Tomato bushy stunt tombusvirus (TBSV) revealed the recruitment of either peroxisomal or endoplasmic reticulum (ER) membranes for replication. In case of Carnation Italian ringspot tombusvirus (CIRV), the mitochondrial membranes supported CIRV replication. In this study, we developed ER and mitochondrion-based in vitro tombusvirus replication assays. Using purified recombinant TBSV and CIRV replication proteins, we showed that TBSV could use the purified yeast ER and mitochondrial preparations for complete viral RNA replication, while CIRV preferentially replicated in the mitochondrial membranes. The viral RNA became partly RNase resistant after ∼40 to 60 min of incubation in the purified ER and mitochondrial preparations, suggesting that assembly of TBSV and CIRV replicases could take place in the purified ER and mitochondrial membranes in vitro. Using chimeric and heterologous combinations of replication proteins, we showed that multiple domains within the replication proteins are involved in determining the efficiency of tombusvirus replication in the two subcellular membranes. Altogether, we demonstrated that TBSV is less limited while CIRV is more restricted in utilizing various intracellular membranes for replication. Overall, the current work provides evidence that tombusvirus replication could occur in vitro in isolated subcellular membranes, suggesting that tombusviruses have the ability to utilize alternative organellar membranes during infection that could increase the chance of mixed virus replication and rapid evolution during coinfection. PMID:22973028

  1. VDAC electronics: 2. A new, anaerobic mechanism of generation of the membrane potentials in mitochondria.

    PubMed

    Lemeshko, Victor V

    2014-07-01

    Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC-HK and the ANT (adenine nucleotide translocator)-CK-VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT-CK-VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC-HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to -160mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen. PMID:24565793

  2. Inhibition of mitochondrial membrane permeability as a putative pharmacological target for cardioprotection

    PubMed Central

    Morin, Didier; Assaly, Rana; Paradis, Stéphanie; Berdeaux, Alain

    2009-01-01

    Myocardial ischemia-reperfusion injury is a major cause of morbidity and mortality in developed countries. To date, the only treatment of complete ischemia is to restore blood flow; thus the search for new cardioprotective approaches is absolutely necessary to reduce the mortality associated with myocardial ischemia. Ischemia has long been considered to result in necrotic tissue damage but the reduction in oxygen supply can also lead to apoptosis. Therefore, in the last few years, mitochondria have become the subject of growing interest in myocardial ischemia-reperfusion since they are strongly involved in the regulation of the apoptotic process. Indeed, during ischemia-reperfusion, pathological signals converge in the mitochondria to induce permeabilization of the mitochondrial membrane. Two classes of mechanisms, which are not mutually exclusive, emerged to explain mitochondrial membrane permeabilization. The first occurs via a non-specific channel known as the mitochondrial permeability transition pore (mPTP) in the inner and the outer membranes causing disruption of the impermeability of the inner membrane, and ultimately complete inhibition of mitochondrial function. The second mechanism, involving only the outer membrane, induces the release of cell death effectors. Thus, drugs able to block or to limit mitochondrial membrane permeabilization may be cytoprotective during ischemia-reperfusion. The objective of this review is to examine the pharmacological strategies capable of inhibiting mitochondrial membrane permeabilization induced by myocardial ischemia-reperfusion. PMID:19835566

  3. Reconstitution of the native mitochondrial outer membrane in planar bilayers. Comparison with the outer membrane in a patch pipette and effect of aluminum compounds.

    PubMed

    Mirzabekov, T; Ballarin, C; Nicolini, M; Zatta, P; Sorgato, M C

    1993-04-01

    Detergent-free rat brain outer mitochondrial membranes were incorporated in planar lipid bilayers in the presence of an osmotic gradient, and studied at high (1 M KCl) and low (150 mM KCl) ionic strength solutions. By comparison, the main outer mitochondrial membrane protein, VDAC, extracted from rat liver with Triton X-100, was also studied in 150 mM KCl. In 1 M KCl, brain outer membranes gave rise to electrical patterns which resembled very closely those widely described for detergent-extracted VDAC, with transitions to several subconducting states upon increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, however, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mM KCl, bilayers containing only one channel had a conductance of 700 +/- 23 pS for rat brain outer membranes, and 890 +/- 29 pS for rat liver VDAC. Use of a fast time resolution setup allowed demonstration of open-close transitions in the millisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference higher than approx. +/- 60 mV induced an almost irreversible decrease of the single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the behavior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Biophys. J. 20:311-319). The neurotoxicological action of aluminum was studied in single outer membrane channels from rat brain mitochondria. We found that microM concentrations of Al Cl3 and aluminum lactate decreased the conductance by about 50%, when the applied potential difference was positive relative to the side of the metal addition. PMID:7685821

  4. HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

    PubMed Central

    Lecoeur, H; Borgne-Sanchez, A; Chaloin, O; El-Khoury, R; Brabant, M; Langonné, A; Porceddu, M; Brière, J-J; Buron, N; Rebouillat, D; Péchoux, C; Deniaud, A; Brenner, C; Briand, J-P; Muller, S; Rustin, P; Jacotot, E

    2012-01-01

    The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor. PMID:22419111

  5. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

  6. Isoliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells.

    PubMed

    Jung, Jae In; Lim, Soon Sung; Choi, Hyun Ju; Cho, Han Jin; Shin, Hyun-Kyung; Kim, Eun Ji; Chung, Won-Yoon; Park, Kwang-Kyun; Park, Jung Han Yoon

    2006-10-01

    Isoliquiritigenin (ISL), a simple chalcone derivative, 4,2',4'-trihydroxychalcone, found in licorice, shallot and bean sprouts, has been reported to have chemoprotective effects. To examine the effects of ISL on the growth of prostate cancer cells, we cultured MAT-LyLu (MLL) rat and DU145 human prostate cancer cells with various concentrations (0-20 micromol/L) of ISL. Treatment of the cells with increasing concentrations of ISL led to dose-dependent decreases in the viable cell numbers in both DU145 and MLL cells (P<.05). Hoechst 33258 dye staining of condensed nuclei and annexin V binding to surface phosphatidylserine revealed increased numbers of apoptotic cells after ISL treatment. Western blot analysis revealed that ISL increased the levels of membrane-bound Fas ligand (FasL), Fas, cleaved casapse-8, truncated Bid (tBid), Bax and Bad in DU145 cells (P<.05). Isoliquiritigenin increased the percentage of cells with depolarized mitochondrial membranes, in a concentration-dependent manner (P<.05). Isoliquiritigenin induced the release of cytochrome c and Smac/Diablo from the mitochondria into the cytoplasm (P<.05). Isoliquiritigenin dose-dependently increased the levels of cleaved caspase-9, caspase-7, caspase-3 and poly(ADP-ribose) polymerase (P<.05). The present results indicate that ISL inhibits prostate cancer cell growth by the induction of apoptosis, which is mediated through mitochondrial events, which are associated with an evident disruption of the mitochondrial membrane potential, and the release of cytochrome c and Smac/Diablo, and the activation of caspase-9. PMID:16517140

  7. Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane.

    PubMed

    Kenwood, Brandon M; Weaver, Janelle L; Bajwa, Amandeep; Poon, Ivan K; Byrne, Frances L; Murrow, Beverley A; Calderone, Joseph A; Huang, Liping; Divakaruni, Ajit S; Tomsig, Jose L; Okabe, Kohki; Lo, Ryan H; Cameron Coleman, G; Columbus, Linda; Yan, Zhen; Saucerman, Jeffrey J; Smith, Jeffrey S; Holmes, Jeffrey W; Lynch, Kevin R; Ravichandran, Kodi S; Uchiyama, Seiichi; Santos, Webster L; Rogers, George W; Okusa, Mark D; Bayliss, Douglas A; Hoehn, Kyle L

    2014-04-01

    Dysregulation of oxidative phosphorylation is associated with increased mitochondrial reactive oxygen species production and some of the most prevalent human diseases including obesity, cancer, diabetes, neurodegeneration, and heart disease. Chemical 'mitochondrial uncouplers' are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. Mitochondrial uncouplers also lessen the proton motive force across the mitochondrial inner membrane and thereby increase the rate of mitochondrial respiration while decreasing production of reactive oxygen species. Thus, mitochondrial uncouplers are valuable chemical tools that enable the measurement of maximal mitochondrial respiration and they have been used therapeutically to decrease mitochondrial reactive oxygen species production. However, the most widely used protonophore uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol have off-target activity at other membranes that lead to a range of undesired effects including plasma membrane depolarization, mitochondrial inhibition, and cytotoxicity. These unwanted properties interfere with the measurement of mitochondrial function and result in a narrow therapeutic index that limits their usefulness in the clinic. To identify new mitochondrial uncouplers that lack off-target activity at the plasma membrane we screened a small molecule chemical library. Herein we report the identification and validation of a novel mitochondrial protonophore uncoupler (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine, named BAM15, that does not depolarize the plasma membrane. Compared to FCCP, an uncoupler of equal potency, BAM15 treatment of cultured cells stimulates a higher maximum rate of mitochondrial respiration and is less cytotoxic. Furthermore, BAM15 is bioactive in vivo and dose

  8. Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity - A flow cytometric study.

    PubMed

    Nagy, Szabolcs Tamás; Kakasi, Balázs; Pál, László; Havasi, Máté; Bercsényi, Miklós; Husvéth, Ferenc

    2016-06-01

    Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques. PMID:27165524

  9. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

    PubMed

    Mishra, Suman; Mishra, Rajnikant

    2016-05-01

    The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis. PMID:26922180

  10. Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations.

    PubMed

    Rottenberg, H

    1984-01-01

    The uptake and binding of the lipophilic cations ethidium+, tetraphenylphosphonium+ (TPP+), triphenylmethylphosphonium+ (TPMP+), and tetraphenylarsonium+ (TPA+) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K'o) and the internal matrix volume and matrix face of the inner membrane (K'i) were determined and were utilized to estimate the membrane potential delta psi from the cation accumulation factor Rc according to the relation delta psi = RT/ZF ln [(RcVo - K'o)/(Vi + K'i)] where Vo and Vi are the volume of the external medium and the mitochondrial matrix, respectively, and Rc is the ratio of the cation content of the mitochondria and the medium. The values of delta psi estimated from this equation are in remarkably good agreement with those estimated from the distribution of 86Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations. PMID:6492133

  11. Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

    PubMed Central

    He, S; Fox, T D

    1997-01-01

    To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p. Images PMID:9285818

  12. How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane?

    PubMed Central

    Rapaport, Doron

    2005-01-01

    A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed β-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted β-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit β-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 β-barrel structure. PMID:16260501

  13. Toxoplasma gondii ROP18: potential to manipulate host cell mitochondrial apoptosis.

    PubMed

    Wu, Liang; Wang, Xiao; Li, Yunhui; Liu, Yuan; Su, Danhua; Fu, Tao; Guo, Fei; Gu, Liangping; Jiang, Xugan; Chen, Shengxia; Cao, Jianping

    2016-06-01

    Toxoplasma gondii is an obligate intracellular parasite that may manipulate host cell mitochondrial apoptosis pathways. In our experiment, 293T cells were transfected with the p3×FLAG-CMV-Myc-ROP18 vector and expressed the ROP18-Myc fusion protein. Cell apoptosis was induced by 0.5 μg/mL actinomycin D (ActD) and was detected by Annexin V-FITC/PI assay. The cell mitochondrial membrane potential was determined by JC-1. Cytochrome c (Cyto-c) from mitochondria and the cytoplasm was measured by Western blot. The Bcl-2 and Bax coding gene expression levels were detected by real-time PCR. We found, in vitro, that T. gondii ROP18 significantly suppressed 293T cell apoptosis induced by ActD and maintained mitochondrial membrane potential and integrity, thereby preventing the release of Cyto-c from mitochondria into the cytoplasm. The ratio of Bcl-2/Bax in ROP18-overexpressing cells was significantly higher than that of the negative control. Therefore, we speculate that ROP18 could suppress host cell apoptosis via the mitochondrial apoptosis pathway in vitro. PMID:27021182

  14. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    PubMed

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. PMID:22001671

  15. Calpeptin, not calpain, directly inhibits an ion channel of the inner mitochondrial membrane.

    PubMed

    Derksen, Maria; Vorwerk, Christian; Siemen, Detlef

    2016-05-01

    The permeability transition pore (PTP) of inner mitochondrial membranes is a large conductance pathway for ions up to 1500 Da which opening is responsible for ion equilibration and loss of membrane potential in apoptosis and thus in several neurodegenerative diseases. The PTP can be regulated by the Ca(2+)-activated mitochondrial K channel (BK). Calpains are Ca(2+)-activated cystein proteases; calpeptin is an inhibitor of calpains. We wondered whether calpain or calpeptin can modulate activity of PTP or BK. Patch clamp experiments were performed on mitoplasts of rat liver (PTP) and of an astrocytoma cell line (BK). Channel-independent open probability (P o) was determined (PTP) and, taking into account the number of open levels, NPo by single channel analysis (BK). We find that PTP in the presence of Ca(2+) (200 μM) is uninfluenced by calpain (13 nM) and shows insignificant decrease by the calpain inhibitor calpeptin (1 μM). The NPo of the BK is insensitive to calpain (54 nM), too. However, it is significantly and reversibly inhibited by the calpain inhibitor calpeptin (IC50 = 42 μM). The results agree with calpeptin-induced activation of the PTP via inhibition of the BK. Screening experiments with respirometry show calpeptin effects, fitting to inhibition of the BK by calpeptin, and strong inhibition of state 3 respiration. PMID:26108743

  16. Effect of the triaminopyridine flupirtine on calcium uptake, membrane potential and ATP synthesis in rat heart mitochondria

    PubMed Central

    Zimmer, Guido; Balakirev, Maxim; Zwicker, Klaus; Hofmann, Michael; Woodcock, Barry G; Pergande, Gabriela

    1998-01-01

    Flupirtine is an analgesic agent which exhibits neuronal cytoprotective activity and may have value in the treatment of conditions involving cell injury and apoptosis. Since flupirtine has no action on known receptor sites we have investigated the effect of this drug on mitochondrial membrane potential, and the changes in intramitochondrial calcium concentration in particular.The findings show that flupirtine increases Ca2+ uptake in mitochondria in vitro. At clinically relevant flupirtine concentrations, corresponding to flupirtine levels in vitro of 0.2 to 10 nmol mg−1 mitochondrial protein, there was a 2 to 3 fold increase in mitochondrial calcium levels (P<0.01). At supra-physiological flupirtine concentrations of 20 nmol mg−1 mitochondrial protein and above, the mitochondrial calcium concentrations were indistinguishable from those in untreated mitochondria.Mitochondrial membrane potential closely paralleled the changes in mitochondrial calcium levels showing a 20% (P<0.01) increase when the flupirtine concentration was raised from 0.2 nmol to 10 nmol mg−1 mitochondrial protein and a return to control values at 20 nmol mg−1 protein.The increase in mitochondrial calcium uptake and membrane potential were accompanied by an increase in mitochondrial ATP synthesis (30%; P<0.05) and a similar percentage reduction in mitochondrial volume.Calcium at 80 and 160 nmol mg−1 mitochondrial protein decreased ATP synthesis by 20–25% (P<0.001). This decrease was prevented or diminished if flupirtine at 10 nmol mg−1 protein was added before the addition of calcium.Since intracellular levels of flupirtine in intact cells never exceeded 10 nmol mg−1 mitochondrial protein, these findings are supportive evidence for an in vivo cytoprotective action of flupirtine at the mitochondrial level. PMID:9559899

  17. Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p

    SciTech Connect

    Josyula, Ratnakar; Jin, Zhongmin; McCombs, Deborah; DeLucas, Lawrence; Sha, Bingdong

    2006-02-01

    Tim44p is an essential mitochondrial peripheral membrane protein. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6{sub 3}22, with unit-cell parameters a = 124.25, c = 77.83 Å. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.

  18. Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes*

    PubMed Central

    Bajaj, Rakhi; Munari, Francesca; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import. PMID:25349212

  19. Cellular membrane potentials induced by alternating fields

    PubMed Central

    Grosse, Constantino; Schwan, Herman P.

    1992-01-01

    Membrane potentials induced by external alternating fields are usually derived assuming that the membrane is insulating, that the cell has no surface conductance, and that the potentials are everywhere solutions of the Laplace equation. This traditional approach is reexamined taking into account membrane conductance, surface admittance, and space charge effects. We find that whenever the conductivity of the medium outside the cell is low, large corrections are needed. Thus, in most of the cases where cells are manipulated by external fields (pore formation, cell fusion, cell rotation, dielectrophoresis) the field applied to the cell membrane is significantly reduced, sometimes practically abolished. This could have a strong bearing on present theories of pore formation, and of the influence of weak electric fields on membranes. PMID:19431866

  20. Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS.

    PubMed

    Gubbens, Jacob; Slijper, Monique; de Kruijff, Ben; de Kroon, Anton I P M

    2008-12-01

    Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins. PMID:18817900

  1. Membrane potential changes during chemokinesis in Paramecium.

    PubMed

    Van Houten, J

    1979-06-01

    Intracellular recordings show that (i) paramecia hyperpolarize slightly in attractants and depolarize in repellents that depend on the avoiding reaction (an abrupt change of swimming direction), and (ii) paramecia more strongly hyperpolarize in repellents and more strongly depolarize in attractants that depend on changes of swimming velocity. These membrane potential changes are in agreement with a hypothesis of membrane potential control of chemokinesis in Paramecium. PMID:572085

  2. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. PMID:27053724

  3. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

    PubMed Central

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-01-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  4. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    PubMed

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  5. Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

    PubMed Central

    Merkwirth, Carsten; Morbin, Michela; Brönneke, Hella S.; Jordan, Sabine D.; Rugarli, Elena I.; Langer, Thomas

    2012-01-01

    Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies

  6. Bcl-xL Blocks a Mitochondrial Inner Membrane Channel and Prevents Ca2+ Overload-Mediated Cell Death

    PubMed Central

    Tornero, Daniel; Posadas, Inmaculada; Ceña, Valentín

    2011-01-01

    Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca2+ is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-xL on Ca2+ overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-xL neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca2+-mediated mitochondrial multiple conductance channel opening. Bcl-xL neuroprotective actions were antagonized by the Bcl-xL inhibitor ABT-737 and potentiated by the Ca2+ chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death. PMID:21674052

  7. Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

    2001-06-01

    An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

  8. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study.

    PubMed

    Picard, Martin; White, Kathryn; Turnbull, Douglass M

    2013-01-15

    Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology. PMID

  9. A novel import route for an N-anchor mitochondrial outer membrane protein aided by the TIM23 complex.

    PubMed

    Song, Jiyao; Tamura, Yasushi; Yoshihisa, Tohru; Endo, Toshiya

    2014-06-01

    The membrane topology of Om45 in the yeast mitochondrial outer membrane (OM) is under debate. Here, we confirm that Om45 is anchored to the OM from the intermembrane space (IMS) by its N-terminal hydrophobic segment. We show that import of Om45 requires the presequence receptors, Tom20 and Tom22, and the import channel of Tom40. Unlike any of the known OM proteins, Om45 import requires the TIM23 complex in the inner membrane, a translocator for presequence-containing proteins, and the membrane potential (ΔΨ). Therefore, Om45 is anchored to the OM via the IMS by a novel import pathway involving the TIM23 complex. PMID:24781694

  10. Probing membrane potential with nonlinear optics.

    PubMed Central

    Bouevitch, O; Lewis, A; Pinevsky, I; Wuskell, J P; Loew, L M

    1993-01-01

    The nonlinear optical phenomenon of second harmonic generation is shown to have intrinsic sensitivity to the voltage across a biological membrane. Our results demonstrate that this second order nonlinear optical process can be used to monitor membrane voltage with excellent signal to noise and other crucial advantages. These advantages suggest extensive use of this novel approach as an important new tool in elucidating membrane potential changes in biological systems. For this first demonstration of the effect we use a chiral styryl dye which exhibits gigantic second harmonic signals. Possible mechanisms of the voltage dependence of the second harmonic signal are discussed. PMID:8218895

  11. Does membrane fatty acid composition modulate mitochondrial functions and their thermal sensitivities?

    PubMed

    Lemieux, H; Blier, P U; Tardif, J-C

    2008-01-01

    We investigated the effect of modifying fatty acid modification of heart mitochondrial membranes by dietary intervention on the functions and thermal sensitivity of electron transport system complexes embedded in the inner mitochondrial membrane. Four groups of rats were fed diets differing in their fat (coconut, olive or fish oil) and antioxidant (fish oil with or without probucol) contents. After 16 weeks of feeding, the coconut and olive oil groups had lower long-chain n-3 polyunsaturated fatty acids contents and a lower unsaturation index compared to both fish oil groups. These differences in fatty acid composition were not related to any differences in the mitochondrial respiration rate induced at Complexes I, II or IV, or to differences in their thermal sensitivity. The coconut oil group showed a lower mitochondrial affinity for pyruvate at 5 degrees C (k(mapp)=6.4+/-1.8) compared to any other groups (k(mapp)=3.8+/-0.5; 4.7+/-0.8; 3.6+/-1.1, for olive, fish oil and fish oil and probucol groups, respectively). At least in rat heart, our results do not support a major impact of the fatty acid composition of the mitochondrial membrane on the function of mitochondrial enzymatic complexes or on their temperature sensitivity. PMID:17993286

  12. Glucocorticoid-induced alterations in mitochondrial membrane properties and respiration in childhood acute lymphoblastic leukemia.

    PubMed

    Eberhart, Karin; Rainer, Johannes; Bindreither, Daniel; Ritter, Ireen; Gnaiger, Erich; Kofler, Reinhard; Oefner, Peter J; Renner, Kathrin

    2011-06-01

    Mitochondria are signal-integrating organelles involved in cell death induction. Mitochondrial alterations and reduction in energy metabolism have been previously reported in the context of glucocorticoid (GC)-triggered apoptosis, although the mechanism is not yet clarified. We analyzed mitochondrial function in a GC-sensitive precursor B-cell acute lymphoblastic leukemia (ALL) model as well as in GC-sensitive and GC-resistant T-ALL model systems. Respiratory activity was preserved in intact GC-sensitive cells up to 24h under treatment with 100 nM dexamethasone before depression of mitochondrial respiration occurred. Severe repression of mitochondrial respiratory function was observed after permeabilization of the cell membrane and provision of exogenous substrates. Several mitochondrial metabolite and protein transporters and two subunits of the ATP synthase were downregulated in the T-ALL and in the precursor B-ALL model at the gene expression level under dexamethasone treatment. These data could partly be confirmed in ALL lymphoblasts from patients, dependent on the molecular abnormality in the ALL cells. GC-resistant cell lines did not show any of these defects after dexamethasone treatment. In conclusion, in GC-sensitive ALL cells, dexamethasone induces changes in membrane properties that together with the reduced expression of mitochondrial transporters of substrates and proteins may lead to repressed mitochondrial respiratory activity and lower ATP levels that contribute to GC-induced apoptosis. PMID:21237131

  13. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    PubMed

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-Ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. PMID:26752045

  14. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana

    PubMed Central

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence. PMID:26752045

  15. Mitochondrial Peroxiredoxin III is a Potential Target for Cancer Therapy

    PubMed Central

    Song, In-Sung; Kim, Hyoung-Kyu; Jeong, Seung-Hun; Lee, Sung-Ryul; Kim, Nari; Rhee, Byoung Doo; Ko, Kyung Soo; Han, Jin

    2011-01-01

    Mitochondria are involved either directly or indirectly in oncogenesis and the alteration of metabolism in cancer cells. Cancer cells contain large numbers of abnormal mitochondria and produce large amounts of reactive oxygen species (ROS). Oxidative stress is caused by an imbalance between the production of ROS and the antioxidant capacity of the cell. Several cancer therapies, such as chemotherapeutic drugs and radiation, disrupt mitochondrial homeostasis and release cytochrome c, leading to apoptosome formation, which activates the intrinsic pathway. This is modulated by the extent of mitochondrial oxidative stress. The peroxiredoxin (Prx) system is a cellular defense system against oxidative stress, and mitochondria in cancer cells are known to contain high levels of Prx III. Here, we review accumulating evidence suggesting that mitochondrial oxidative stress is involved in cancer, and discuss the role of the mitochondrial Prx III antioxidant system as a potential target for cancer therapy. We hope that this review will provide the basis for new strategic approaches in the development of effective cancer treatments. PMID:22072940

  16. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II.

    PubMed

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  17. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

    PubMed Central

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

  18. Induction of rat hepatic mitochondrial membrane permeability transition pore opening by leaf extract of Olax subscorpioidea

    PubMed Central

    Adegbite, Oluwatobi Samuel; Akinsanya, Yetunde Ifeoma; Kukoyi, Ayobami Jahdahunsi; Iyanda-Joel, Wisdom O.; Daniel, Oluwatoyin O.; Adebayo, Abiodun Humphrey

    2015-01-01

    Background: The induction of the mitochondrial membrane permeability transition (MMPT) pore has been implicated in the cascade of events involved in apoptosis (programmed cell death). Olax subscorpioidea is traditionally used for the treatment of several diseases and infection. However, its role on MMPT is not yet established. This study was aimed at evaluating the effects of varying concentrations of the methanol leaf extract of O. subscorpioidea (MEOS) on MMPT pore opening, mitochondrial adenosine triphosphatase (ATPase), and mitochondrial lipid peroxidation. Materials and Methods: Opening of the pore was spectrophotometrically assayed under succinate-energized conditions. Results: In the absence of triggering agent (calcium), MEOS induced MMPT pore opening by 350, 612, 827, 845% at 36, 60, 86 and 112 μg/ml, respectively. MEOS further induced MMPT pore opening in the presence of a triggering agent by 866, 905, 831, 840, 949% at 12, 36, 60, 86 and 112 μg/ml, respectively. The extract significantly induced mitochondrial membrane lipid peroxidation in all the concentration used. MEOS also significantly increased mitochondrial ATP hydrolysis by mitochondrial ATPase in all concentration of the extract used. Conclusion: It may be deduced from this results, that MEOS contains certain bioactive components that may find use in pathological conditions that require an enhanced rate of apoptosis. PMID:26109790

  19. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    PubMed Central

    Clémençon, Benjamin

    2012-01-01

    The existence of a mitochondrial interactosome (MI) has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK) in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp) and inorganic phosphate (PiC) carriers as well as the VDAC (or mitochondrial porin) catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1) under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003. PMID:22408429

  20. Heinrich Wieland--prize lecture. Transport of proteins across mitochondrial membranes.

    PubMed

    Neupert, W

    1994-03-01

    The vast majority of proteins comprising the mitochondrion are encoded by nuclear genes, synthesized on ribosomes in the cytosol, and translocated into the various mitochondrial subcompartments. During this process proteins must cross the lipid membranes of the mitochondrion without interfering with the integrity or functions of the organelle. In recent years an approach combining biochemical, molecular, genetic, and morphological methodology has provided insights into various aspects of this complex process of intracellular protein sorting. In particular, a greater understanding of the molecular specificity and mechanism of targeting of mitochondrial preproteins has been reached, as a protein complex of the outer membrane which facilitates recognition and initial membrane insertion has been identified and characterized. Furthermore, pathways and components involved in the translocation of pre-proteins across the two mitochondrial membranes are being dissected and defined. The energetics of translocation and the processes of unfolding and folding of proteins during transmembrane transfer are closely linked to the function of a host of proteins known as heat-shock proteins or molecular chaperones, present both outside and inside the mitochondrion. In addition, the analysis of the process of folding of polypeptides in the mitochondrial matrix has allowed novel and unexpected insights into general pathways of protein folding assisted by folding factors. Pathways of sorting of proteins to the four different mitochondrial subcompartments--the outer membrane (OM), intermembrane space, inner membrane (IM) and matrix--are only partly understood and reveal an amazing complexity and variation. Many additional protein factors are involved in these latter processes, a few of which have been analyzed, such as cytochrome c heme lyase and cytochrome c1 heme lyase, enzymes that catalyze the covalent addition of the heme group to cytochrome c and c1 preproteins, and the

  1. Quantitative mitochondrial redox imaging of breast cancer metastatic potential

    NASA Astrophysics Data System (ADS)

    Xu, He N.; Nioka, Shoko; Glickson, Jerry D.; Chance, Britton; Li, Lin Z.

    2010-05-01

    Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp/(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

  2. Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1.

    PubMed

    Vanwalleghem, Gilles; Fontaine, Frédéric; Lecordier, Laurence; Tebabi, Patricia; Klewe, Kristoffer; Nolan, Derek P; Yamaryo-Botté, Yoshiki; Botté, Cyrille; Kremer, Anneke; Burkard, Gabriela Schumann; Rassow, Joachim; Roditi, Isabel; Pérez-Morga, David; Pays, Etienne

    2015-01-01

    Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion. PMID:26307671

  3. Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1

    PubMed Central

    Vanwalleghem, Gilles; Fontaine, Frédéric; Lecordier, Laurence; Tebabi, Patricia; Klewe, Kristoffer; Nolan, Derek P.; Yamaryo-Botté, Yoshiki; Botté, Cyrille; Kremer, Anneke; Burkard, Gabriela Schumann; Rassow, Joachim; Roditi, Isabel; Pérez-Morga, David; Pays, Etienne

    2015-01-01

    Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion. PMID:26307671

  4. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

  5. Mitochondrial outer membrane forms bridge between two mitochondria in Arabidopsis thaliana.

    PubMed

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Tsutsumi, Nobuhiro; Arimura, Shin-Ichi

    2016-05-01

    Mitochondria are double-membrane organelles that move around and change their shapes dynamically. In plants, the dynamics of the outer membrane is not well understood. We recently demonstrated that mitochondria had tubular protrusions of the outer membrane with little or no matrix, called MOPs (mitochondrial outer-membrane protrusions; MOPs). Here we show that a MOP can form a bridge between two mitochondria in Arabidopsis thaliana. The bridge does not appear to involve the inner membranes. Live imaging revealed stretching of the MOP bridge, demonstrating the flexibility of the outer membrane. Mitochondria frequently undergo fission and fusion. These observations raise the possibility that MOPs bridges have a role in these processes. PMID:27031262

  6. Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

    PubMed

    Motz, Christian; Martin, Heiko; Krimmer, Thomas; Rassow, Joachim

    2002-11-01

    The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane. PMID:12419260

  7. A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

    PubMed Central

    Otera, Hidenori; Taira, Yohsuke; Horie, Chika; Suzuki, Yurina; Suzuki, Hiroyuki; Setoguchi, Kiyoko; Kato, Hiroki; Oka, Toshihiko; Mihara, Katsuyoshi

    2007-01-01

    The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs. PMID:18158327

  8. A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments.

    PubMed

    Otera, Hidenori; Taira, Yohsuke; Horie, Chika; Suzuki, Yurina; Suzuki, Hiroyuki; Setoguchi, Kiyoko; Kato, Hiroki; Oka, Toshihiko; Mihara, Katsuyoshi

    2007-12-31

    The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component-depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail-anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs. PMID:18158327

  9. Large conductance channel in plasma membranes of astrocytic cells is functionally related to mitochondrial VDAC-channels.

    PubMed

    Guibert, B; Dermietzel, R; Siemen, D

    1998-03-01

    Large conductance anion channels with similar electrophysiological characteristics were found in plasma membranes and in outer mitochondrial membranes of various cell types. Although their large conductance and their peculiar voltage dependence point to a close relation, it was questioned whether they belong to the same family. We therefore compared some biochemical features of a plasmalemmal channel with those known from the mitochondrial channel. Current events were recorded from excised patches of plasma membranes of a rat astrocytic cell line (RGCN). The underlying channels exhibited a conductance of 401 +/- 50 pS. Open probability was highest between +/- 10 mV and gradually approached zero beyond +/- 25 mV. Activity as induced by voltage ramps between +/- 40 mV appeared after a delay of up to several min. The delay could be reduced by bathing either side of the patch in an acidic Ringer solution (pH 6.2). 1 mM Al3+ increased the open time at potentials more positive than 20 mV. 10 mM dextran sulfate (MW 8000) caused reversible flickering, increasing the closed probability. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) also caused a reversible flickering into the closed state, reducing the apparent single channel amplitude by up to 70% at 0.5 mM DIDS. Application of 5 mM ATP resulted in reversible blockade; ATP was more effective from the outside than from the inside (blocking activity 65% vs. 16% of the patches). We conclude that the large conductance anion channel from astrocytic cells displays electrophysiological and pharmacological characteristics that resemble those of VDAC (Voltage Dependent Anion Channel) from the outer mitochondrial membrane. PMID:9611779

  10. Control of the ornithine cycle in Neurospora crassa by the mitochondrial membrane.

    PubMed Central

    Davis, R H; Ristow, J L

    1983-01-01

    In Neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol. Ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline. Neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro. Nevertheless, when arginine is added to cells and cytosolic ornithine increases as arginine degradation begins, the rate of citrulline synthesis drops immediately to about 20% of normal (B. J. Bowman and R. H. Davis, Bacteriol. 130:285-291, 1977). We have studied this phenomenon in citrulline-accumulating strains carrying the arg-1 mutation. Citrulline accumulation is blocked when arginine is added to an arg-1 strain but not to an arg-1 strain carrying a mutation conferring insensitivity of intramitochondrial ornithine synthesis to arginine. Thus, ornithine is evidently unable to enter mitochondria in normal (feedback-sensitive) cells. Other experiments show that cytosolic ornithine enters mitochondria readily except when arginine or other basic amino acids are present at high levels in the cells. We conclude that in N. crassa, the mitochondrial membrane has evolved as a secondary site of feedback inhibition in arginine synthesis and that this prevents a wasteful cycling of catabolic ornithine back through the anabolic pathway. This is compared to the quite different mechanism by which the yeast Saccharomyces cerevisiae prevents a futile ornithine cycle. PMID:6222031

  11. Making heads or tails of mitochondrial membranes in longevity and aging: a role for comparative studies

    PubMed Central

    2014-01-01

    Mitochondria play vital roles in metabolic energy transduction, intermediate molecule metabolism, metal ion homeostasis, programmed cell death and regulation of the production of reactive oxygen species. As a result of their broad range of functions, mitochondria have been strongly implicated in aging and longevity. Numerous studies show that aging and decreased lifespan are also associated with high reactive oxygen species production by mitochondria, increased mitochondrial DNA and protein damage, and with changes in the fatty acid composition of mitochondrial membranes. It is possible that the extent of fatty acid unsaturation of the mitochondrial membrane determines susceptibility to lipid oxidative damage and downstream protein and genome toxicity, thereby acting as a determinant of aging and lifespan. Reviewing the vast number of comparative studies on mitochondrial membrane composition, metabolism and lifespan reveals some evidence that lipid unsaturation ratios may correlate with lifespan. However, we caution against simply relating these two traits. They may be correlative but have no functional relation. We discuss an important methodology for body mass and phylogenetic correction in comparative studies. PMID:24588808

  12. Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

    SciTech Connect

    Moepert, Kristin; Hajek, Petr; Frank, Stephan; Chen, Christiane; Kaufmann, Joerg; Santel, Ansgar

    2009-08-01

    RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with 'bulge'-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous 'fusion' and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.

  13. Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane.

    PubMed

    Console, Lara; Giangregorio, Nicola; Indiveri, Cesare; Tonazzi, Annamaria

    2014-09-01

    Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane. PMID:24898781

  14. Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation.

    PubMed Central

    Brand, M D; Felber, S M

    1984-01-01

    The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements. PMID:6696741

  15. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    PubMed

    Szarka, András; Horemans, Nele; Passarella, Salvatore; Tarcsay, Akos; Orsi, Ferenc; Salgó, András; Bánhegyi, Gábor

    2008-10-01

    Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism. PMID:18600345

  16. Top Down Proteomics of Human Membrane Proteins from Enriched Mitochondrial Fractions

    PubMed Central

    Catherman, Adam D.; Li, Mingxi; Tran, John C.; Durbin, Kenneth R.; Compton, Philip D.; Early, Bryan P.; Thomas, Paul M.; Kelleher, Neil L.

    2013-01-01

    The interrogation of intact integral membrane proteins has long been a challenge for biological mass spectrometry. Here, we demonstrate the application of Top Down mass spectrometry to whole membrane proteins below 60 kDa with up to 8 transmembrane helices. Analysis of enriched mitochondrial membrane preparations from human cells yielded identification of 83 integral membrane proteins, along with 163 membrane-associated or soluble proteins, with a median q value of 3 × 10−10. An analysis of matching fragment ions demonstrated that significantly more fragment ions were found within transmembrane domains than would be expected based upon the observed protein sequence. Forty-six proteins from the complexes of oxidative phosphorylation were identified which exemplifies the increasing ability of Top Down Proteomics to provide extensive coverage in a biological network. PMID:23305238

  17. The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane.

    PubMed

    Meisinger, Chris; Rissler, Michael; Chacinska, Agnieszka; Szklarz, Luiza K Sanjuán; Milenkovic, Dusanka; Kozjak, Vera; Schönfisch, Birgit; Lohaus, Christiane; Meyer, Helmut E; Yaffe, Michael P; Guiard, Bernard; Wiedemann, Nils; Pfanner, Nikolaus

    2004-07-01

    The biogenesis of mitochondrial outer membrane proteins involves the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The two known subunits of the SAM complex, Mas37 and Sam50, are required for assembly of the abundant outer membrane proteins porin and Tom40. We have identified an unexpected subunit of the SAM complex, Mdm10, which is involved in maintenance of mitochondrial morphology. Mitochondria lacking Mdm10 are selectively impaired in the final steps of the assembly pathway of Tom40, including the association of Tom40 with the receptor Tom22 and small Tom proteins, while the biogenesis of porin is not affected. Yeast mutants of TOM40, MAS37, and SAM50 also show aberrant mitochondrial morphology. We conclude that Mdm10 plays a specific role in the biogenesis of the TOM complex, indicating a connection between the mitochondrial protein assembly apparatus and the machinery for maintenance of mitochondrial morphology. PMID:15239954

  18. Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

    PubMed Central

    Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

    2013-01-01

    Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

  19. Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes

    PubMed Central

    Krumpe, Katrin; Frumkin, Idan; Herzig, Yonatan; Rimon, Nitzan; Özbalci, Cagakan; Brügger, Britta; Rapaport, Doron; Schuldiner, Maya

    2012-01-01

    Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in ∆spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in ∆spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell. PMID:22918956

  20. Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

    PubMed

    Elustondo, Pia A; Angelova, Plamena R; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y; Pavlov, Evgeny V

    2013-01-01

    Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

  1. Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch.

    PubMed

    Karo, Jaanus; Peterson, Pearu; Vendelin, Marko

    2012-03-01

    Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies. PMID:22241474

  2. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  3. Mitochondrial Respiratory Pathways Inhibition in Rhizopus oryzae Potentiates Activity of Posaconazole and Itraconazole via Apoptosis

    PubMed Central

    Shirazi, Fazal; Kontoyiannis, Dimitrios P.

    2013-01-01

    The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents. PMID:23696824

  4. Mitochondrial respiratory pathways inhibition in Rhizopus oryzae potentiates activity of posaconazole and itraconazole via apoptosis.

    PubMed

    Shirazi, Fazal; Kontoyiannis, Dimitrios P

    2013-01-01

    The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents. PMID:23696824

  5. Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

    PubMed Central

    Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

    2005-01-01

    DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

  6. σ-1 Receptor at the Mitochondrial-Associated Endoplasmic Reticulum Membrane Is Responsible for Mitochondrial Metabolic Regulation

    PubMed Central

    Marriott, Karla-Sue C.; Prasad, Manoj; Thapliyal, Veena

    2012-01-01

    The mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a small section of the outer mitochondrial membrane tethered to the ER by lipid and protein filaments. One such MAM protein is the σ-1 receptor, which contributes to multiple signaling pathways. We found that short interfering RNA-mediated knockdown of σ-1 reduced pregnenolone synthesis by 95% without affecting expression of the inner mitochondrial membrane resident enzyme, 3-β-hydroxysteroid dehydrogenase 2. To explore the underlying mechanism of this effect, we generated a series of σ-receptor ligands: 5,6-dimethoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-1), 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamide (KSCM-5), and 6-methoxy-3-methyl-N-phenyl-N-(3-(piperidin-1-yl) propyl)benzofuran-2-carboxamide (KSCM-11) specifically bound to σ-1 in the nanomolar range, whereas KSCM-5 and KSCM-11 also bound to σ-2. Treatment of cells with the KSCM ligands led to decreased cell viability, with KSCM-5 having the most potent effect followed by KSCM-11. KSCM-1 increased σ-1 expression by 4-fold and progesterone synthesis, whereas the other compounds decreased progesterone synthesis. These differences probably are caused by ligand molecular structure. For example, KSCM-1 has two methoxy substituents at C-5 and C-6 of the benzofuran ring, whereas KSCM-11 has one at C-6. KSCM ligands or σ-1 knockdown did not alter the expression of ER resident enzymes that synthesize steroids. However, coimmunoprecipitation of the σ-1 receptor pulled down voltage-dependent anion channel 2 (VDAC2), whose expression was enhanced by KSCM-1. VDAC2 plays a key role in cholesterol transport into the mitochondria, suggesting that the σ-1 receptor at the MAM coordinates with steroidogenic acute regulatory protein for cholesterol trafficking into the mitochondria for metabolic regulation. PMID:22923735

  7. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

    PubMed Central

    Yu, Seong-Woon; Wang, Yingfei; Frydenlund, Didrik S; Ottersen, Ole Petter; Dawson, Valina L; Dawson, Ted M

    2009-01-01

    Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders. PMID:19863494

  8. Stability of membrane potential in heart mitochondria: Single mitochondrion imaging

    SciTech Connect

    Uechi, Yukiko; Yoshioka, Hisashi; Morikawa, Daisuke; Ohta, Yoshihiro . E-mail: ohta@cc.tuat.ac.jp

    2006-06-16

    Mitochondrial membrane potential ({delta}{psi} {sub m}) plays an important role in cellular activity. Although {delta}{psi} {sub m} of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of {delta}{psi} {sub m}. The current study is performed to investigate the factors that stabilize {delta}{psi} {sub m} in cells by observing {delta}{psi} {sub m} of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of {delta}{psi} {sub m} in cells.

  9. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

    PubMed

    Stiller, Sebastian B; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G; Vent-Schmidt, Jens; Horvath, Susanne E; Frazier, Ann E; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils

    2016-05-10

    The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane. PMID:27166948

  10. Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart.

    PubMed

    Malekova, Lubica; Kominkova, Viera; Ferko, Miroslav; Stefanik, Peter; Krizanova, Olga; Ziegelhöffer, Attila; Szewczyk, Adam; Ondrias, Karol

    2007-01-01

    The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects. PMID:17123460

  11. A Novel DNA-Binding Protein Bound to the Mitochondrial Inner Membrane Restores the Null Mutation of Mitochondrial Histone Abf2p in Saccharomyces cerevisiae

    PubMed Central

    Cho, Jae Hyoung; Ha, Sang Jin; Kao, Ling Rong; Megraw, Timothy L.; Chae, Chi-Bom

    1998-01-01

    The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes. PMID:9742088

  12. Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde.

    PubMed

    Genaro-Mattos, Thiago C; Appolinário, Patricia P; Mugnol, Katia C U; Bloch, Carlos; Nantes, Iseli L; Di Mascio, Paolo; Miyamoto, Sayuri

    2013-10-21

    Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli. PMID:24059586

  13. Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

    PubMed Central

    Schafer, Blanca; Quispe, Joel; Choudhary, Vineet; Chipuk, Jerry E.; Ajero, Teddy G.; Du, Han; Schneiter, Roger

    2009-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin. PMID:19244344

  14. Fluctuations of the proton-electromotive force across the inner mitochondrial membrane

    NASA Astrophysics Data System (ADS)

    Procopio, Joaquim; Fornés, José A.

    1997-05-01

    The intermembrane mitochondrial space (IMMS) is delimited by the inner and outer mitochondrial membranes and defines a region of molecular dimension where fluctuations of the number of free protons and of transmembrane voltage can give rise to fluctuations in the proton-electromotive force EPMF across the inner mitochondrial membrane (IMM). We have applied the fluctuation-dissipation theorem to an electrical equivalent circuit consisting of a resistor Rm in parallel with a capacitor Cm representing the passive electrical properties of the IMM, in series with another capacitor Cb representing the proton-buffering power of the IMMS fluid. An access resistance Ra was defined as a link between the capacitor Cb and the membrane. Average EPMF fluctuations across the IMM were calculated for different assumptions concerning the intermembrane space dimensions. The calculated average EPMF fluctuations were in the vicinity of 100 mV for relaxation times in the few-microseconds range. The corresponding fluctuational protonic free energy is about 10 kJ/mole, which is comparable to the binding energy for protons in different transporters. This suggests that fluctuations in EPMF can be of relevance in the universe of forces influencing the molecular machinery embedded in the IMM.

  15. ANT2-defective fibroblasts exhibit normal mitochondrial bioenergetics

    PubMed Central

    Prabhu, Dolly; Goldstein, Amy C.; El-Khoury, Riyad; Rak, Malgorzata; Edmunds, Lia; Rustin, Pierre; Vockley, Jerry; Schiff, Manuel

    2015-01-01

    Adenine nucleotide translocase 2 (ANT2) transports glycolytic ATP across the inner mitochondrial membrane. Patients with ANT2 deletion were recently reported. We aimed at characterizing mitochondrial functions in ANT2-defective fibroblasts. In spite of ANT2 expression in fibroblasts, we observed no difference between ANT2-defective and control fibroblasts for mitochondrial respiration, respiratory chain activities, mitochondrial membrane potential and intracellular ATP levels. This indicates that ANT2 insufficiency does not alter fibroblast basal mitochondrial bioenergetics. PMID:26000237

  16. High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice

    PubMed Central

    Kahle, M.; Schäfer, A.; Seelig, A.; Schultheiß, J.; Wu, M.; Aichler, M.; Leonhardt, J.; Rathkolb, B.; Rozman, J.; Sarioglu, H.; Hauck, S.M.; Ueffing, M.; Wolf, E.; Kastenmueller, G.; Adamski, J.; Walch, A.; Hrabé de Angelis, M.; Neschen, S.

    2014-01-01

    Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid

  17. Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

    PubMed Central

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-01-01

    The pathophysiology of glomerular lesions of membranous nephropathy (MN), including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII) at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2) externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease. PMID:26137589

  18. Aspirin, acetaminophen and proton transport through phospholipid bilayers and mitochondrial membranes.

    PubMed

    Gutknecht, J

    1992-09-01

    Mechanisms of proton transport were investigated in planar phospholipid bilayer membranes exposed to aspirin (acetylsalicylic acid), acetaminophen (4-acetamidophenol), benzoic acid and three aspirin metabolites (salicylic acid, gentisic acid and salicyluric acid). The objectives were to characterize the conductances and permeabilities of these weak acids in lipid bilayer membranes and then predict their effects on mitochondrial membranes. Of the compounds tested only aspirin, benzoate and salicylate caused significant increases in membrane conductance. The conductance was due mainly to proton current at low pH and to weak acid anion current at neutral pH. Analysis of the concentration and pH dependence suggests that these weak acids act as HA-2-type proton carriers when pH approximately pK and as lipid soluble anions at neutral pH. Salicylate is much more potent than aspirin and benzoate because salicylate contains an internal hydrogen bond which delocalizes the negative charge and increases the permeability of the anion. Model calculations for mitochondria suggest that salicylate causes net H+ uptake by a cyclic process of HA influx and A- efflux. This model can explain the salicylate-induced uncoupling and swelling observed in isolated mitochondria. Since ingested aspirin breaks down rapidly to form salicylate, these results may clarify the mechanisms of aspirin toxicity in humans. The results may also help to explain why the ingestion of aspirin but not acetaminophen is associated with Reye's syndrome, a disease characterized by impaired energy metabolism and mitochondrial swelling. PMID:1334228

  19. The Self-Interaction of a Nodavirus Replicase Is Enhanced by Mitochondrial Membrane Lipids

    PubMed Central

    Qiu, Yang; Wang, Zhaowei; Liu, Yongxiang; Han, Yajuan; Miao, Meng; Qi, Nan; Yang, Jie; Xia, Hongjie; Li, Xiaofeng; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2014-01-01

    RNA replication of positive-strand (+)RNA viruses requires the protein-protein interactions among viral replicases and the association of viral replicases with intracellular membranes. Protein A from Wuhan nodavirus (WhNV), which closely associate with mitochondrial membranes, is the sole replicase required for viral RNA replication. Here, we studied the direct effects of mitochondrial membrane lipids (MMLs) on WhNV protein A activity in vitro. Our investigations revealed the self-interaction of WhNV protein A is accomplished via two different patterns (i.e., homotypic and heterotypic self-interactions via different interfaces). MMLs stimulated the protein A self-interaction, and this stimulation exhibited selectivity for specific phospholipids. Moreover, we found that specific phospholipids differently favor the two self-interaction patterns. Furthermore, manipulating specific phospholipid metabolism affected protein A self-interaction and the activity of protein A to replicate RNA in cells. Taken together, our findings reveal the direct effects of membrane lipids on a nodaviral RNA replicase. PMID:24586921

  20. The transport machinery for the import of preproteins across the outer mitochondrial membrane.

    PubMed

    Ryan, M T; Wagner, R; Pfanner, N

    2000-01-01

    In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria. PMID:10661891

  1. The Mitochondrial ADP/ATP Carrier Associates with the Inner Membrane Presequence Translocase in a Stoichiometric Manner*

    PubMed Central

    Mehnert, Carola S.; Rampelt, Heike; Gebert, Michael; Oeljeklaus, Silke; Schrempp, Sandra G.; Kochbeck, Lioba; Guiard, Bernard; Warscheid, Bettina; van der Laan, Martin

    2014-01-01

    The majority of mitochondrial proteins are synthesized with amino-terminal signal sequences. The presequence translocase of the inner membrane (TIM23 complex) mediates the import of these preproteins. The essential TIM23 core complex closely cooperates with partner protein complexes like the presequence translocase-associated import motor and the respiratory chain. The inner mitochondrial membrane also contains a large number of metabolite carriers, but their association with preprotein translocases has been controversial. We performed a comprehensive analysis of the TIM23 interactome based on stable isotope labeling with amino acids in cell culture. Subsequent biochemical studies on identified partner proteins showed that the mitochondrial ADP/ATP carrier associates with the membrane-embedded core of the TIM23 complex in a stoichiometric manner, revealing an unexpected connection of mitochondrial protein biogenesis to metabolite transport. Our data indicate that direct TIM23-AAC coupling may support preprotein import into mitochondria when respiratory activity is low. PMID:25124039

  2. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

    PubMed Central

    Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

    2011-01-01

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

  3. A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates.

    PubMed

    Schülke, N; Sepuri, N B; Gordon, D M; Saxena, S; Dancis, A; Pain, D

    1999-08-01

    Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively "zippered" together, forming long stretches in which they are in close contact (Schülke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery. PMID:10428870

  4. Importing Mitochondrial Proteins: Machineries and Mechanisms

    PubMed Central

    Chacinska, Agnieszka; Koehler, Carla M.; Milenkovic, Dusanka; Lithgow, Trevor; Pfanner, Nikolaus

    2014-01-01

    Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis. PMID:19703392

  5. Novel ligands that target the mitochondrial membrane protein mitoNEET

    PubMed Central

    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  6. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    PubMed

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. PMID:26947183

  7. Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

    SciTech Connect

    Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

    2004-12-17

    Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

  8. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers

    PubMed Central

    2004-01-01

    CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP–Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP–Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway. PMID:15270716

  9. Plasma membrane oxidoreductase activity in cultured cells in relation to mitochondrial function and oxidative stress.

    PubMed

    Deleonardi, Giulia; Biondi, Annalisa; D'Aurelio, Marilena; Pich, Milena Merlo; Stankov, Karmen; Falasca, Anna; Formiggini, Gabriella; Bovina, Carla; Romeo, Giovanni; Lenaz, Giorgio

    2004-01-01

    Dichlorophenol indophenol (DCIP) reduction by intracellualr pyridine nucleotides was investigated in two different lines of cultured cells characterized by enhanced production of reacive oxygen species (ROS) with respect to suitable controls. The first line denominated XTC-UC1 was derived from a metastasis of an oxyphilic thyroid tumor characterized by mitochondrial hyperplasia and compared with a line (B-CPAP) derived from a papillary thyroid carcinoma with normal mitochondrial mass. The second line (170 MN) was a cybrid line derived from rho0 cells from an osteosarcoma line (143B) fused with platelets from a patient with a nucleotide 9957 mutation in mitochondrial DNA (encoding for cytochrome c oxidase subunit III) in comparison with the parent 143B line. The experimental lines had no major decreases of electron transfer activities with respect to the controls; both of them, however, exhibited an increased peroxide production. The XTC-UC1 cell line exhibited enhanced activity with respect to control of dicoumarol-sensitive DCIP reduction, identified with membrane bound DT-diaphorase, whereas dicoumarol insensitive DCIP reduction was not significantly changed. On the other hand the mtDNA mutated cybrids exhibited a strong increase of both dicoumarol sensitive and insensitive DCIP reduction. The results suggest that enhanced oxidative stress and not deficient respiratory activity per se is the stimulus triggering over-expression of plasma membrane oxidative enzymes. PMID:15706061

  10. TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila

    PubMed Central

    Lin, Ran; Rittenhouse, Danielle; Sweeney, Katelyn; Potluri, Prasanth; Wallace, Douglas C.

    2015-01-01

    The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs. PMID:26241038

  11. Genetically encoded fluorescent sensors of membrane potential

    PubMed Central

    Baker, B. J.; Mutoh, H.; Dimitrov, D.; Akemann, W.; Perron, A.; Iwamoto, Y.; Jin, L.; Cohen, L. B.; Isacoff, E. Y.; Pieribone, V. A.; Hughes, T.; Knöpfel, T.

    2009-01-01

    Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments. PMID:18679801

  12. Imaging Mitochondrial Redox Potential and Its Possible Link to Tumor Metastatic Potential

    PubMed Central

    Li, Lin Z.

    2012-01-01

    Cellular redox states can regulate cell metabolism, growth, differentiation, motility, apoptosis, signaling pathways, and gene expressions etc. Growing body of literature suggest importance of redox status for cancer progression. While most studies on redox state were done on cells and tissue lysates, it is important to understand the role of redox state in tissue in vivo/ex vivo and image its heterogeneity. Redox scanning is a clinically-translatable method for imaging tissue mitochondrial redox potential with a submillimeter resolution. Redox scanning data in mouse models of human cancers demonstrate a correlation between mitochondrial redox state and tumor metastatic potential. I will discuss the significance of this correlation and possible directions for future research. PMID:22895837

  13. D-AKAP1a is a signal-anchored protein in the mitochondrial outer membrane.

    PubMed

    Jun, Yong-Woo; Park, Heeju; Lee, You-Kyung; Kaang, Bong-Kiun; Lee, Jin-A; Jang, Deok-Jin

    2016-04-01

    Dual A-kinase anchoring protein 1a (D-AKAP1a, AKAP1) regulates cAMP signaling in mitochondria. However, it is not clear how D-AKAP1a is associated with mitochondria. In this study, we show that D-AKAP1a is a transmembrane protein in the mitochondrial outer membrane (MOM). We revealed that the N-terminus of D-AKAP1a is exposed to the intermembrane space of mitochondria and that its C-terminus is located on the cytoplasmic side of the MOM. Moderate hydrophobicity and the positively charged flanking residues of the transmembrane domain of D-AKAP1a were important for targeting. Taken together, D-AKAP1a can be classified as a signal-anchored protein in the MOM. Our topological study provides valuable information about the molecular and cellular mechanisms of mitochondrial targeting of AKAP1. PMID:26950402

  14. Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin.

    PubMed

    Perevoshchikova, Irina V; Zorov, Savva D; Kotova, Elena A; Zorov, Dmitry B; Antonenko, Yuri N

    2010-06-01

    Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria. PMID:20412805

  15. Consequences of defective vitamin A transportation on mitochondrial membrane integrity during protein depletion.

    PubMed

    Olowookere, J O

    1986-01-01

    The relationships between the structural integrity and functionality of rat liver mitochondrial membranes, and different levels of dietary protein and vitamin A transportation during protein depletion in animals have been investigated. Although the vitamin A content of the protein-depleted diet was 1680 +/- 35 IU/kg diet, and that of the control diet was 1,650 +/- 30 IU/kg diet, the vitamin A content of the liver of depleted rats was reduced to 16.7% of controls. The hepatic mitochondria of rats fed a protein-depleted diet showed excessive passive swelling (about 3-fold of controls) in isotonic solutions. Whereas a seemingly inverse relationship existed between the vitamin A content of the liver and the osmotic behaviour of hepatic mitochondria of rats fed a protein-depleted diet, there is a direct relationship between their hepatic mitochondrial vitamin A and the respiratory control ratio. The implications of these observations are discussed. PMID:3717896

  16. The membrane dipole potential in a total membrane potential model. Applications to hydrophobic ion interactions with membranes.

    PubMed Central

    Flewelling, R F; Hubbell, W L

    1986-01-01

    The total potential energy profile for hydrophobic ion interactions with lipid bilayers can be written as the sum of four terms: the electrical Born, image and dipole contributions, and a neutral energy term. We introduce a specific model for the membrane dipole potential, treating it as a two-dimensional array of point dipoles located near each membrane-water interface. Together with specific theoretical models for the other energy terms, a total potential profile is developed that successfully describes the complete set of thermodynamic parameters for binding and translocation for the two hydrophobic ion structural analogues, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-). A reasonable fit to the data is possible if the dipole potential energy has a magnitude of 5.5 + 0.5 kcal/mol (240 + 20 mV), positive inside, and if the neutral energy contribution for TPP+ and TPB- is -7.0 + 1.0 kcal/mol. These results may also have important implications for small ion interactions with membranes and the energetics of charged groups in membrane proteins. PMID:3955184

  17. Vanadate induces necrotic death in neonatal rat cardiomyocytes through mitochondrial membrane depolarization.

    PubMed

    Soares, Sandra Sofia; Henao, Fernando; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2008-03-01

    Besides the well-known inotropic effects of vanadium in cardiac muscle, previous studies have shown that vanadate can stimulate cell growth or induce cell death. In this work, we studied the toxicity to neonatal rat ventricular myocytes (cardiomyocytes) of two vanadate solutions containing different oligovanadates distribution, decavanadate (containing decameric vanadate, V 10) and metavanadate (containing monomeric vanadate and also di-, tetra-, and pentavanadate). Incubation for 24 h with decavanadate or metavanadate induced necrotic cell death of cardiomyocytes, without significant caspase-3 activation. Only 10 microM total vanadium of either decavanadate (1 microM V 10) or metavanadate (10 microM total vanadium) was needed to produce 50% loss of cell viability after 24 h (assessed with MTT and propidium iodide assays). Atomic absorption spectroscopy showed that vanadium accumulation in cardiomyocytes after 24 h was the same when incubation was done with decavanadate or metavanadate. A decrease of 75% of the rate of mitochondrial superoxide anion generation, monitored with dihydroethidium, and a sustained rise of cytosolic calcium (monitored with Fura-2-loaded cardiomyocytes) was observed after 24 h of incubation of cardiomyocytes with decavanadate or metavanadate concentrations close to those inducing 50% loss of cell viability produced. In addition, mitochondrial membrane depolarization within cardiomyocytes, monitored with tetramethylrhodamine ethyl esther or with 3,3',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, were observed after only 6 h of incubation with decavanadate or metavanadate. The concentration needed for 50% mitochondrial depolarization was 6.5 +/- 1 microM total vanadium for both decavanadate (0.65 microM V 10) and metavanadate. In conclusion, mitochondrial membrane depolarization was an early event in decavanadate- and monovanadate-induced necrotic cell death of cardiomyocytes. PMID:18251508

  18. Targeting and insertion of the cholesterol-binding translocator protein into the outer mitochondrial membrane.

    PubMed

    Rone, Malena B; Liu, Jun; Blonder, Josip; Ye, Xiaoying; Veenstra, Timothy D; Young, Jason C; Papadopoulos, Vassilios

    2009-07-28

    Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP. PMID:19552401

  19. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  20. Complexes of the outer mitochondrial membrane protein mitoNEET with resveratrol-3-sulfate.

    PubMed

    Arif, Waqar; Xu, Shu; Isailovic, Dragan; Geldenhuys, Werner J; Carroll, Richard T; Funk, Max O

    2011-06-28

    Binding of the thiazolidinedione antidiabetic drug pioglitazone led to the discovery of a novel outer mitochondrial membrane protein of unknown function called mitoNEET. The protein is homodimeric and contains a uniquely ligated two iron-two sulfur cluster in each of its two cytosolic domains. Electrospray ionization mass spectrometry was employed to characterize solutions of the soluble cytosolic domain (amino acids 32--108) of the protein. Ions characteristic of dimers containing the cofactors were readily detected under native conditions. mitoNEET responded to exposure to solutions at low pH by dissociation to give monomers that retained the cofactor, followed by dissociation of the cofactor in a concerted fashion. mitoNEET formed complexes with resveratrol-3-sulfate, one of the primary metabolites of the natural product resveratrol. Resveratrol itself showed no tendency to interact with mitoNEET. The formation of complexes was evident in both electrospray ionization mass spectrometry and isothermal titration calorimetry measurements. Up to eight molecules of the compound associated with the dimeric form of the protein in a sequential fashion. Dissociation constants determined by micorcalorimetry were in the range 5-16 μM for the various binding sites. The only other known naturally occurring binding partner for mitoNEET at present is NADPH. It is very interesting that the iron-sulfur cluster containing protein interacts with two potentially redox active substances at the surface of mitochondria. These findings provide a new direction for research into two poorly understood, yet biomedically relevant, species. PMID:21591687

  1. Cell membrane potentials induced during exposure to EMP fields

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1994-09-01

    Internal current densities and electric fields induced in the human body during exposure to EMP fields are reviewed and used to predict resulting cell membrane potentials. Using several different approaches, membrane potentials of about 100 mV are predicted. These values are comparable to the static membrane potentials maintained by cells as a part of normal physiological function, but the EMP-induced potentials persist for only about 10 ns. Possible biological implications of EMP-induced membrane potentials including conformational changes and electroporation are discussed.

  2. Game and players: mitochondrial apoptosis and the therapeutic potential of ursodeoxycholic acid.

    PubMed

    Solá, Susana; Aranha, Márcia M; Steer, Clifford J; Rodrigues, Cecília M P

    2007-07-01

    Apoptosis represents a universal and exquisitely efficient cellular suicide pathway essential for a variety of normal biological processes ranging from embryonic development to ageing. In fact, tissue homeostasis is dependent on the perfect balance between positive and negative signals that determines the decision between life and death. Therefore, any imbalance can result in a wide range of pathologic disorders associated with unwanted apoptosis or cell growth. During the apoptotic process, the molecular players interact closely with each other in ways relevant to accelerate or interrupt the cellular death process. In addition, two major pathways of apoptosis activation have been recognized as the "intrinsic" mitochondrial pathway and the "extrinsic" death receptor pathway. Although these pathways act independently to initiate apoptosis, a delicate balance and cross-talk between the extrinsic and intrinsic pathways is thought to occur in many cell types. Interestingly, we have shown that ursodeoxycholic acid (UDCA), an endogenous hydrophilic bile acid, is a potent inhibitor of apoptosis by either stabilizing the mitochondrial membrane or modulating the expression of specific upstream targets. Herein, we review the main effectors involved in the death machinery, describe how they interact to regulate apoptosis, and discuss the main pathways that control cell death and survival. Further, we address multiple interesting targets as well as the potential application of UDCA as a therapeutic modality for apoptosis-related disorders. PMID:17489439

  3. Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration.

    PubMed

    Miyake, Takahito; Shirakawa, Hisashi; Nakagawa, Takayuki; Kaneko, Shuji

    2015-10-01

    Microglia, the resident immune cells in the brain, survey the environment of the healthy brain. Microglial migration is essential for many physiological and pathophysiological processes. Although microglia express some members of the transient receptor potential (TRP) channel family, there is little knowledge regarding the physiological roles of TRP channels in microglia. Here, we explored the role of TRP vanilloid 1 (TRPV1), a channel opened by capsaicin, heat, protons, and endovanilloids, in microglia. We found that application of capsaicin induced concentration-dependent migration in microglia derived from wild-type mice but not in those derived from TRPV1 knockout (TRPV1-KO) mice. Capsaicin-induced microglial migration was significantly inhibited by co-application of the TRPV1 blocker SB366791 and the Ca(2+) chelator BAPTA-AM. Using RT-PCR and immunocytochemistry, we validated that TRPV1 was expressed in microglia. Electrophysiological recording, intracellular Ca(2+) imaging, and immunocytochemistry indicated that TRPV1 was localized primarily in intracellular organelles. Treatment with capsaicin induced an increase in intramitochondrial Ca(2+) concentrations and mitochondrial depolarization. Furthermore, microglia derived from TRPV1-KO mice showed delayed Ca(2+) efflux compared with microglia derived from wild-type mice. Capsaicin-induced microglial migration was inhibited by membrane-permeable antioxidants and MAPK inhibitors, suggesting that mitochondrial TRPV1 activation induced Ca(2+) -dependent production of ROS followed by MAPK activation, which correlated with an augmented migration of microglia. Moreover, a mixture of three endovanilloids augmented microglial migration via TRPV1 activation. Together, these results indicate that mitochondrial TRPV1 plays an important role in inducing microglial migration. Activation of TRPV1 triggers an increase in intramitochondrial Ca(2+) concentration and following depolarization of mitochondria, which results in mt

  4. Sensitivity of inhibition of rat liver mitochondrial outer-membrane carnitine palmitoyltransferase by malonyl-CoA to chemical- and temperature-induced changes in membrane fluidity.

    PubMed Central

    Kolodziej, M P; Zammit, V A

    1990-01-01

    We have tested the possibility that alterations in the fluidity of the outer membrane of rat liver mitochondria could result in changes in the sensitivity of overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA [Zammit (1986) Biochem. Soc. Trans. 14. 676-679]. The sensitivity of CPT I to malonyl-CoA inhibition was measured by using highly purified mitochondrial outer membranes prepared from fed or 48 h-starved rats in the presence and absence of agents that increase membrane fluidity by perturbing membrane lipid order [benzyl alcohol, isoamyl alcohol (3-methylbutan-l-ol) and 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylpropyl)octanoate (A2C)]. All these agents resulted in marked decreases in the ability of malonyl-CoA to inhibit CPT I. This effect was accompanied by a modest increase in the absolute activity of CPT I in the absence of malonyl-CoA when the short-chain alcohols were used, but not when A2C was used, suggesting that the effect of increased membrane fluidity to decrease the malonyl-CoA sensitivity of CPT I may occur independently from other actions that may affect more directly the active site of the enzyme. In confirmation of the potential importance of fluidity changes, we showed that a marked increase in sensitivity of CPT I to malonyl-CoA could be produced when assays were performed at lower temperatures than those normally employed. These observations are discussed in the context of the slowness of the changes in CPT I sensitivity to malonyl-CoA inhibition that are induced by physiological perturbations. PMID:2268270

  5. Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca(2+) accumulation.

    PubMed

    Moysés, Danuza Nogueira; Barrabin, Hector

    2004-06-01

    Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca(2+) uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13+/-0.23 nmol Ca(2+) mg x protein(-1) min(-1). It is completely abolished by the H(+) ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 microM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca(2+) transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H(+) gradient can be used to promote Ca(2+) uptake by the mitochondria. PMID:15178471

  6. Effect of EMP fields on cell membrane potentials

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1993-06-01

    A simple model is presented for cell membrane potentials induced during exposure to electromagnetic pulse (EMP). Using calculated values of internal electric field strength induced during EMP exposure, the model predicts that cell membrane potentials of about 100 mV may be induced for time frames on the order of 10 ns. Possible biological effects of these potentials including electroporation area discussed.

  7. Antifungal Action of Methylene Blue Involves Mitochondrial Dysfunction and Disruption of Redox and Membrane Homeostasis in C. albicans.

    PubMed

    Ansari, Moiz A; Fatima, Zeeshan; Hameed, Saif

    2016-01-01

    Candida albicans is known to cause infections ranging from superficial and systemic in immunocompromised person. In this study, we explored that the antifungal action of Methylene blue (MB) is mediated through mitochondrial dysfunction and disruption of redox and membrane homeostasis against C. albicans. We demonstrated that MB displayed its antifungal potential against C. albicans and two clinical isolates tested. We also showed that MB is effective against two non- albicans species as well. Notably, the antifungal effect of MB seems to be independent of the major drug efflux pumps transporter activity. We explored that MB treated Candida cells were sensitive on non-fermentable carbon source leading us to propose that MB inhibits mitochondria. This sensitive phenotype was reinforced with the fact that sensitivity of Candida cells to MB could be rescued upon the supplementation of ascorbic acid, an antioxidant. This clearly suggests that disturbances in redox status are linked with MB action. We further demonstrated that Candida cells were susceptible to membrane perturbing agent viz. SDS which was additionally confirmed by transmission electron micrographs showing disruption of membrane integrity. Moreover, the ergosterol levels were significantly decreased by 66% suggesting lipid compositional changes due to MB. Furthermore, we could demonstrate that MB inhibits the yeast to hyphal transition in C. albicans which is one of the major virulence attribute in most of the hyphal inducing conditions. Taken together, the data generated from present study clearly establishes MB as promising antifungal agent that could be efficiently employed in strategies to treat Candida infections. PMID:27006725

  8. Antifungal Action of Methylene Blue Involves Mitochondrial Dysfunction and Disruption of Redox and Membrane Homeostasis in C. albicans

    PubMed Central

    Ansari, Moiz A.; Fatima, Zeeshan; Hameed, Saif

    2016-01-01

    Candida albicans is known to cause infections ranging from superficial and systemic in immunocompromised person. In this study, we explored that the antifungal action of Methylene blue (MB) is mediated through mitochondrial dysfunction and disruption of redox and membrane homeostasis against C. albicans. We demonstrated that MB displayed its antifungal potential against C. albicans and two clinical isolates tested. We also showed that MB is effective against two non- albicans species as well. Notably, the antifungal effect of MB seems to be independent of the major drug efflux pumps transporter activity. We explored that MB treated Candida cells were sensitive on non-fermentable carbon source leading us to propose that MB inhibits mitochondria. This sensitive phenotype was reinforced with the fact that sensitivity of Candida cells to MB could be rescued upon the supplementation of ascorbic acid, an antioxidant. This clearly suggests that disturbances in redox status are linked with MB action. We further demonstrated that Candida cells were susceptible to membrane perturbing agent viz. SDS which was additionally confirmed by transmission electron micrographs showing disruption of membrane integrity. Moreover, the ergosterol levels were significantly decreased by 66% suggesting lipid compositional changes due to MB. Furthermore, we could demonstrate that MB inhibits the yeast to hyphal transition in C. albicans which is one of the major virulence attribute in most of the hyphal inducing conditions. Taken together, the data generated from present study clearly establishes MB as promising antifungal agent that could be efficiently employed in strategies to treat Candida infections. PMID:27006725

  9. Changes in interfacial potentials induced by carbonylcyanide phenylhydrazone uncouplers: possible role in inhibition of mitochondrial oxygen consumption and other transport processes.

    PubMed

    Reyes, J; Benos, D J

    1984-01-01

    The charged and uncharged forms of carbonylcyanide phenylhydrazone uncouplers bind to phosphatidylcholine monolayers in a dose-dependent fashion, inducing changes in the interfacial potential of these model membranes. The interfacial potential change produced by the charged uncoupler is composed of a double-layer potential and an internal electrostatic potential (boundary and/or dipole). Changes in double-layer potential induced by the uncouplers in mitochondrial membranes can explain both the inhibition of oxygen consumption (QO2) caused by the uncouplers and the competition shown by succinate when mitochondria are respiring in the presence of rotenone. From these results and from dose-response curves of QO2 versus uncoupler concentrations, we conclude that 1 microM is an upper limit for free uncoupler concentration in the medium to avoid unwanted side effects during cell physiology studies that require total mitochondrial uncoupling. PMID:6748952

  10. Correlation between the rate of proteolysis of mitochondrial translation products and fluidity of the mitochondrial inner membrane in Saccharomyces cerevisiae yeast. Alteration of the rate of proteolysis under glucose repression.

    PubMed Central

    Luzikov, V N; Novikova, L A; Tikhonov, A N; Zubatov, A S

    1983-01-01

    Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids. PMID:6354177

  11. The reaction pathway of membrane-bound rat liver mitochondrial monoamine oxidase

    PubMed Central

    Houslay, Miles D.; Tipton, Keith F.

    1973-01-01

    1. A preparation of a partly purified mitochondrial outer-membrane fraction suitable for kinetic investigations of monoamine oxidase is described. 2. An apparatus suitable for varying the O2 concentration in a spectrophotometer cuvette is described. 3. The reaction catalysed by the membrane-bound enzyme is shown to proceed by a double-displacement (Ping Pong) mechanism, and a formal mechanism is proposed. 4. KCN, NaN3, benzyl cyanide and 4-cyanophenol are shown to be reversible inhibitors of the enzyme. 5. The non-linear reciprocal plot obtained with impure preparations of benzylamine, which is typical of high substrate inhibition, is shown to be due to aldehyde contamination of the substrate. PMID:4778271

  12. Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

    PubMed Central

    Klein, Astrid; Israel, Lars; Lackey, Sebastian W.K.; Nargang, Frank E.; Imhof, Axel; Baumeister, Wolfgang

    2012-01-01

    The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase. PMID:23128244

  13. [Mitochondrial dynamics: a potential new therapeutic target for heart failure].

    PubMed

    Kuzmicic, Jovan; Del Campo, Andrea; López-Crisosto, Camila; Morales, Pablo E; Pennanen, Christian; Bravo-Sagua, Roberto; Hechenleitner, Jonathan; Zepeda, Ramiro; Castro, Pablo F; Verdejo, Hugo E; Parra, Valentina; Chiong, Mario; Lavandero, Sergio

    2011-10-01

    Mitochondria are dynamic organelles able to vary their morphology between elongated interconnected mitochondrial networks and fragmented disconnected arrays, through events of mitochondrial fusion and fission, respectively. These events allow the transmission of signaling messengers and exchange of metabolites within the cell. They have also been implicated in a variety of biological processes including embryonic development, metabolism, apoptosis, and autophagy. Although the majority of these studies have been confined to noncardiac cells, emerging evidence suggests that changes in mitochondrial morphology could participate in cardiac development, the response to ischemia-reperfusion injury, heart failure, and diabetes mellitus. In this article, we review how the mitochondrial dynamics are altered in different cardiac pathologies, with special emphasis on heart failure, and how this knowledge may provide new therapeutic targets for treating cardiovascular diseases. PMID:21820793

  14. Versatile Membrane Deformation Potential of Activated Pacsin

    PubMed Central

    Byrnes, Laura J.; Sondermann, Holger

    2012-01-01

    Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pacsin isoform 1 (pacsin-1) has reduced activity compared to its isolated F-BAR domain, implicating an inhibitory role for its C-terminal Src homology 3 (SH3) domain. Here we show that the autoinhibitory, intramolecular interactions in pacsin-1 can be released upon binding to the entire proline-rich domain (PRD) of dynamin-1, resulting in potent membrane deformation activity that is distinct from the isolated F-BAR domain. Most strikingly, we observe the generation of small, homogenous vesicles with the activated protein complex under certain experimental conditions. In addition, liposomes prepared with different methods yield distinct membrane deformation morphologies of BAR domain proteins and apparent activation barriers to pacsin-1's activity. Theoretical free energy calculations suggest bimodality of the protein-membrane system as a possible source for the different outcomes, which could account for the coexistence of energetically equivalent membrane structures induced by BAR domain-containing proteins in vitro. Taken together, our results suggest a versatile role for pacsin-1 in sculpting cellular membranes that is likely dependent both on protein structure and membrane properties. PMID:23236520

  15. Peripheral-type benzodiazepine receptors are highly concentrated in mitochondrial membranes of rat testicular interstitial cells.

    PubMed

    Calvo, D J; Ritta, M N; Calandra, R S; Medina, J H

    1990-10-01

    The binding of 3H-RO 5-4864 to the peripheral-type benzodiazepine receptors (PBZDR) in rat testicular interstitial cells (TIC) was characterized. The binding was saturable, reversible and showed a single high-affinity (Kd = 5.02 +/- 0.86 nM) class of binding sites. The maximal binding capacity (Bmax) in crude mitochondrial fractions (77.6 +/- 9.1 pmol/mg protein) represents the highest density of PBZDR in tissues thus far studied. In comparison with the crude mitochondrial fraction the subcellular fractionation of TIC revealed a 2-fold enrichment of 3H-RO 5-4864 binding sites to the purified mitochondria (Bmax = 140 +/- 23 pmol/mg protein). The ability of various drugs to displace 3H-RO 5-4864 from TIC binding sites was examined and the inhibition constants (Ki) for RO 5-4864, PK 11195, diazepam and flunitrazepam were 3.5, 4.4, 159, and 353 nM, respectively, whereas clonazepam and RO 15-1788 were inefficient in displacing 3H-RO 5-4864 (Ki greater than 10 microM). This pharmacological profile is characteristic of PBZDR described in other tissues. In conclusion, rat TIC possess a very high concentration of PBZDR primarily associated with mitochondrial membranes. PMID:2175849

  16. Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins

    PubMed Central

    Nijtmans, Leo G.J.; de Jong, Liesbeth; Artal Sanz, Marta; Coates, Philip J.; Berden, Jan A.; Willem Back, Jaap; Muijsers, Anton O.; van der Spek, Hans; Grivell, Les A.

    2000-01-01

    Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse–chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins. PMID:10835343

  17. Effects of Insecticides on the Fluidity of Mitochondrial Membranes of the Diamondback Moth, Plutella xylostella, Resistant and Susceptible to Avermectin

    PubMed Central

    Hu, J.; Liang, P.; Shi, X.; Gao, X.

    2008-01-01

    The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25°C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1×10-4 mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25°C and no difference was observed when the temperature was more than 25°C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain PMID:20345311

  18. Effects of curcumin on cancer cell mitochondrial function and potential monitoring with ¹⁸F-FDG uptake.

    PubMed

    Jung, Kyung-Ho; Lee, Jin Hee; Park, Jin Won; Moon, Seung-Hwan; Cho, Young Seok; Choe, Yearn Seong; Lee, Kyung-Han

    2016-02-01

    A better understanding of how curcumin influences cancer cell biology could help devise new strategies to enhance its antitumor effect. Many curcumin actions are proposed to occur by targeting mitochondrial function, among which glucose metabolism and reactive oxygen species (ROS) production are pivotal. However, little is known of how curcumin influences cancer cell glucose metabolism. We thus evaluated the effect of curcumin on cancer cell glucose metabolism and mitochondrial function, and further investigated whether these responses could be modified to enhance the anticancer potency of the compound. MCF-7 breast cancer cells treated with curcumin were measured for 18F-fluorodeoxyglucose (18F‑FDG) uptake, lactate production, hexokinase activity, oxygen consumption rate (OCR), ROS production and mitochondrial membrane potential (MMP). Activation of signaling pathways was evaluated by western blots, and cell survival was assessed with sulforhodamine B assays. Curcumin stimulated a 3.6-fold increase of 18F-FDG uptake in MCF-7 cells, along with augmented hexokinase activity and lactate efflux. This was accompanied by significantly suppressed cellular OCR, consistent with a metabolic shift to glycolytic flux. Inhibiting this metabolic response with 2-deoxyglucose (2-DG) blocked curcumin-induced mTOR activation and resulted in a greater anti-proliferative effect. Curcumin-induced MMP depolarization led to reduced ROS production, which may hinder the anticancer effect of the compound. Intracellular ROS was completely restored by adding Cu2+, which can bind and modify the curcumin's physico-chemical property, and this resulted in a marked potentiation of its anti-proliferative effect. Thus, curcumin suppresses cancer cell MMP and ROS generation, and this response is accompanied by stimulated 18F-FDG uptake via shifting of metabolism from mitochondrial respiration to glycolytic flux. These mitochondrial and metabolic responses may provide potential targets that can

  19. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

    PubMed

    Clinton, Ryan W; Francy, Christopher A; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction. PMID:26578514

  20. Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites.

    PubMed

    Park, Jae-Sook; Thorsness, Mary K; Policastro, Robert; McGoldrick, Luke L; Hollingsworth, Nancy M; Thorsness, Peter E; Neiman, Aaron M

    2016-08-01

    The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum-mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome-mitochondrion contacts and to the nuclear-vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc. PMID:27280386

  1. Ethanol influences on Bax associations with mitochondrial membrane proteins in neonatal rat cerebellum.

    PubMed

    Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

    2013-02-01

    These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure and focused on interactions between proapoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that, following ethanol exposure, Bax proapoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least 2 h postexposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment and found such interactions were altered by ethanol treatment, but only at 2 h postexposure and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax but not by a Bax channel blocker. Therefore, we conclude that, at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex and not channel formation independent of PTP constituents. PMID:22767450

  2. Evidence that Synthesis of the Saccharomyces cerevisiae Mitochondrially Encoded Ribosomal Protein Var1p May Be Membrane Localized

    PubMed Central

    Fiori, Alessandro; Mason, Thomas L.; Fox, Thomas D.

    2003-01-01

    The 5′-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle. VAR1 is the only yeast mitochondrial gene encoding a major soluble protein. A chimeric mRNA bearing the VAR1 untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface. We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane. PMID:12796311

  3. Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins*

    PubMed Central

    Becker, Thomas; Horvath, Susanne E.; Böttinger, Lena; Gebert, Natalia; Daum, Günther; Pfanner, Nikolaus

    2013-01-01

    The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex. PMID:23625917

  4. Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA.

    PubMed

    Tan, An S; Baty, James W; Dong, Lan-Feng; Bezawork-Geleta, Ayenachew; Endaya, Berwini; Goodwin, Jacob; Bajzikova, Martina; Kovarova, Jaromira; Peterka, Martin; Yan, Bing; Pesdar, Elham Alizadeh; Sobol, Margarita; Filimonenko, Anatolyj; Stuart, Shani; Vondrusova, Magdalena; Kluckova, Katarina; Sachaphibulkij, Karishma; Rohlena, Jakub; Hozak, Pavel; Truksa, Jaroslav; Eccles, David; Haupt, Larisa M; Griffiths, Lyn R; Neuzil, Jiri; Berridge, Michael V

    2015-01-01

    We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells. PMID:25565207

  5. Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

    PubMed

    Narayan, Shoba; Devi, R S; Devi, C S Shyamala

    2007-11-20

    Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa. PMID:17719569

  6. Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function.

    PubMed

    Martin, Laura A; Kennedy, Barry E; Karten, Barbara

    2016-04-01

    Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology. PMID:25425472

  7. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

    PubMed

    Rogers, Carlyle; Davis, Barbara; Neufer, P Darrell; Murphy, Michael P; Anderson, Ethan J; Robidoux, Jacques

    2014-02-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels. PMID:24269897

  8. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    PubMed Central

    Mukherjee, Sutapa; Samanta, Luna; Roy, Anita; Bhanja, Shravani; Chainy, Gagan B. N.

    2014-01-01

    Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation. PMID:24987693

  9. Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

    PubMed Central

    Wrobel, Lidia; Trojanowska, Agata; Sztolsztener, Malgorzata E.; Chacinska, Agnieszka

    2013-01-01

    The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria. PMID:23283984

  10. Acute mitochondrial dysfunction after blast exposure: potential role of mitochondrial glutamate oxaloacetate transaminase.

    PubMed

    Arun, Peethambaran; Abu-Taleb, Rania; Oguntayo, Samuel; Wang, Ying; Valiyaveettil, Manojkumar; Long, Joseph B; Nambiar, Madhusoodana P

    2013-10-01

    Use of improvised explosive devices has significantly increased the incidence of traumatic brain injury (TBI) and associated neuropsychiatric deficits in the recent wars in Iraq and Afghanistan. Acute deleterious effects of single and repeated blast exposure can lead to long-term neurobiological effects and neuropsychiatric deficits. Using in vitro and in vivo shock tube models of blast-induced TBI, we studied changes in mitochondrial energy metabolism after blast exposure. Single and repeated blast exposures in vitro resulted in significant decreases in neuronal adenosine triphosphate (ATP) levels at 6 h post-blast that returned towards normal levels by 24 h. Similar changes in ATP also were observed in the cerebral cortices of mice subjected to single and repeated blast exposures. In neurons, mitochondrial glutamate oxaloacetate transaminase (GOT2) plays a critical role in metabolism and energy production. Proteomic analysis of brain cortices showed a significant decrease in GOT2 levels 6 h after repeated blast exposures, which was further confirmed by Western blotting. Western blot analysis of GOT2 and pyruvate dehydrogenase in the cortex showed direct correlation only between GOT2 and ATP levels. Activity of GOT2 in the isolated cortical mitochondria also showed significant decrease at 6 h supporting the results of proteomic and Western blot analyses. Knowing the significant role of GOT2 in the neuronal mitochondrial energy metabolism, it is quite likely that the down regulation of GOT2 after blast exposure is playing a significant role in mitochondrial dysfunction after blast exposure. PMID:23600763

  11. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins

    PubMed Central

    Gasanov, Sardar E.; Shrivastava, Indira H.; Israilov, Firuz S.; Kim, Aleksandr A.; Rylova, Kamila A.; Zhang, Boris; Dagda, Ruben K.

    2015-01-01

    Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity. PMID:26091109

  12. Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease.

    PubMed

    Banerjee, Kalpita; Munshi, Soumyabrata; Xu, Hui; Frank, David E; Chen, Huan-Lian; Chu, Charleen T; Yang, Jiwon; Cho, Sunghee; Kagan, Valerian E; Denton, Travis T; Tyurina, Yulia Y; Jiang, Jian Fei; Gibson, Gary E

    2016-06-01

    Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23-53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches. PMID:26923918

  13. Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

    PubMed Central

    Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

    2011-01-01

    The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

  14. Distinct effects of TRAIL on the mitochondrial network in human cancer cells and normal cells: role of plasma membrane depolarization

    PubMed Central

    Suzuki-Karasaki, Yoshihiro; Fujiwara, Kyoko; Saito, Kosuke; Suzuki-Karasaki, Miki; Ochiai, Toyoko; Soma, Masayoshi

    2015-01-01

    Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a promising anticancer drug due to its tumor-selective cytotoxicity. Here we report that TRAIL exhibits distinct effects on the mitochondrial networks in malignant cells and normal cells. Live-cell imaging revealed that multiple human cancer cell lines and normal cells exhibited two different modes of mitochondrial responses in response to TRAIL and death receptor agonists. Mitochondria within tumor cells became fragmented into punctate and clustered in response to toxic stimuli. The mitochondrial fragmentation was observed at 4 h, then became more pronounced over time, and associated with apoptotic cell death. In contrast, mitochondria within normal cells such as melanocytes and fibroblasts became only modestly truncated, even when they were treated with toxic stimuli. Although TRAIL activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, inhibition of this process by Drp1 knockdown or with the Drp1 inhibitor mdivi-1, potentiated TRAIL-induced apoptosis, mitochondrial fragmentation, and clustering. Moreover, mitochondrial reactive oxygen species (ROS)-mediated depolarization accelerated mitochondrial network abnormalities in tumor cells, but not in normal cells, and TRAIL caused higher levels of mitochondrial ROS accumulation and depolarization in malignant cells than in normal cells. Our findings suggest that tumor cells are more prone than normal cells to oxidative stress and depolarization, thereby being more vulnerable to mitochondrial network abnormalities and that this vulnerability may be relevant to the tumor-targeting killing by TRAIL. PMID:26057632

  15. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    SciTech Connect

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  16. Therapeutic Modulation of Apoptosis: Targeting the BCL-2 Family at the Interface of the Mitochondrial Membrane

    PubMed Central

    Nemec, Kathleen N.

    2008-01-01

    A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease. PMID:18972587

  17. "Eye of tiger sign" mimic in an adolescent boy with mitochondrial membrane protein associated neurodegeneration (MPAN).

    PubMed

    Yoganathan, Sangeetha; Sudhakar, Sniya Valsa; Thomas, Maya; Dutta, Atanu Kumar; Danda, Sumita

    2016-05-01

    Neurodegeneration with brain iron accumulation (NBIA) refers to an inherited heterogeneous group of disorders pathologically characterized by focal brain iron deposition. Clinical phenotype, imaging findings and genotype are variable among the different types of this disorder. In this case report, we describe the imaging finding of an adolescent boy with mitochondrial membrane protein associated neurodegeneration (MPAN), a subentity of NBIA. Magnetic resonance imaging of brain revealed hypointensity of globi pallidi with medial medullary lamina appearing as a hyperintense streak in T2 weighted images. Mild cerebellar atrophy in T2 weighted images and blooming of substantia nigra and globi pallidi in susceptibility weighted images were also observed. Imaging findings in patients with MPAN mimics the eye of tiger appearance in patients with pantothenate kinase associated neurodegeneration. Classical phenotype and eye of tiger sign mimic in imaging of patients with NBIA should raise the suspect for MPAN. Genetic studies helps in the confirmation of diagnosis of this neurodegenerative disorder. PMID:26602591

  18. Hydrolysis of ITP generates a membrane potential in submitochondrial particles.

    PubMed

    Sorgato, M C; Galiazzo, F; Valente, M; Cavallini, L; Ferguson, S J

    1982-08-20

    ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential. PMID:6214275

  19. Mitochondrial flashes: new insights into mitochondrial ROS signalling and beyond.

    PubMed

    Hou, Tingting; Wang, Xianhua; Ma, Qi; Cheng, Heping

    2014-09-01

    Respiratory mitochondria undergo stochastic, intermittent bursts of superoxide production accompanied by transient depolarization of the mitochondrial membrane potential and reversible opening of the membrane permeability transition pore. These discrete events were named 'superoxide flashes' for the reactive oxygen species (ROS) signal involved, and 'mitochondrial flashes' (mitoflashes) for the entirety of the multifaceted and intertwined mitochondrial processes. In contrast to the flashless basal ROS production of 'homeostatic ROS' for redox regulation, bursting ROS production during mitoflashes may provide 'signalling ROS' at the organelle level, fulfilling distinctly different cell functions. Mounting evidence indicates that mitoflash frequency is richly regulated over a broad range, and represents a novel, universal, and 'digital' readout of mitochondrial functional status and of the mitochondrial stress response. An emerging view is that mitoflashes participate in vital processes including metabolism, cell differentiation, the stress response and ageing. These recent advances shed new light on the role of mitochondrial functional dynamics in health and disease. PMID:25038239

  20. Metabolically derived potential on the outer membrane of mitochondria: a computational model.

    PubMed Central

    Lemeshko, S V; Lemeshko, V V

    2000-01-01

    The outer mitochondrial membrane (OMM) is permeable to various small substances because of the presence of a voltage-dependent anion channel (VDAC). The voltage dependence of VDAC's permeability is puzzling, because the existence of membrane potential on the OMM has never been shown. We propose that steady-state metabolically derived potential (MDP) may be generated on the OMM as the result of the difference in its permeability restriction for various charged metabolites. To demonstrate the possibility of MDP generation, two models were considered: a liposomal model and a simplified cell model with a creatine kinase energy channeling system. Quantitative computational analysis of the simplified cell model shows that a MDP of up to -5 mV, in addition to the Donnan potential, may be generated at high workloads, even if the OMM is highly permeable to small inorganic ions, including potassium. Calculations show that MDP and DeltapH, generated on the OMM, depend on the cytoplasmic pH and energy demand rate. Computational modeling suggests that MDP may be important for cell energy metabolism regulation in multiple ways, including VDAC's permeability modulation and the effect of electrodynamic compartmentation. The osmotic pressure difference between the mitochondrial intermembrane space and the cytoplasm, as related to the electrodynamic compartmentation effects, might explain the morphological changes in mitochondria under intense workloads. PMID:11106589

  1. Specific ion effects on membrane potential and the permselectivity of ion exchange membranes.

    PubMed

    Geise, Geoffrey M; Cassady, Harrison J; Paul, Donald R; Logan, Bruce E; Hickner, Michael A

    2014-10-21

    Membrane potential and permselectivity are critical parameters for a variety of electrochemically-driven separation and energy technologies. An electric potential is developed when a membrane separates electrolyte solutions of different concentrations, and a permselective membrane allows specific species to be transported while restricting the passage of other species. Ion exchange membranes are commonly used in applications that require advanced ionic electrolytes and span technologies such as alkaline batteries to ammonium bicarbonate reverse electrodialysis, but membranes are often only characterized in sodium chloride solutions. Our goal in this work was to better understand membrane behaviour in aqueous ammonium bicarbonate, which is of interest for closed-loop energy generation processes. Here we characterized the permselectivity of four commercial ion exchange membranes in aqueous solutions of sodium chloride, ammonium chloride, sodium bicarbonate, and ammonium bicarbonate. This stepwise approach, using four different ions in aqueous solution, was used to better understand how these specific ions affect ion transport in ion exchange membranes. Characterization of cation and anion exchange membrane permselectivity, using these ions, is discussed from the perspective of the difference in the physical chemistry of the hydrated ions, along with an accompanying re-derivation and examination of the basic equations that describe membrane potential. In general, permselectivity was highest in sodium chloride and lowest in ammonium bicarbonate solutions, and the nature of both the counter- and co-ions appeared to influence measured permselectivity. The counter-ion type influences the binding affinity between counter-ions and polymer fixed charge groups, and higher binding affinity between fixed charge sites and counter-ions within the membrane decreases the effective membrane charge density. As a result permselectivity decreases. The charge density and polarizability

  2. The iPLA(2)γ is identified as the membrane potential sensitive phospholipase in liver mitochondria.

    PubMed

    Rauckhorst, Adam J; Pfeiffer, Douglas R; Broekemeier, Kimberly M

    2015-08-19

    Previous reports from our lab identified a mitochondrial calcium-independent phospholipase A2 activity that is activated when the mitochondrial membrane potential is decreased. This activity was demonstrated to influence occurrence of the permeability transition. Originally, this activity was ascribed to the iPLA2β protein. Recently, both iPLA2β and iPLA2γ knock out mice have been generated. It has been shown by others that the iPLA2γ plays a significant role in progression of the permeability transition. In this paper, using the iPLA2β and iPLA2γ knock out mice we show that the membrane potential sensitive activity is the iPLA2γ. PMID:26206229

  3. Understanding the molecular mechanism of protein translocation across the mitochondrial inner membrane: still a long way to go.

    PubMed

    Marom, Milit; Azem, Abdussalam; Mokranjac, Dejana

    2011-03-01

    In order to reach the final place of their function, approximately half of the proteins in any eukaryotic cell have to be transported across or into one of the membranes in the cell. In this article, we present an overview of our current knowledge concerning the structural properties of the TIM23 complex and their relationship with the molecular mechanism of protein transport across the mitochondrial inner membrane. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes. PMID:20646995

  4. Membrane Transport of Singlet Oxygen Monitored by Dipole Potential Measurements

    PubMed Central

    Sokolov, Valerij S.; Pohl, Peter

    2009-01-01

    Abstract The efficiency of photodynamic reactions depends on 1), the penetration depth of the photosensitizer into the membrane and 2), the sidedness of the target. Molecules which are susceptible to singlet oxygen (1O2) experience less damage when separated from the photosensitizer by the membrane. Since 1O2 lifetime in the membrane environment is orders of magnitude longer than the time required for nonexcited oxygen (O2) to cross the membrane, this observation suggests that differences between the permeabilities or membrane partition of 1O2 and O2 exist. We investigated this hypothesis by releasing 1O2 at one side of a planar membrane while monitoring the kinetics of target damage at the opposite side of the same membrane. Damage to the target, represented by dipole-modifying molecules (phloretin or phlorizin), was indicated by changes in the interleaflet dipole potential difference Δϕb. A simple analytical model allowed estimation of the 1O2 interleaflet concentration difference from the rate at which Δϕb changed. It confirmed that the lower limit of 1O2 permeability is ∼2 cm/s; i.e., it roughly matches O2 permeability as predicted by Overton's rule. Consequently, the membrane cannot act as a barrier to 1O2 diffusion. Differences in the reaction rates at the cytoplasmic and extracellular membrane leaflets may be attributed only to 1O2 quenchers inside the membrane. PMID:18931253

  5. Aluminum chloride and membrane potentials of barley root cells

    SciTech Connect

    Etherton, B.; Shane, M.

    1986-04-01

    Aluminum chloride at pH 4 hyperpolarizes the membrane potentials of barley root epidermal cells. The authors tested to see whether this hyperpolarization could be caused by an aluminum induced alteration of the permeability of the membrane to potassium or sodium ions by measuring the effect of .04 mM aluminum ions (the Ca/sup + +/ conc. was 0.1 mM) on the membrane potential changes induced by changing the potassium or sodium concentrations in the medium bathing the roots. Aluminum ions did not change the magnitude of potassium or sodium induced changes in membrane potentials but significantly altered the rates of potassium and sodium induced changes of the potential. The results indicate that aluminum ions did not change sodium or potassium ion permeabilities of barley root cells.

  6. Respiratory activity and mitochondrial membrane associated with fruit senescence in postharvest peaches in response to UV-C treatment.

    PubMed

    Yang, Zhenfeng; Cao, Shifeng; Su, Xinguo; Jiang, Yueming

    2014-10-15

    The effect of 3.0kJ/m(2) ultraviolet-C (UV-C) treatment on respiratory activity and mitochondrial membrane associated with fruit senescence in peach fruit stored at 20°C for 8days was investigated. UV-C treatment could reduce senescence development, as evidenced by higher fruit firmness due to inhibition of respiration rate via reducing succinic dehydrogenase and cytochrome C oxidase activity. Meanwhile, the activities of superoxide dismutase, catalase and ascorbate peroxidase in the UV-C-treated fruit were much higher than those in control fruit, resulting in lower levels of superoxide radicals (O2(-)) and hydrogen peroxide (H2O2). In addition, this treatment maintained a higher level of mitochondrial membrane fluidity and inhibited opening of mitochondrial permeability transition pore. Our results suggest that the induction of antioxidant enzymes to scavenge O2(-) and H2O2 by UV-C treatment was associated with the maintenance of mitochondrial membrane integrity, which also played an important role in senescence retardation in peach fruit. PMID:24837916

  7. The use of cardiolipin-containing liposomes as a model system to study the interaction between proteins and the inner mitochondrial membrane.

    PubMed

    Marom, Milit; Azem, Abdussalam

    2013-01-01

    The interaction of proteins with biological membranes is a key factor in their biogenesis and proper function. Hence, unraveling the properties of this interaction is very important and constitutes an essential step in deciphering the structural and functional characteristics of a membrane protein. Here we describe the use of cardiolipin-containing liposomes to analyze the interaction of the import protein Tim44 with the inner mitochondrial membrane. Using this system we showed that Tim44 is peripherally attached to the membrane and we detected the membrane binding site of the protein. The cardiolipin-containing liposomes serve as an excellent in vitro model system to the inner mitochondrial membrane and thus provide a good tool to analyze the interaction of various mitochondrial proteins with the inner membrane. PMID:23996176

  8. Steady-state coupling of four membrane systems in mitochondrial oxidative phosphorylation.

    PubMed

    Hill, T L

    1979-05-01

    According to Alexandre, Reynafarje, and Lehninger, four different membrane systems are involved, with definite stoichiometry, in the mitochondrial synthesis of ATP by electron transport, via proton transport. We adopt this model and pursue some of its thermodynamic consequences. At steady state, each of the four systems must have the same flux J through the membrane and the overall thermodynamic force X for oxidative phosphorylation is the sum of the four separate forces. From these properties, using an empirical linear flux-force relation for each system, it is easy to obtain J as a function of X. In turn, X depends on the inside [NAD+]/[NADH] and the outside [ATP]/[ADP][Pi] quotients (and on the pH inside). Thus, J is related to these quotients. The relationship we derive is similar to that described by Erecińska and Wilson, as deduced from a quite different model of oxidative phosphorylation. Proton transport is involved explicitly in three of the four systems of the present model. However, because of the steady-state stoichiometric coupling of the four systems, proton transport does not appear in the overall reaction. On the other hand, Erecińska and Wilson use, in their model, a direct connection between electron transport and ATP synthesis. The present paper demonstrates that J can be related to the quotients mentioned above without this direct connection. PMID:287064

  9. Multistep assembly of the protein import channel of the mitochondrial outer membrane.

    PubMed

    Model, K; Meisinger, C; Prinz, T; Wiedemann, N; Truscott, K N; Pfanner, N; Ryan, M T

    2001-04-01

    Proteins targeted to mitochondria are transported into the organelle through a high molecular weight complex called the translocase of the outer mitochondrial membrane (TOM). At the core of this machinery is a multisubunit general import pore (GIP) of 400 kDa. Here we report the assembly of the yeast GIP that involves two successive intermediates of 250 kDa and 100 kDa. The precursor of the channel-lining Tom40 is first targeted to the membrane via the receptor proteins Tom20 and Tom22; it then assembles with Tom5 to form the 250 kDa intermediate exposed to the intermembrane space. The 250 kDa intermediate is followed by the formation of the 100 kDa intermediate that associates with Tom6. Maturation to the 400 kDa complex occurs by association of Tom7 and Tom22. Tom7 functions by promoting both the dissociation of the 400 kDa complex and the transition from the 100 kDa intermediate to the mature complex. These results indicate that the dynamic conversion between the 400 kDa complex and the 100 kDa late intermediate allows integration of new precursor subunits into pre-existing complexes. PMID:11276259

  10. Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane.

    PubMed

    Diekert, K; de Kroon, A I; Ahting, U; Niggemeyer, B; Neupert, W; de Kruijff, B; Lill, R

    2001-10-15

    The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress. PMID:11598006

  11. Triphenylmethylphosphonium cation distribution as a measure of hormone-induced alterations in white adipocyte membrane potential

    SciTech Connect

    Vallano, M.L.; Sonenberg, M.

    1982-01-01

    Triphenylmethylphosphonium (TPMP+) partitions into the mitochondrial and cytosolic compartments in the rat white adipocyte in a potential-dependent fashion. The relationship between (/sup 3/H)TPMP+ distribution, intracellular cAMP generation and lipolysis in response to hormones and cAMP-mimetic compounds was examined. Half-maximal (/sup 3/H)TPMP+ efflux and glycerol release were produced by 15 and 9 nM adrenocorticotropin, 170 and 110 nM 1-epinephrine, 70 and 27 microM isobutylmethylxanthine and 800 and 750 microM dibutyryl cAMP, respectively. Hormone-stimulated cAMP generation was also correlated with (/sup 3/H)TPMP+ efflux and lipolysis in terms of concentration dependency. In kinetic experiments, glycerol release and (/sup 3/H)TPMP+ efflux in response to adrenocorticotropin or cholera toxin proceeded over a similar time course, whereas an earlier rise in cAMP generation was detected. The depolarizing effect of lipolytic compounds was localized to the mitochondrial compartment. When cells were incubated in elevated-(K+)0 buffer, the stimulatory effect of dibutyryl cAMP on (/sup 3/H)TPMP+ efflux and lipolysis persisted, suggesting that maintenance of the plasma membrane potential is not critical for demonstration of these responses. When the extracellular concentration of serum albumin, which provides binding sites for free fatty acids, was increased from 1 to 3%, an increase in glycerol release and a decrease in (/sup 3/H)TPMP+ efflux was observed. We suggest that intracellular free fatty acid accumulation in response to lipolytic agents causes dissipation of the mitochondrial membrane potential and efflux of (/sup 3/H)TPMP+ from the organelle and cell.

  12. Sodium and potassium conductance changes during a membrane action potential.

    PubMed

    Bezanilla, F; Rojas, E; Taylor, R E

    1970-12-01

    1. A method for turning a membrane potential control system on and off in less than 10 musec is described. This method was used to record membrane currents in perfused giant axons from Dosidicus gigas and Loligo forbesi after turning on the voltage clamp system at various times during the course of a membrane action potential.2. The membrane current measured just after the capacity charging transient was found to have an almost linear relation to the controlled membrane potential.3. The total membrane conductance taken from these current-voltage curves was found to have a time course during the action potential similar to that found by Cole & Curtis (1939).4. The instantaneous current voltage curves were linear enough to make it possible to obtain a good estimate of the individual sodium and potassium channel conductances, either algebraically or by clamping to the sodium, or potassium, reversal potentials. Good general agreement was obtained with the predictions of the Hodgkin-Huxley equations.5. We consider these results to constitute the first direct experimental demonstration of the conductance changes to sodium and potassium during the course of an action potential. PMID:5505231

  13. Potential efficacy of mitochondrial genes for animal DNA barcoding: a case study using eutherian mammals

    PubMed Central

    2011-01-01

    Background A well-informed choice of genetic locus is central to the efficacy of DNA barcoding. Current DNA barcoding in animals involves the use of the 5' half of the mitochondrial cytochrome oxidase 1 gene (CO1) to diagnose and delimit species. However, there is no compelling a priori reason for the exclusive focus on this region, and it has been shown that it performs poorly for certain animal groups. To explore alternative mitochondrial barcoding regions, we compared the efficacy of the universal CO1 barcoding region with the other mitochondrial protein-coding genes in eutherian mammals. Four criteria were used for this comparison: the number of recovered species, sequence variability within and between species, resolution to taxonomic levels above that of species, and the degree of mutational saturation. Results Based on 1,179 mitochondrial genomes of eutherians, we found that the universal CO1 barcoding region is a good representative of mitochondrial genes as a whole because the high species-recovery rate (> 90%) was similar to that of other mitochondrial genes, and there were no significant differences in intra- or interspecific variability among genes. However, an overlap between intra- and interspecific variability was still problematic for all mitochondrial genes. Our results also demonstrated that any choice of mitochondrial gene for DNA barcoding failed to offer significant resolution at higher taxonomic levels. Conclusions We suggest that the CO1 barcoding region, the universal DNA barcode, is preferred among the mitochondrial protein-coding genes as a molecular diagnostic at least for eutherian species identification. Nevertheless, DNA barcoding with this marker may still be problematic for certain eutherian taxa and our approach can be used to test potential barcoding loci for such groups. PMID:21276253

  14. Human mitochondrial DNA nucleoids are linked to protein folding machinery and metabolic enzymes at the mitochondrial inner membrane.

    PubMed

    Wang, Yousong; Bogenhagen, Daniel F

    2006-09-01

    Mitochondrial DNA (mtDNA) is packaged into bacterial nucleoid-like structures, each containing several mtDNA molecules. The distribution of nucleoids during mitochondrial fission and fusion events and during cytokinesis is important to the segregation of mitochondrial genomes in heteroplasmic cells bearing a mixture of wild-type and mutant mtDNA molecules. We report fractionation of HeLa cell mtDNA nucleoids into two subsets of complexes that differ in their sedimentation velocity and their association with cytoskeletal proteins. Pulse labeling studies indicated that newly replicated mtDNA molecules are evenly represented in the rapidly and slowly sedimenting fractions. Slowly sedimenting nucleoids were immunoaffinity purified using antibodies to either of two abundant mtDNA-binding proteins, TFAM or mtSSB. These two different immunoaffinity procedures yielded very similar sets of proteins, with 21 proteins in common, including most of the proteins previously shown to play roles in mtDNA replication and transcription. In addition to previously identified mitochondrial proteins, multiple peptides were observed for one novel DNA metabolic protein, the DEAH-box helicase DHX30. Antibodies raised against a recombinant fragment of this protein confirmed the mitochondrial localization of a specific isoform of DHX30. PMID:16825194

  15. Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

    PubMed Central

    Alfahel, E; Korngreen, A; Parola, A H; Priel, Z

    1996-01-01

    To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways. PMID:8789123

  16. Membrane potential shapes regulation of dopamine transporter trafficking at the plasma membrane

    PubMed Central

    Richardson, Ben D.; Saha, Kaustuv; Krout, Danielle; Cabrera, Elizabeth; Felts, Bruce; Henry, L. Keith; Swant, Jarod; Zou, Mu-Fa; Newman, Amy Hauck; Khoshbouei, Habibeh

    2016-01-01

    The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels. PMID:26804245

  17. Inhibition of mitochondrial respiration by nitric oxide is independent of membrane fluidity modulation or oxidation of sulfhydryl groups.

    PubMed

    Pérez-Rojas, Jazmin M; Muriel, Pablo

    2005-01-01

    Nitric oxide (NO) modulates the fluidity of a variety of membranes. Thus, the aim of the present work was to study if the inhibitory effect of NO on mitochondrial respiration is associated with its effects on membrane fluidity. Liver mitochondria and an inner mitochondrial membrane fraction (IMMF) were isolated from male Wistar rats by differential centrifugation. Oxygen consumption was measured polarographically and fluidity by the fluorescence polarization method. S-nitroso-N-acetylpenicillamine (SNAP) was used as a NO donor. It was observed that NO decreased IMMF fluidity and oxygen consumption in a concentration dependent fashion. However, SAM a fluidizing agent that prevented the decrement in fluidity produced by SNAP, failed to preserve oxygen consumption. Protection of sulfhydryl groups with dithiotreitol was utilized to evaluate the role of oxidation of these groups on IMMF respiration. Incubation with dithiotreitol did not preserve IMMF oxygen consumption. The data shown herein suggest that NO inhibits the respiratory chain by a mechanism not involving the modulation of membrane fluidity or the oxidation of sulfhydryl groups. Thus, it seems that the mechanism by which NO modulates mitochondrial respiration is by cytochrome oxidase inhibition, because (as reported by others) low concentrations of NO specifically inhibit reversibly cytochrome oxidase in competition with oxygen. PMID:16167323

  18. Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    PubMed Central

    GAO, SUJIE; CHEN, XUEBO; JIN, HONGYONG; REN, SHENGNAN; LIU, ZHUO; FANG, XUEDONG; ZHANG, GUIZHEN

    2016-01-01

    ErbB2 is known to upregulate glycolysis in breast cancer, however, the precise mechanisms remain unclear. In the present study, ErbB2 upregulated Hexokinase II (HK II) activity by increasing the binding of HK II to the mitochondrial outer membrane. Dysregulated glucose metabolism in high ErbB2-expressing breast cancer cells induces susceptibility to glucose starvation and glycolysis inhibition. Additionally, HK II has a tendency to dissociate from the mitochondria outer membrane in ErbB2-overexpressing cells following treatment with the HK II inhibitor, 3-BrPA. Furthermore, 3-BrPA treatment results in decreased mitochondria membrane potential and release of cytochrome c into cytoplasm in ErbB2-overexpressing cells, leading to activation of the mitochondrial apoptotic signaling pathway. In summary, the results demonstrate a novel mechanism for ErbB2-activated glycolysis and reveal that 3-BrPA is effective in reducing ErbB2-positive breast cancer cell viability by targeting HK II in vitro and in vivo. PMID:26893781

  19. Direct measurements of membrane potential and membrane resistance of human red cells

    PubMed Central

    Lassen, U. V.; Sten-Knudsen, O.

    1968-01-01

    1. In order to evaluate the membrane potentials calculated from the distribution of chloride ions in human red cells and plasma, it is desirable to have a direct measurement of the transmembrane potential of these cells. 2. A method has been devised for introducing a capillary micro-electrode into human red cells. The method allows simultaneous measurements of potential and membrane resistance with only one micro-electrode located in the cell. 3. Upon impalement of single cells in plasma, a scatter of membrane potentials and of resistance values was obtained. The potential drop never exceeded -14 mV and the maximum resistances were about 7 Ω. cm2. Positive potentials were obtained on impalement of red cell aggregates. 4. Arguments are given to support the view that it is in these cells which suffer least damage from the impalement that maximum values of membrane potentials and resistances are observed. The errors caused by the change in the liquid junction during the impalement have been estimated. 5. As judged from this study, it seems permissible under normal conditions to calculate the membrane potential of the red cell from the chloride concentrations in plasma and in intracellular water. PMID:5649641

  20. Toward Better Genetically Encoded Sensors of Membrane Potential.

    PubMed

    Storace, Douglas; Sepehri Rad, Masoud; Kang, BokEum; Cohen, Lawrence B; Hughes, Thom; Baker, Bradley J

    2016-05-01

    Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions. Optical signals of membrane potential are useful because membrane potential changes are a direct sign of both synaptic and action potentials. Here we describe recent improvements in the in vitro and in vivo signal size and kinetics of genetically encoded voltage indicators (GEVIs) and discuss their relationship to alternative sensors of neural activity. PMID:27130905

  1. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes.

    PubMed

    Magrì, Andrea; Di Rosa, Maria Carmela; Tomasello, Marianna Flora; Guarino, Francesca; Reina, Simona; Messina, Angela; De Pinto, Vito

    2016-06-01

    Cu/Zn Superoxide Dismutase (SOD1), the most important antioxidant defense against ROS in eukaryotic cells, localizes in cytosol and intermembrane space of mitochondria (IMS). Several evidences show a SOD1 intersection with both fermentative and respiratory metabolism. The Voltage Dependent Anion Channel (VDAC) is the main pore-forming protein in the mitochondrial outer membrane (MOM), and is considered the gatekeeper of mitochondrial metabolism. Saccharomyces cerevisiae lacking VDAC1 (Δpor1) is a very convenient model system, since it shows an impaired growth rate on non-fermentable carbon source. Transformation of Δpor1 yeast with human SOD1 completely restores the cell growth deficit in non-fermentative conditions and re-establishes the physiological levels of ROS, as well as the mitochondrial membrane potential. No similar result was found upon yeast SOD1 overexpression. A previous report highlighted the action of SOD1 as a transcription factor. Quantitative Real-Time PCR showed that β-barrel outer-membrane encoding-genes por2, tom40, sam50 are induced by hSOD1, but the same effect was not obtained in Δpor1Δpor2 yeast, indicating a crucial function for yVDAC2. Since the lack of VDAC1 in yeast can be considered a stress factor for the cell, hSOD1 could relieve it stimulating the expression of genes bringing to the recovery of the MOM function. Our results suggest a direct influence of SOD1 on VDAC. PMID:26947057

  2. The clinical maze of mitochondrial neurology

    PubMed Central

    DiMauro, Salvatore; Schon, Eric A.; Carelli, Valerio; Hirano, Michio

    2014-01-01

    Mitochondrial diseases involve the respiratory chain, which is under the dual control of nuclear and mitochondrial DNA (mtDNA). The complexity of mitochondrial genetics provides one explanation for the clinical heterogeneity of mitochondrial diseases, but our understanding of disease pathogenesis remains limited. Classification of Mendelian mitochondrial encephalomyopathies has been laborious, but whole-exome sequencing studies have revealed unexpected molecular aetiologies for both typical and atypical mitochondrial disease phenotypes. Mendelian mitochondrial defects can affect five components of mitochondrial biology: subunits of respiratory chain complexes (direct hits); mitochondrial assembly proteins; mtDNA translation; phospholipid composition of the inner mitochondrial membrane; or mitochondrial dynamics. A sixth category—defects of mtDNA maintenance—combines features of Mendelian and mitochondrial genetics. Genetic defects in mitochondrial dynamics are especially important in neurology as they cause optic atrophy, hereditary spastic paraplegia, and Charcot–Marie–Tooth disease. Therapy is inadequate and mostly palliative, but promising new avenues are being identified. Here, we review current knowledge on the genetics and pathogenesis of the six categories of mitochondrial disorders outlined above, focusing on their salient clinical manifestations and highlighting novel clinical entities. An outline of diagnostic clues for the various forms of mitochondrial disease, as well as potential therapeutic strategies, is also discussed. PMID:23835535

  3. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    SciTech Connect

    Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N.

    1995-11-01

    Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display and early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90{degrees} light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly, a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. 33 refs., 5 figs., 1 tab.

  4. Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

    PubMed

    Mikkelsen, R B; Wallach, D F

    1976-05-21

    (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone. PMID:6061

  5. Descending Vasa Recta Endothelial Membrane Potential Response Requires Pericyte Communication

    PubMed Central

    Zhang, Zhong; Payne, Kristie; Pallone, Thomas L.

    2016-01-01

    Using dual-cell electrophysiological recording, we examined the routes for equilibration of membrane potential between the pericytes and endothelia that comprise the descending vasa recta (DVR) wall. We measured equilibration between pericytes in intact vessels, between pericytes and endothelium in intact vessels and between pericytes physically separated from the endothelium. Dual pericyte recording on the abluminal surface of DVR showed that both resting potential and subsequent time-dependent voltage fluctuations after vasoconstrictor stimulation remained closely equilibrated, regardless of the agonist employed (angiotensin II, vasopressin or endothelin 1). When pericytes where removed from the vessel wall but retained physical contact with one another, membrane potential responses were also highly coordinated. In contrast, responses of pericytes varied independently when they were isolated from both the endothelium and from contact with one another. When pericytes and endothelium were in contact, their resting potentials were similar and their temporal responses to stimulation were highly coordinated. After completely isolating pericytes from the endothelium, their mean resting potentials became discordant. Finally, complete endothelial isolation eliminated all membrane potential responses to angiotensin II. We conclude that cell-to-cell transmission through the endothelium is not needed for pericytes to equilibrate their membrane potentials. AngII dependent responses of DVR endothelia may originate from gap junction coupling to pericytes rather than via receptor dependent signaling in the endothelium, per se. PMID:27171211

  6. Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.

    PubMed

    Sinzel, Monika; Tan, Tao; Wendling, Philipp; Kalbacher, Hubert; Özbalci, Cagakan; Chelius, Xenia; Westermann, Benedikt; Brügger, Britta; Rapaport, Doron; Dimmer, Kai Stefan

    2016-07-01

    Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER-mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES-related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane. PMID:27226123

  7. Helicobacter pylori VacA Toxin/Subunit p34: Targeting of an Anion Channel to the Inner Mitochondrial Membrane

    PubMed Central

    Harsman, Anke; Papatheodorou, Panagiotis; Reljic, Boris; Dian-Lothrop, Elke A.; Galmiche, Antoine; Kepp, Oliver; Becker, Lars; Günnewig, Kathrin; Wagner, Richard; Rassow, Joachim

    2010-01-01

    The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% β-strands, similar to pore-forming β-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal. PMID:20442789

  8. Inhibition of mitochondrial permeability transition by low pH is associated with less extensive membrane protein thiol oxidation.

    PubMed

    Teixeira, B M; Kowaltowski, A J; Castilho, R F; Vercesi, A E

    1999-12-01

    Ca2+ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H2O2 production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca2+ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues. PMID:10841269

  9. Genome-Wide Screens in Saccharomyces cerevisiae Highlight a Role for Cardiolipin in Biogenesis of Mitochondrial Outer Membrane Multispan Proteins

    PubMed Central

    Sauerwald, Julia; Jores, Tobias; Eisenberg-Bord, Michal; Chuartzman, Silvia Gabriela

    2015-01-01

    A special group of mitochondrial outer membrane (MOM) proteins spans the membrane several times via multiple helical segments. Such multispan proteins are synthesized on cytosolic ribosomes before their targeting to mitochondria and insertion into the MOM. Previous work recognized the import receptor Tom70 and the mitochondrial import (MIM) complex, both residents of the MOM, as required for optimal biogenesis of these proteins. However, their involvement is not sufficient to explain either the entire import pathway or its regulation. To identify additional factors that are involved in the biogenesis of MOM multispan proteins, we performed complementary high-throughput visual and growth screens in Saccharomyces cerevisiae. Cardiolipin (CL) synthase (Crd1) appeared as a candidate in both screens. Our results indeed demonstrate lower steady-state levels of the multispan proteins Ugo1, Scm4, and Om14 in mitochondria from crd1Δ cells. Importantly, MOM single-span proteins were not affected by this mutation. Furthermore, organelles lacking Crd1 had a lower in vitro capacity to import newly synthesized Ugo1 and Scm4 molecules. Crd1, which is located in the mitochondrial inner membrane, condenses phosphatidylglycerol together with CDP-diacylglycerol to obtain de novo synthesized CL molecules. Hence, our findings suggest that CL is an important component in the biogenesis of MOM multispan proteins. PMID:26149385

  10. Homodimeric Intrinsic Membrane Proteins. Identification and Modulation of Interactions between Mitochondrial Transporter (Carrier) Subunits

    PubMed Central

    Wohlrab, Hartmut

    2010-01-01

    Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic pathways within the matrix and the cytosol with transport/exchange of metabolites and inorganic ions. Their strict control of these fluxes is required for oxidative phosphorylation. Understanding the ternary complex transport mechanism with which most of these transporters function requires an accounting of the number and interactions of their subunits. The phosphate transporter (PTP, Mir1p) subunit readily forms homodimers with intersubunit affinities changeable by mutations. Cys28, likely at the subunit interface, is a site for mutations yielding transport inhibition or a channel-like transport mode. Such mutations yield a small increase or decrease in affinity between the subunits. The PTP inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP mutations that yield the highest (40%) and the lowest (2%) liposome incorporation efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p) subunit has an almost 2-fold lower affinity than the PTP subunit. The Oac1p, dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even lower affinities. These results form a firm basis for detailed studies to establish the effect of subunit affinities on transport mode and activity and for the identification of the mechanism that prevents formation of heterodimers that surely will negatively impact oxidative phosphorylation and ATP levels with serious consequences for the cell. PMID:20171189

  11. SOM 1, a small new gene required for mitochondrial inner membrane peptidase function in Saccharomyces cerevisiae.

    PubMed

    Esser, K; Pratje, E; Michaelis, G

    1996-09-25

    IMP1 encodes a subunit of the mitochondrial inner membrane peptidase responsible for the proteolytic processing of cytochrome oxidase subunit 2 (Cox2) and cytochrome b2 (Cytb2). The molecular defect in an imp1 mutation and the characterisation of a high-copy-number suppressor is described. A deletion of the suppressor region causes respiration deficiency. The DNA sequence revealed three very small overlapping ORFs. Constructs which carried termination codons within the ORFs or lacked ATG initiation codons still retained complementing activity on a high-copy-number plasmid. Nevertheless, the possibility that the suppressor acts at DNA or RNA level could be excluded. Subcloning of the ORFs, complementation analysis in low-copy-number plasmids and transcript mapping identified the 222 bp ORF as the suppressor gene designated SOM1. The SOM1 gene is transcribed into a 375 bp polyadenylated RNA and the deduced amino acid sequence predicts a small protein of 8.4 kDa with no significant sequence similarity to known proteins. In the som1 deletion mutant, proteolytic processing of the Cox2 precursor is prevented and Cytb2 is strongly reduced. SOM1 represents a new small gene which encodes a novel factor that is essential for the correct function of the Imp1 peptidase and/or the protein sorting machinery. PMID:8879245

  12. GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

    PubMed

    Shiromoto, Yusuke; Kuramochi-Miyagawa, Satomi; Daiba, Akito; Chuma, Shinichiro; Katanaya, Ami; Katsumata, Akiko; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakamura, Toshinobu; Yoshinaga, Koichi; Asada, Noriko; Nakamura, Shota; Yasunaga, Teruo; Kojima-Kita, Kanako; Itou, Daisuke; Kimura, Tohru; Nakano, Toru

    2013-06-01

    piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis. PMID:23611983

  13. A Potential Role for Bat Tail Membranes in Flight Control

    PubMed Central

    Gardiner, James D.; Dimitriadis, Grigorios; Codd, Jonathan R.; Nudds, Robert L.

    2011-01-01

    Wind tunnel tests conducted on a model based on the long-eared bat Plecotus auritus indicated that the positioning of the tail membrane (uropatagium) can significantly influence flight control. Adjusting tail position by increasing the angle of the legs ventrally relative to the body has a two-fold effect; increasing leg-induced wing camber (i.e., locally increased camber of the inner wing surface) and increasing the angle of attack of the tail membrane. We also used our model to examine the effects of flying with and without a tail membrane. For the bat model with a tail membrane increasing leg angle increased the lift, drag and pitching moment (nose-down) produced. However, removing the tail membrane significantly reduced the change in pitching moment with increasing leg angle, but it had no significant effect on the level of lift produced. The drag on the model also significantly increased with the removal of the tail membrane. The tail membrane, therefore, is potentially important for controlling the level of pitching moment produced by bats and an aid to flight control, specifically improving agility and manoeuvrability. Although the tail of bats is different from that of birds, in that it is only divided from the wings by the legs, it nonetheless, may, in addition to its prey capturing function, fulfil a similar role in aiding flight control. PMID:21479137

  14. Mitochondrial Ca2+ uptake from plasma membrane Cav3.2 protein channels contributes to ischemic toxicity in PC12 cells.

    PubMed

    Gouriou, Yves; Bijlenga, Philippe; Demaurex, Nicolas

    2013-05-01

    T-type Ca(2+) channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca(2+) elevations during anoxia. To test this hypothesis, we measured the Ca(2+) concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca(2+) elevations that were reduced by Cav3.2 inhibition (50 μm Ni(2+)) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca(2+) elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni(2+) and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni(2+) and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca(2+) load actively extruded by plasma membrane Ca(2+) pumps. The cytosolic Ca(2+) elevations were paralleled by mitochondrial Ca(2+) elevations that were also increased by Cav3.2 overexpression and decreased by Ni(2+) but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca(2+) uniporter, the major mitochondrial Ca(2+) uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca(2+) elevations. Selective activation of T-type Ca(2+) channels evoked both cytosolic and mitochondrial Ca(2+) elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca(2+) ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca(2+) overload. PMID:23508951

  15. Mitochondrial Ca2+ Uptake from Plasma Membrane Cav3.2 Protein Channels Contributes to Ischemic Toxicity in PC12 Cells*

    PubMed Central

    Gouriou, Yves; Bijlenga, Philippe; Demaurex, Nicolas

    2013-01-01

    T-type Ca2+ channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca2+ elevations during anoxia. To test this hypothesis, we measured the Ca2+ concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca2+ elevations that were reduced by Cav3.2 inhibition (50 μm Ni2+) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca2+ elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni2+ and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni2+ and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca2+ load actively extruded by plasma membrane Ca2+ pumps. The cytosolic Ca2+ elevations were paralleled by mitochondrial Ca2+ elevations that were also increased by Cav3.2 overexpression and decreased by Ni2+ but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca2+ uniporter, the major mitochondrial Ca2+ uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca2+ elevations. Selective activation of T-type Ca2+ channels evoked both cytosolic and mitochondrial Ca2+ elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca2+ ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca2+ overload. PMID:23508951

  16. Membrane, action, and oscillatory potentials in simulated protocells.

    PubMed

    Przybylski, A T; Stratten, W P; Syren, R M; Fox, S W

    1982-12-01

    Electrical membrane potentials, oscillations, and action potentials are observed in proteinoid microspheres impaled with (3 M KC1) microelectrodes. Although effects are of greater magnitude when the vesicles contain glycerol and natural or synthetic lecithin, the results in the purely synthetic thermal protein structures are substantial, attaining 20 mV amplitude in some cases. The results add the property of electrical potential to the other known properties of proteinoid microspheres, in their role as models for protocells. PMID:7162535

  17. Membrane, action, and oscillatory potentials in simulated protocells

    NASA Astrophysics Data System (ADS)

    Przybylski, Aleksander T.; Stratten, Wilford P.; Syren, Robert M.; Fox, Sidney W.

    1982-12-01

    Electrical membrane potentials, oscillations, and action potentials are observed in proteinoid microspheres impaled with (3 M KCl) microelectrodes. Although effects are of greater magnitude when the vesicles contain glycerol and natural or synthetic lecithin, the results in the purely synthetic thermal protein structures are substantial, attaining 20 mV amplitude in some cases. The results add the property of electrical potential to the other known properties of proteinoid microspheres, in their role as models for protocells.

  18. Membrane, action, and oscillatory potentials in simulated protocells

    NASA Technical Reports Server (NTRS)

    Syren, R. M.; Fox, S. W.; Przybylski, A. T.; Stratten, W. P.

    1982-01-01

    Electrical membrane potentials, oscillations, and action potentials are observed in proteinoid microspheres impaled with (3 M KCl) microelectrodes. Although effects are of greater magnitude when the vesicles contain glycerol and natural or synthetic lecithin, the results in the purely synthetic thermal protein structures are substantial, attaining 20 mV amplitude in some cases. The results add the property of electrical potential to the other known properties of proteinoid microspheres, in their role as models for protocells.

  19. Ion separations based on electrical potentials nanoporous and microporous membranes

    NASA Astrophysics Data System (ADS)

    Armstrong, Jason

    This dissertation examines several types of ion separations in nanometer to micrometer pores in membranes. Membranes provide an attractive platform for ion separations, primarily because they operate continuously (i.e. not in a batch mode), and small pores offer the potential for ion separation based on charge and electrophoretic mobility differences. Initial studies employed charged, nanoporous membranes to separate monovalent and divalent ions. Adsorption of polyelectrolyte multilayers in nanoporous membranes afforded control over the surface charge and pore radii in track-etched membranes, and electrostatic ion-exclusion, particularly for divalent ions, occurred in these membranes because the electrical double layer filled the entire nanopore. Initial experiments employed adsorption of (PSS/PAH) multilayers in the 50-nm diameter pores of PCTE membranes to give a K+/Mg2+ selectivity of ~10 in pressure-driven dead-end filtration. Adsorption of (PSS/PAH) 1 films in 30-nm pores gave a similar K+/Mg2+ selectivity with a simpler modification procedure. Separations utilizing (PSS/PAH)1 films in 30-nm pores showed the lowest ion rejections with high ion concentrations, consistent with enhanced screening of the electrical double layer at high ionic strength. However, solutions with < 5 mM ionic strength exhibited essentially 100% Mg2+ rejections (the Mg2+ concentration in the permeate was below the method detection limit). Moreover, K+ rejections increased in the presence of Mg2+, which may stem from Mg2+-adsorption within the PEM and increased surface charge. Finally, separation of Br- and SO42- with a PSS1-modified, 30-nm PCTE membrane validated the exclusion mechanism for anions. The average Br-/SO42- selectivity was 3.4 +/- 0.8 for a solution containing 0.5 mM NaBr and 0.5 mM Na2SO4. The low selectivity in this case likely stems from a relatively large pore. The membranes used for the separation of monovalent and divalent ions also facilitated separation of

  20. Targeting and assembly of rat mitochondrial translocase of outer membrane 22 (TOM22) into the TOM complex.

    PubMed

    Nakamura, Yasuhiko; Suzuki, Hiroyuki; Sakaguchi, Masao; Mihara, Katsuyoshi

    2004-05-14

    Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic alpha-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20. PMID:14985332

  1. Characterization of a Leishmania Stage Specific Mitochondrial Membrane Protein that Enhances the Activity of Cytochrome C Oxidase and Its Role in Virulence

    PubMed Central

    Dey, Ranadhir; Meneses, Claudio; Salotra, Poonam; Kamhawi, Shaden; Nakhasi, Hira L.; Duncan, Robert

    2010-01-01

    Summary Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27kDa protein encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-imunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene deleted parasites (Ldp27−/−) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27−/− parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. PMID:20497506

  2. Structural and functional changes in gill mitochondrial membranes from the Mediterranean mussel Mytilus galloprovincialis exposed to tri-n-butyltin.

    PubMed

    Fiorini, Rosamaria; Pagliarani, Alessandra; Nesci, Salvatore; Pirini, Maurizio; Tucci, Elisabetta; Ventrella, Vittoria

    2012-04-01

    The use of tributyltin (TBT) as a biocide in antifouling paints leads to a ruinous input of this contaminant in the aquatic environment. Human exposure to TBT mainly occurs through ingestion of contaminated seafood such as filter-feeding mollusks. Tributyltin is known to act as a membrane-active toxicant on several targets, but especially on the mitochondria, and by several mechanisms. The effects of tributyltin on fatty acid composition, on Mg-adenosine triphosphatase (ATPase) activities, and on the membrane physical state were investigated in gill mitochondrial membranes from cultivated mussels Mytilus galloprovincialis exposed to 0.5 µg/L and 1.0 µg/L TBT and unexposed for 120 h. The higher TBT exposure dose induced a decrease in the total and n-3 polyunsaturated fatty acids (PUFAs), especially 22:6 n-3, and an activation of the oligomycin-sensitive Mg-ATPase. Both TBT concentrations decreased mitochondrial membrane polarity detected by Laurdan steady-state fluorescence spectroscopy. These findings may help cast light on the multiple modes of action of this toxicant. PMID:22374617

  3. Identification of mammalian TOM22 as a subunit of the preprotein translocase of the mitochondrial outer membrane.

    PubMed

    Saeki, K; Suzuki, H; Tsuneoka, M; Maeda, M; Iwamoto, R; Hasuwa, H; Shida, S; Takahashi, T; Sakaguchi, M; Endo, T; Miura, Y; Mekada, E; Mihara, K

    2000-10-13

    A mitochondrial outer membrane protein of approximately 22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19-20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the approximately 400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in Deltatom22 yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex. PMID:10900208

  4. Potential impact of human mitochondrial replacement on global policy regarding germline gene modification.

    PubMed

    Ishii, Tetsuya

    2014-08-01

    Previous discussions regarding human germline gene modification led to a global consensus that no germline should undergo genetic modification. However, the UK Human Fertilisation and Embryology Authority, having conducted at the UK Government's request a scientific review and a wide public consultation, provided advice to the Government on the pros and cons of Parliament's lifting a ban on altering mitochondrial DNA content of human oocytes and embryos, so as to permit the prevention of maternal transmission of mitochondrial diseases. In this commentary, relevant ethical and biomedical issues are examined and requirements for proceeding with this novel procedure are suggested. Additionally, potentially significant impacts of the UK legalization on global policy concerning germline gene modification are discussed in the context of recent advances in genome-editing technology. It is concluded that international harmonization is needed, as well as further ethical and practical consideration, prior to the legalization of human mitochondrial replacement. PMID:24832374

  5. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    PubMed

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic

  6. MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on opposite membrane surfaces in mitochondrial inner membrane of Neurospora crassa.

    PubMed

    Klanner, C; Prokisch, H; Langer, T

    2001-09-01

    Eukaryotic AAA proteases form a conserved family of membrane-embedded ATP-dependent proteases but have been analyzed functionally only in the yeast Saccharomyces cerevisiae. Here, we have identified two novel members of this protein family in the filamentous fungus Neurospora crassa, which were termed MAP-1 and IAP-1. Both proteins are localized to the inner membrane of mitochondria. They are part of two similar-sized high molecular mass complexes, but expose their catalytic sites to opposite membrane surfaces, namely, the intermembrane and the matrix space. Disruption of iap-1 by repeat-induced point mutation caused a slow growth phenotype at high temperature and stabilization of a misfolded inner membrane protein against degradation. IAP-1 could partially substitute for functions of its yeast homolog Yme1, demonstrating functional conservation. However, respiratory growth at 37 degrees C was not restored. Our results identify two components of the quality control system of the mitochondrial inner membrane in N. crassa and suggest that AAA proteases with catalytic sites exposed to opposite membrane surfaces are present in mitochondria of all eukaryotic cells. PMID:11553723

  7. Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex.

    PubMed Central

    Kassenbrock, C K; Cao, W; Douglas, M G

    1993-01-01

    To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p. Images PMID:8344244

  8. Endophilin B2 promotes inner mitochondrial membrane degradation by forming heterodimers with Endophilin B1 during mitophagy

    PubMed Central

    Wang, Yi-Han; Wang, Jiu-Qiang; Wang, Qiaochu; Wang, Yun; Guo, Caixia; Chen, Quan; Chai, Tuanyao; Tang, Tie-Shan

    2016-01-01

    Mitochondrial sequestration by autophagosomes is a key step in mitophagy while the mechanisms mediating this process are not fully understood. It has been reported that Endophilin B1 (EB1) promotes mitochondrial sequestration by binding and shaping membrane. However, the role of EB1 homolog Endophilin B2 (EB2) in mitophagy remains unclear. Here we report that EB2 plays an indispensable role in mitochondria sequestration and inner mitochondrial membrane (IMM) protein degradation during mitophagy. Similar to EB1, EB2 aggregates into foci and then translocates to damaged mitochondria. Loss of either EB2 and/or EB1 significantly enervates the foci translocation to fragmented mitochondria and IMM degradation, and the EB1/EB2 heterodimer formed by EB1/EB2 interaction promotes the above process. We noticed that, it is the dimer domain of EB2 but not that of EB1 mediating the heterodimer formation, manifesting the importance of EB2 in mitophagy. Furthermore, we demonstrate that the EB foci formation is closely regulated by the PINK1-Parkin signaling pathway. From these results, we propose that EB1/EB2 heterodimers may serve as linkers between damaged mitochondria and phagophores during mitophagy. PMID:27112121

  9. Stability of Mitochondrial Membrane Proteins in Terrestrial Vertebrates Predicts Aerobic Capacity and Longevity

    PubMed Central

    Kitazoe, Yasuhiro; Kishino, Hirohisa; Hasegawa, Masami; Matsui, Atsushi; Lane, Nick; Tanaka, Masashi

    2011-01-01

    The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5–10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs. PMID:21824868

  10. Zero-current potentials in a large membrane channel: a simple theory accounts for complex behavior.

    PubMed Central

    Zambrowicz, E B; Colombini, M

    1993-01-01

    Flow of ions through large channels is complex because both cations and anions can penetrate and multiple ions can be in the channel at the same time. A modification of the fixed-charge membrane theory of Teorell was reported (Peng, S., E. Blachly-Dyson, M. Forte, and M. Colombini. 1992. Biophys. J. 62:123-135) in which the channel is divided into two compartments: a relatively charged cylindrical shell of solution adjacent to the wall of the pore and a relatively neutral central cylinder of solution. The zero-current (reversal) potential results in current flow in opposite directions in these two compartments. This description accounted rather well for the observed reversal potential changes following site-directed mutations. Here we report the results of systematic tests of this simple theory with the mitochondrial channel, VDAC (isolated from Neurospora crassa), reconstituted into planar phospholipid membranes. The variation of the observed reversal potential with transmembrane activity ratio, ionic strength, ion mobility ratio, and net charge on the wall of the pore are accounted for reasonably well. The Goldman-Hodgkin-Katz theory fails to account for the observations. PMID:7694668

  11. Assembly of the mitochondrial membrane system. XVIII. Genetic loci on mitochondrial DNA involved in cytochrome b biosynthesis.

    PubMed

    Tzagoloff, A; Foury, F; Akai, A

    1976-11-24

    1. Fourteen cytoplasmic mutants of Saccharomyces cerevisiae with a specific deficiency of cytochrome b have been studied. The mutations have been shown to occur in two separate genetic loci, COB 1 and COB 2. These loci can be distinguished by mit- X mit- crosses. Pairwise crosses of cytochrome b mutants belonging to different loci yield 4-6% wild type recombinants corresponding to recombinational frequencies of 8-12%. In intra-locus crosses, the recombinational frequencies range from 1% to less than 0.01%. The two loci can also be distinguished by mit- X rho- crosses. Twenty rho- testers have been isolated of which ten preferentially restore mutations in COB 1 and ten others in COB 2. 2. The COB 1 and COB 2 loci have been localized on mitochondrial DNA between the two antibiotic resistance loci OLI 1 and OLI 2 in the order OLI 2-COB 2-COB 1-OLI 1. The results of mit- X mit- and mit- X rho- crosses have also been used to map the cytochrome b mutations relative to each other. The maps obtained by the two independent methods are in good agreement. 3. Mutations in COB 1 have been found to be linked to the OLI1 locus in some but not in other strains of S. cervisiae. This evidence suggests that there may be a spacer region between the two loci whose length varies from strain to strain. 4. Two mutations in COB 2 have been found to cause a loss of a mitochondrial translation product corresponding to the cytochrome b apoprotein. Instead of the wild type protein the mutants have a new low-molecular weight product which is probably a fragment of cytochrome b. The fact that the mutations revert suggests that they are nonsense mutations in the structural gene of cytochrome b. PMID:796670

  12. Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid.

    PubMed Central

    Guzmán, M; Geelen, M J

    1992-01-01

    A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed. PMID:1332675

  13. Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

    PubMed

    Yu, Ying; Yang, Runmei; Zhao, Xiuyun; Qin, Dandan; Liu, Zhaoyang; Liu, Fang; Song, Xin; Li, Liqin; Feng, Renqing; Gao, Nannan

    2016-05-01

    To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis. PMID:27055473

  14. Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: roles of membrane proteins, phospholipids and their fatty acid compositions.

    PubMed

    Kraffe, Edouard; Marty, Yanic; Guderley, Helga

    2007-01-01

    Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5 degrees C to 15 degrees C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15 degrees C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5 degrees C to 15 degrees C, the maximal oxidative capacity of mitochondria measured at 15 degrees C increased slightly, but rates at both assay temperatures (5 degrees C and 15 degrees C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15 degrees C were expressed relative to cytochrome a and c(1) levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15 degrees C decreased the ratio of

  15. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics

    PubMed Central

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  16. Novel super-resolution capable mitochondrial probe, MitoRed AIE, enables assessment of real-time molecular mitochondrial dynamics.

    PubMed

    Lo, Camden Yeung-Wah; Chen, Sijie; Creed, Sarah Jayne; Kang, Miaomiao; Zhao, Na; Tang, Ben Zhong; Elgass, Kirstin Diana

    2016-01-01

    Mitochondria and mitochondrial dynamics play vital roles in health and disease. With the intricate nanometer-scale structure and rapid dynamics of mitochondria, super-resolution microscopy techniques possess great un-tapped potential to significantly contribute to understanding mitochondrial biology and kinetics. Here we present a novel mitochondrial probe (MitoRed AIE) suitable for live mitochondrial dynamics imaging and single particle tracking (SPT), together with a multi-dimensional data analysis approach to assess local mitochondrial (membrane) fluidity. The MitoRed AIE probe localizes primarily to mitochondrial membranes, with 95 ms fluorophore on-time delivering 106 photons/ms, characteristics which we exploit to demonstrate live cell 100 fps 3D time-lapse tracking of mitochondria. Combining our experimental and analytical approaches, we uncover mitochondrial dynamics at unprecedented time scales. This approach opens up a new regime into high spatio-temporal resolution dynamics in many areas of mitochondrial biology. PMID:27492961

  17. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

    PubMed Central

    Sawada, Kazutaka

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation. PMID:26839744

  18. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation.

    PubMed

    Sawada, Kazutaka; Kitagaki, Hiroshi

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation. PMID:26839744

  19. VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

    PubMed

    Lemeshko, Victor V

    2016-07-01

    Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death. PMID:27085978

  20. Tuning the membrane surface potential for efficient toxin import

    PubMed Central

    Zakharov, Stanislav D.; Rokitskaya, Tatyana I.; Shapovalov, Vladimir L.; Antonenko, Yuri N.; Cramer, William A.

    2002-01-01

    Membrane surface electrostatic interactions impose structural constraints on imported proteins. An unprecedented sensitive dependence on these constraints was seen in the voltage-gated import and channel formation by the C-terminal pore-forming domain of the bacteriocin, colicin E1. At physiological ionic strengths, significant channel current was observed only in a narrow interval of anionic lipid content ([L−]), with the maximum current (Imax) at 25–30 mol% (dioleoyl)-phosphatidylglycerol ([L−]max) corresponding to a surface potential of the lipid bilayer in the absence of protein, ψ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{o}^{max}}}\\end{equation*}\\end{document} = −60 ± 5 mV. Higher ionic strength shifted [L−]max to larger values, but ψ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{o}^{max}}}\\end{equation*}\\end{document} remained approximately constant. It is proposed that the channel current (i) increases and (ii) decreases at |ψo| values <55 mV and >65 mV, because of (i) electrostatic interactions needed for effective insertion of the channel polypeptide and (ii) constraints due to electrostatic forces on the flexibility needed for cooperative insertion into the membrane. The loss of flexibility for |ψo| ≫ 65 mV was demonstrated by the absence of thermally induced intraprotein distance changes of the bound polypeptide. The anionic lipid content, 25–30 mol%, corresponding to the channel current maxima, is similar to that of the target Escherichia coli cytoplasmic membrane and membranes of mesophilic microorganisms. This suggests that one

  1. Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathway

    PubMed Central

    Fernandez-Gomez, Francisco J; Galindo, Maria F; Gómez-Lázaro, Maria; Yuste, Victor J; Comella, Joan X; Aguirre, Norberto; Jordán, Joaquín

    2005-01-01

    Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain-isolated mitochondria. Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. Malonate added to SH-SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH-SY5Y cells overexpressing Bcl-xL. When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl-xL overexpression was not evident anymore. Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate-induced toxicity. PMID:15655518

  2. Branched Chain Amino Acids Induce Apoptosis in Neural Cells without Mitochondrial Membrane Depolarization or Cytochrome c Release: Implications for Neurological Impairment Associated with Maple Syrup Urine Disease

    PubMed Central

    Jouvet, Philippe; Rustin, Pierre; Taylor, Deanna L.; Pocock, Jennifer M.; Felderhoff-Mueser, Ursula; Mazarakis, Nicholas D.; Sarraf, Catherine; Joashi, Umesh; Kozma, Mary; Greenwood, Kirsty; Edwards, A. David; Mehmet, Huseyin

    2000-01-01

    Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain α-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular α-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, α-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their α-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway. PMID:10793161

  3. Mitochondrial network regulation and its potential interference with inflammatory signals in pancreatic beta cells.

    PubMed

    Baltrusch, Simone

    2016-04-01

    Mitochondria fulfil multiple tasks in nutrient metabolism, energy production, redox homeostasis and stress response, and are essential for pancreatic beta cell function. The dynamism and health of the mitochondrial network is regulated by fission- and fusion-triggering factors and by a quality control system that removes dysfunctional organelles. Alongside the role of mitochondria in regulating apoptotic cell death mediated primarily via production of reactive oxygen species and release of cytochrome c, there is evidence of other links between mitochondria and inflammation that have implications for cell viability. This review briefly outlines two pathways that are potentially vital for pancreatic beta cell function. The first concerns the regulation of Parkin, a protein that acts, not only as a central player in regulating mitophagy, but also as an activator of the NF-ĸB pathway. The fact that expression of optic atrophy protein 1 (OPA1), a mitochondrial fusion inducer and master mitochondrial cristae biogenetic factor, is increased following NF-ĸB activation highlights a point of mitochondrial control that might be influenced by TNFα signalling. A second axis of interest is suggested by IL-6-mediated upregulation of the fission inducer FIS1 alongside downregulation of mitofusin 2 (MFN2), a guard of mitochondrial fusion and metabolism and an inhibitor of apoptosis. This review summarises a presentation given at the 'Islet inflammation in type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied two other reviews on topics from this symposium (by Marc Donath, DOI: 10.1007/s00125-016-3873-z , and Jerry Nadler and colleagues, DOI: 10.1007/s00125-016-3890-y ) and a commentary by the Session Chair, Piero Marchetti (DOI: 10.1007/s00125-016-3875-x ). PMID:26873508

  4. Mitochondrial oncobioenergetic index: A potential biomarker to predict progression from indolent to aggressive prostate cancer

    PubMed Central

    Vayalil, Praveen K.; Landar, Aimee

    2015-01-01

    Mitochondrial function is influenced by alterations in oncogenes and tumor suppressor genes and changes in the microenvironment occurring during tumorigenesis. Therefore, we hypothesized that mitochondrial function will be stably and dynamically altered at each stage of the prostate tumor development. We tested this hypothesis in RWPE-1 cells and its tumorigenic clones with progressive malignant characteristics (RWPE-1 < WPE-NA22 < WPE-NB14 < WPE-NB11 < WPE-NB26) using high-throughput respirometry. Our studies demonstrate that mitochondrial content do not change with increasing malignancy. In premalignant cells (WPE-NA22 and WPE-NB14), OXPHOS is elevated in presence of glucose or glutamine alone or in combination compared to RWPE-1 cells and decreases with increasing malignancy. Glutamine maintained higher OXPHOS than glucose and suggests that it may be an important substrate for the growth and proliferation of prostate epithelial cells. Glycolysis significantly increases with malignancy and follow a classical Warburg phenomenon. Fatty acid oxidation (FAO) is significantly lower in tumorigenic clones and invasive WPE-NB26 does not utilize FAO at all. In this paper, we introduce for the first time the mitochondrial oncobioenergetic index (MOBI), a mathematical representation of oncobioenergetic profile of a cancer cell, which increases significantly upon transformation into localized premalignant form and rapidly falls below the normal as they become aggressive in prostate tumorigenesis. We have validated this in five prostate cancer cell lines and MOBI appears to be not related to androgen dependence or mitochondrial content, but rather dependent on the stage of the cancer. Altogether, we propose that MOBI could be a potential biomarker to distinguish aggressive cancer from that of indolent disease. PMID:26515588

  5. Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission

    PubMed Central

    Stepanyants, Natalia; Macdonald, Patrick J.; Francy, Christopher A.; Mears, Jason A.; Qi, Xin; Ramachandran, Rajesh

    2015-01-01

    Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division. PMID:26157169

  6. Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

    PubMed

    Hoffmann, A; Hildebrandt, V; Heberle, J; Büldt, G

    1994-09-27

    The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism. PMID:7937771

  7. New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

    PubMed Central

    Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

    2014-01-01

    Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

  8. Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

    PubMed Central

    Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

    2011-01-01

    Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent. PMID:21274005

  9. The Amniotic Membrane: Development and Potential Applications - A Review.

    PubMed

    Favaron, P O; Carvalho, R C; Borghesi, J; Anunciação, A R A; Miglino, M A

    2015-12-01

    Foetal membranes are essential tissues for embryonic development, playing important roles related to protection, breathing, nutrition and excretion. The amnion is the innermost extraembryonic membrane, which surrounds the foetus, forming an amniotic sac that contains the amniotic fluid (AF). In recent years, the amniotic membrane has emerged as a potential tool for clinical applications and has been primarily used in medicine in order to stimulate the healing of skin and corneal diseases. It has also been used in vaginal reconstructive surgery, repair of abdominal hernia, prevention of surgical adhesions and pericardium closure. More recently, it has been used in regenerative medicine because the amniotic-derived stem cells as well as AF-derived cells exhibit cellular plasticity, angiogenic, cytoprotective, immunosuppressive properties, antitumoural potential and the ability to generate induced pluripotent stem cells. These features make them a promising source of stem cells for cell therapy and tissue engineering. In this review, we discussed the development of the amnion, AF and amniotic cavity in different species, as well as the applicability of stem cells from the amnion and AF in cellular therapy. PMID:26510939

  10. Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss

    SciTech Connect

    Izquierdo-Vega, Jeannett A.; Sanchez-Gutierrez, Manuel; Razo, Luz Maria del

    2008-08-01

    Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential ({delta}{psi}{sub m}). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O{sub 2}{sup {center_dot}}{sup -}), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) {delta}{psi}{sub m}, vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity ({approx} 33%), accompanied with a significant increase in the generation of O{sub 2}{sup {center_dot}} ({approx} 40%), a significant decrease in {delta}{psi}{sub m} ({approx} 33%), and a significant increase in lipid peroxidation concentration ({approx} 50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility.

  11. Latent progenitor cells as potential regulators for tympanic membrane regeneration

    NASA Astrophysics Data System (ADS)

    Kim, Seung Won; Kim, Jangho; Seonwoo, Hoon; Jang, Kyung-Jin; Kim, Yeon Ju; Lim, Hye Jin; Lim, Ki-Taek; Tian, Chunjie; Chung, Jong Hoon; Choung, Yun-Hoon

    2015-06-01

    Tympanic membrane (TM) perforation, in particular chronic otitis media, is one of the most common clinical problems in the world and can present with sensorineural healing loss. Here, we explored an approach for TM regeneration where the latent progenitor or stem cells within TM epithelial layers may play an important regulatory role. We showed that potential TM stem cells present highly positive staining for epithelial stem cell markers in all areas of normal TM tissue. Additionally, they are present at high levels in perforated TMs, especially in proximity to the holes, regardless of acute or chronic status, suggesting that TM stem cells may be a potential factor for TM regeneration. Our study suggests that latent TM stem cells could be potential regulators of regeneration, which provides a new insight into this clinically important process and a potential target for new therapies for chronic otitis media and other eardrum injuries.

  12. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

    PubMed Central

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-01-01

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  13. Thermoelectric Potential of Polymer-Scaffolded Ionic Liquid Membranes

    NASA Astrophysics Data System (ADS)

    Datta, R. S.; Said, S. M.; Sahamir, S. R.; Karim, M. R.; Sabri, M. F. M.; Nakajo, T.; Kubouchi, M.; Hayashi, K.; Miyazaki, Y.

    2014-06-01

    Organic thin films have been viewed as potential thermoelectric (TE) materials, given their ease of fabrication, flexibility, cost effectiveness, and low thermal conductivity. However, their intrinsically low electrical conductivity is a main drawback which results in a relatively lower TE figure of merit for polymer-based TE materials than for inorganic materials. In this paper, a technique to enhance the ion transport properties of polymers through the introduction of ionic liquids is presented. The polymer is in the form of a nanofiber scaffold produced using the electrospinning technique. These fibers were then soaked in different ionic liquids based on substituted imidazolium such as 1-ethyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium bromide. This method was applied to electrospun polyacrylonitrile and a mixture of polyvinyl alcohol and chitosan polymers. The ion transport properties of the membranes have been observed to increase with increasing concentration of ionic liquid, with maximum electrical conductivity of 1.20 × 10-1 S/cm measured at room temperature. Interestingly, the maximum electrical conductivity value surpassed the value of pure ionic liquids. These results indicate that it is possible to significantly improve the electrical conductivity of a polymer membrane through a simple and cost-effective method. This may in turn boost the TE figures of merit of polymer materials, which are well known to be considerably lower than those of inorganic materials. Results in terms of the Seebeck coefficient of the membranes are also presented in this paper to provide an overall representation of the TE potential of the polymer-scaffolded ionic liquid membranes.

  14. Imaging cellular membrane potential through ionization of quantum dots

    NASA Astrophysics Data System (ADS)

    Rowland, Clare E.; Susumu, Kimihiro; Stewart, Michael H.; Oh, Eunkeu; Mäkinen, Antti J.; O'Shaughnessy, Thomas J.; Kushto, Gary; Wolak, Mason A.; Erickson, Jeffrey S.; Efros, Alexander L.; Huston, Alan L.; Delehanty, James B.

    2016-03-01

    Recent interest in quantum dots (QDs) stems from the plethora of potential applications that arises from their tunable absorption and emission profiles, high absorption cross sections, resistance to photobleaching, functionalizable surfaces, and physical robustness. The emergent use of QDs in biological imaging exploits these and other intrinsic properties. For example, quantum confined Stark effect (QCSE), which describes changes in the photoluminescence (PL) of QDs driven by the application of an electric field, provides an inherent means of detecting changes in electric fields by monitoring QD emission and thus points to a ready mean of imaging membrane potential (and action potentials) in electrically active cells. Here we examine the changing PL of various QDs subjected to electric fields comparable to those found across a cellular membrane. By pairing static and timeresolved PL measurements, we attempt to understand the mechanism driving electric-field-induced PL quenching and ultimately conclude that ionization plays a substantial role in initiating PL changes in systems where QCSE has traditionally been credited. Expanding on these findings, we explore the rapidity of response of the QD PL to applied electric fields and demonstrate changes amply able to capture the millisecond timescale of cellular action potentials.

  15. Rasagiline prevents cyclosporine A-sensitive superoxide flashes induced by PK11195, the initial signal of mitochondrial membrane permeabilization and apoptosis.

    PubMed

    Wu, Yuqiu; Shamoto-Nagai, Masayo; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

    2016-05-01

    Rasagiline, a neuroprotective inhibitor of type B monoamine oxidase, prevented PK111195-induced apoptosis in SH-SY5Y cells through inhibition of mitochondrial apoptosis signaling (J Neural Transm 120:1539-1551, 2013, J Neural Transm 122:1399-1407, 2015). This paper presents that PK11195 induced superoxide flashes, the transit production burst, mediated by cyclosporine A-sensitive membrane permeability transition. Rasagiline prevented superoxide flashes, calcium efflux, and cell death by PK11195. Regulation of the initial pore formation at the inner mitochondrial membrane was confirmed as the decisive mechanism of neuroprotection by rasagiline. PMID:26931622

  16. A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.

    PubMed

    Isenmann, S; Khew-Goodall, Y; Gamble, J; Vadas, M; Wattenberg, B W

    1998-07-01

    Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. PMID:9658161

  17. Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport

    PubMed Central

    Rajan, Sreekanth; Choi, Minjoo; Nguyen, Quoc Toan; Ye, Hong; Liu, Wei; Toh, Hui Ting; Kang, CongBao; Kamariah, Neelagandan; Li, Chi; Huang, Huiya; White, Carl; Baek, Kwanghee; Grüber, Gerhard; Yoon, Ho Sup

    2015-01-01

    Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl β-D-Maltoside (OM). The detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) by swapping helices α6-α8 between two monomers. Unlike Bax, a proapoptotic member of the Bcl-2 family, Bcl-xL is not converted to 3DDS homodimer upon binding BH3 peptides and ABT-737, a BH3 mimetic drug. We also designed Bcl-xL mutants which cannot dimerize and show that these mutants reduced mitochondrial calcium uptake in MEF cells. This illustrates the structural plasticity in Bcl-xL providing hints toward the probable molecular mechanism for Bcl-xL to play a regulatory role in mitochondrial calcium ion transport. PMID:26023881

  18. Mitochondrial Cristae: Where Beauty Meets Functionality.

    PubMed

    Cogliati, Sara; Enriquez, Jose A; Scorrano, Luca

    2016-03-01

    Mitochondrial cristae are dynamic bioenergetic compartments whose shape changes under different physiological conditions. Recent discoveries have unveiled the relation between cristae shape and oxidative phosphorylation (OXPHOS) function, suggesting that membrane morphology modulates the organization and function of the OXPHOS system, with a direct impact on cellular metabolism. As a corollary, cristae-shaping proteins have emerged as potential modulators of mitochondrial bioenergetics, a concept confirmed by genetic experiments in mouse models of respiratory chain deficiency. Here, we review our knowledge of mitochondrial ultrastructural organization and how it impacts mitochondrial metabolism. PMID:26857402

  19. Induction of necrosis and apoptosis to KB cancer cells by sanguinarine is associated with reactive oxygen species production and mitochondrial membrane depolarization

    SciTech Connect

    Chang, M.-C.; Chan, C.-P.; Wang, Y.-J.; Lee, P.-H.; Chen, L.-I; Tsai, Y.-L.; Lin, B.-R.; Wang, Y.-L.; Jeng, J.-H. . E-mail: huei@ha.mc.ntu.edu.tw

    2007-01-15

    Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 {mu}M) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (> 52-58%) and growth of KB cancer cells at concentrations higher than 0.5-1 {mu}M. Short-term exposure to sanguinarine (> 0.5 {mu}M) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 {mu}M) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 {mu}M) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death.

  20. Polymeric membrane systems of potential use for battery separators

    NASA Technical Reports Server (NTRS)

    Philipp, W. H.

    1977-01-01

    Two membrane systems were investigated that may have potential use as alkaline battery separators. One system comprises two miscible polymers: a support polymer (e.g., polyvinyl formal) and an ion conductor such as polyacrylic acid. The other system involves a film composed of two immiscible polymers: a conducting polymer (e.g., calcium polyacrylate) suspended in an inert polymer support matrix, polyphenylene oxide. Resistivities in 45-percent potassium hydroxide and qualitative mechanical properties are presented for films comprising various proportions of conducting and support polymers. In terms of these parameters, the results are encouraging for optimum ratios of conducting to support polymers.

  1. Measuring H(+) Pumping and Membrane Potential Formation in Sealed Membrane Vesicle Systems.

    PubMed

    Wielandt, Alex Green; Palmgren, Michael G; Fuglsang, Anja Thoe; Günther-Pomorski, Thomas; Justesen, Bo Højen

    2016-01-01

    The activity of enzymes involved in active transport of matter across lipid bilayers can conveniently be assayed by measuring their consumption of energy, such as ATP hydrolysis, while it is more challenging to directly measure their transport activities as the transported substrate is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes. PMID:26695032

  2. Scanning Ion Conductance Microscopy for living cell membrane potential measurement

    NASA Astrophysics Data System (ADS)

    Panday, Namuna

    Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using fluorescence microscopy. One hypothesis is that these long lasting potential patterns play a vital role in regulating important cell activities such as embryonic patterning, regenerative repair and reduction of cancerous disorganization. We used multifunctional Scanning Ion Conductance Microscopy (SICM) to study these extracellular potential patterns of single cell with higher spatial resolution. To validate this novel technique, we compared the extracellular potential distribution on the fixed HeLa cell surface and Polydimethylsiloxane (PDMS) surface and found significant difference. We then measured the extracellular potential distributions of living melanocytes and melanoma cells and found both the mean magnitude and spatial variation of extracellular potential of the melanoma cells are bigger than those of melanocytes. As compared to the voltage reporter dye based fluorescence microscope method, SICM can achieve quantitative potential measurements of non-labeled living cell membranes with higher spatial resolution.

  3. Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

    SciTech Connect

    Li, Jingzhi; Qian, Xinguo; Hu, Junbin; Sha, Bingdong

    2010-11-03

    The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.

  4. Antihepatocellular Carcinoma Potential of Tetramethylpyrazine Induces Cell Cycle Modulation and Mitochondrial-Dependent Apoptosis: Regulation of p53 Signaling Pathway in HepG2 Cells In Vitro.

    PubMed

    Bi, Lei; Yan, Xiaojing; Chen, Weiping; Gao, Jing; Qian, Lei; Qiu, Shuang

    2016-06-01

    Tetramethylpyrazine (TMP) was originally isolated from a traditional Chinese herbal medicine, Ligusticum chuanxiong In the present study, TMP exhibits potent antitumor activities in vitro. However, the molecular mechanisms remain to be defined. Hence, this study aims to investigate the antiproliferative and apoptotic effects of TMP on HepG2 and elucidate the underlying mechanisms. Analyses using Cell Counting Kit-8 and real-time cell analyzer indicated that TMP significantly inhibited HepG2 cell proliferation. We also observed that TMP induced cell cycle arrest at the G0/G1 checkpoint and apoptosis, using flow cytometry and high-content screening. Furthermore, our results predicted that TMP could directly decrease mitochondrial membrane potential (Δψm), increase the release of cytochrome c, and increase caspase activation, indicating that mitochondrial pathway apoptosis could be the mechanism for TMP within HepG2 cells. Moreover, TMP altered expression of p53 and the Bcl-2/Bax protein ratio, which revealed that TMP induced cell cycle arrest and caspase-dependent mitochondrial apoptosis in HepG2 cells in vitro. These studies provided mechanistic insights into the antitumor properties of TMP, which may be explored as a potential option for treatment of hepatocellular carcinoma. PMID:27179035

  5. Identification of potential mitochondrial CLPXP protease interactors and substrates suggests its central role in energy metabolism

    PubMed Central

    Fischer, Fabian; Langer, Julian D.; Osiewacz, Heinz D.

    2015-01-01

    Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP’s substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes. PMID:26679294

  6. A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

    PubMed Central

    De Stefani, Diego; Raffaello, Anna; Teardo, Enrico; Szabò, Ildikò; Rizzuto, Rosario

    2014-01-01

    Mitochondrial Ca2+ homeostasis plays a key role in the regulation of aerobic metabolism and cell survival1, but the molecular identity of the Ca2+ channel, the mitochondrial calcium uniporter2, was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator3, coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca2+ uptake. MCU overexpression doubled the [Ca2+]mt rise evoked by IP3-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively-charged residues of the putative pore forming region were replaced, had no channel activity and reduced agonist-dependent [Ca2+]mt transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca2+ uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance. PMID:21685888

  7. Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p

    PubMed Central

    Lauffer, Susann; Mäbert, Katrin; Czupalla, Cornelia; Pursche, Theresia; Hoflack, Bernard; Rödel, Gerhard; Krause-Buchholz, Udo

    2012-01-01

    Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites. PMID:22461620

  8. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    SciTech Connect

    Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Helix-Nielsen, Claus

    2011-03-04

    Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

  9. BDE-154 induces mitochondrial permeability transition and impairs mitochondrial bioenergetics.

    PubMed

    Pereira, Lílian Cristina; Miranda, Luiz Felippe Cabral; de Souza, Alecsandra Oliveira; Dorta, Daniel Junqueira

    2014-01-01

    Brominated flame retardants are used in various consumer goods to make these materials difficult to burn. Polybrominated diphenyl ethers (PBDE), which are representative of this class of retardants, consist of two benzene rings linked by an oxygen atom, and contain between 1 and 10 bromine atoms in their chemical structure, with the possibility of up to 209 different congeners. Among these congeners, BDE-154 (hexa-BDE) is persistent in the environment and easy to detect in the biota, but no apparent information regarding the mechanism underlying action and toxicity is available. Mitochondria, as the main energy-producing organelles, play an important role in the maintenance of various cellular functions. Therefore, mitochondria were used in the present study as an experimental model to determine the effects of BDE-154 congener at concentrations ranging from 0.1 μM to 50 μM. Our results demonstrated that BDE-154 interacts with the mitochondrial membrane, preferably by inserting into the hydrophobic core of the mitochondrial membrane, which partially inhibits respiration, dissipates Δψ, and permeabilizes the inner mitochondrial membrane to deplete ATP. These effects are more pronounced at concentrations equal to or higher than 10 μM. Results also showed that BDE-154 did not induce reactive oxygen species (ROS) accumulation within the mitochondria, indicating the absence of oxidative stress. Therefore, BDE-154 impairs mitochondrial bioenergetics and permeabilizes the mitochondrial membrane, potentially leading to cell death but not via mechanisms involving oxidative stress. PMID:24555644

  10. Fouling mitigation of anion exchange membrane by zeta potential control.

    PubMed

    Park, Jin-Soo; Lee, Hong-Joo; Choi, Seok-Ju; Geckeler, Kurt E; Cho, Jaeweon; Moon, Seung-Hyeon

    2003-03-15

    The feasibility of fouling mitigation of anion exchange membranes (AEMs) in the presence of humate was studied by adding three different types of water-soluble polymers, i.e., poly(acrylic acid) (PAA), poly(vinyl alcohol) (PVA), and poly(ethylene imine) (PEI), during electrodialysis (ED) desalination. Measurement of zeta potential of the humate used in this study showed highly negative potential (about -30 mV), implying that the humate had a strong fouling potential on the AEMs in ED. Of the three water-soluble polymers, PEI showed a positive zeta potential (about +14 mV) and is able to form an interpolymer complex with the humate. PAA and PVA hardly formed interpolymer complexes with humate due to electrostatic repulsion. The PEI-humate mixture with a volume ratio of 1:20 (PEI:humate) showed zero zeta potential, and a complexed humate with zero surface charge was formed, resulting in no fouling effects on the AEMs. Accordingly, the desalting ED experiments with PEI showed improved ED performance. Further, black colloids formed in the mixture did not cause the cell resistance to increase. PMID:16256509

  11. Mitochondrial ion channels as therapeutic targets

    PubMed Central

    Peixoto, Pablo M.; Ryu, Shin-Young; Kinnally, Kathleen W.

    2010-01-01

    The study of mitochondrial ion channels changed our perception of these double-wrapped organelles from being just the power house of a cell to the guardian of a cell's fate. Mitochondria communicate with the cell through these special channels. Most of the time, the message is encoded by ion flow across the mitochondrial outer and inner membranes. Potassium, sodium, calcium, protons, nucleotides, and proteins traverse the mitochondrial membranes in an exquisitely regulated manner to control a myriad of processes, from respiration and mitochondrial morphology to cell proliferation and cell death. This review is an update on both well established and putative mitochondrial channels regarding their composition, function, regulation, and therapeutic potential. PMID:20178788

  12. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    PubMed Central

    2011-01-01

    Background Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd2 [S(-)C2, N-dmpa]2 (μ-dppe)Cl2} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. Methods B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Results Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. Conclusions The

  13. Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A.

    PubMed

    Dagda, Ruben K; Barwacz, Chris A; Cribbs, J Thomas; Strack, Stefan

    2005-07-22

    Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B'') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme. PMID:15923182

  14. Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

    SciTech Connect

    Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

    2008-10-01

    Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore.

  15. Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

    PubMed

    Huang, Kai; Zhang, Jingjing; O'Neill, Katelyn L; Gurumurthy, Channabasavaiah B; Quadros, Rolen M; Tu, Yaping; Luo, Xu

    2016-05-27

    The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis. PMID:27053107

  16. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

    PubMed

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-12-15

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration. PMID:22983956

  17. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model

    PubMed Central

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-01-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of neurodegenerative disorders characterized by high brain content of iron and presence of axonal spheroids. Mutations in the PANK2 gene, which encodes pantothenate kinase 2, underlie an autosomal recessive inborn error of coenzyme A metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). PKAN is characterized by dystonia, dysarthria, rigidity and pigmentary retinal degeneration. The pathogenesis of this disorder is poorly understood and, although PANK2 is a mitochondrial protein, perturbations in mitochondrial bioenergetics have not been reported. A knock-out (KO) mouse model of PKAN exhibits retinal degeneration and azoospermia, but lacks any neurological phenotype. The absence of a clinical phenotype has partially been explained by the different cellular localization of the human and murine PANK2 proteins. Here we demonstrate that the mouse Pank2 protein localizes to mitochondria, similar to its human orthologue. Moreover, we show that Pank2-defective neurons derived from KO mice have an altered mitochondrial membrane potential, a defect further corroborated by the observations of swollen mitochondria at the ultra-structural level and by the presence of defective respiration. PMID:22983956

  18. Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development.

    PubMed

    Spinaci, M; De Ambrogi, M; Volpe, S; Galeati, G; Tamanini, C; Seren, E

    2005-07-01

    The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos. PMID:15935852

  19. Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex*

    PubMed Central

    Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.

    2011-01-01

    CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568

  20. Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.

    PubMed

    Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L

    2011-07-22

    CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568

  1. IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention

    PubMed Central

    Pazarentzos, Evangelos; Mahul-Mellier, Anne-Laure; Datler, Christoph; Chaisaklert, Wanwisa; Hwang, Ming-Shih; Kroon, Jan; Qize, Ding; Osborne, Foy; Al-Rubaish, Abdullah; Al-Ali, Amein; Mazarakis, Nicholas D; Aboagye, Eric O; Grimm, Stefan

    2014-01-01

    IκBα resides in the cytosol where it retains the inducible transcription factor NF-κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-κB that accumulate high amounts of mitochondrial IκBα as a NF-κB target gene. 3T3 IκBα−/− cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF-κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF-κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-κB. PMID:25361605

  2. Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

    PubMed

    Zanatta, Ângela; Rodrigues, Marília Danyelle Nunes; Amaral, Alexandre Umpierrez; Souza, Débora Guerini; Quincozes-Santos, André; Wajner, Moacir

    2016-09-01

    Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease. PMID:27161368

  3. Cardiolipin and Mitochondrial Phosphatidylethanolamine Have Overlapping Functions in Mitochondrial Fusion in Saccharomyces cerevisiae*

    PubMed Central

    Joshi, Amit S.; Thompson, Morgan N.; Fei, Naomi; Hüttemann, Maik; Greenberg, Miriam L.

    2012-01-01

    The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TETOFF promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion. PMID:22433850

  4. Mitochondrial ion channels as oncological targets.

    PubMed

    Leanza, L; Zoratti, M; Gulbins, E; Szabo, I

    2014-12-01

    Mitochondria, the key bioenergetic intracellular organelles, harbor a number of proteins with proven or hypothetical ion channel functions. Growing evidence points to the important contribution of these channels to the regulation of mitochondrial function, such as ion homeostasis imbalances profoundly affecting energy transducing processes, reactive oxygen species production and mitochondrial integrity. Given the central role of mitochondria in apoptosis, their ion channels with the potential to compromise mitochondrial function have become promising targets for the treatment of malignancies. Importantly, in vivo evidence demonstrates the involvement of the proton-transporting uncoupling protein, a mitochondrial potassium channel, the outer membrane located porin and the permeability transition pore in tumor progression/control. In this review, we focus on mitochondrial channels that have been assigned a definite role in cell death regulation and possess clear oncological relevance. Overall, based on in vivo and in vitro genetic and pharmacological evidence, mitochondrial ion channels are emerging as promising targets for cancer treatment. PMID:24469031

  5. CCN6 regulates mitochondrial function.

    PubMed

    Patra, Milan; Mahata, Sushil K; Padhan, Deepesh K; Sen, Malini

    2016-07-15

    Despite established links of CCN6, or Wnt induced signaling protein-3 (WISP3), with progressive pseudo rheumatoid dysplasia, functional characterization of CCN6 remains incomplete. In light of the documented negative correlation between accumulation of reactive oxygen species (ROS) and CCN6 expression, we investigated whether CCN6 regulates ROS accumulation through its influence on mitochondrial function. We found that CCN6 localizes to mitochondria, and depletion of CCN6 in the chondrocyte cell line C-28/I2 by using siRNA results in altered mitochondrial electron transport and respiration. Enhanced electron transport chain (ETC) activity of CCN6-depleted cells was reflected by increased mitochondrial ROS levels in association with augmented mitochondrial ATP synthesis, mitochondrial membrane potential and Ca(2+) Additionally, CCN6-depleted cells display ROS-dependent PGC1α (also known as PPARGC1A) induction, which correlates with increased mitochondrial mass and volume density, together with altered mitochondrial morphology. Interestingly, transcription factor Nrf2 (also known as NFE2L2) repressed CCN6 expression. Taken together, our results suggest that CCN6 acts as a molecular brake, which is appropriately balanced by Nrf2, in regulating mitochondrial function. PMID:27252383

  6. [The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens (Leuckart, 1895) on mitochondrial and lipid bilayer membranes].

    PubMed

    Kuchboev, A E; Kazakov, I; Asrarov, M I; Isakova, D T; Azimov, D A; Golovanov, V I

    2007-01-01

    The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens on mitochondrial and lipid bilayer membranes has been studied. The homogenate of P. rufescens affects efficiently the cell energy by the inhibition of the mitochondrial respiration in the metabolic state V3, uncouples oxidative phosphorylation and affects the functions of mitochondria at the level of cyclosporine A-sensitive pore by making it highly permeable. Moreover, the nematode homogenate at the concentration of 1 mkg/ml increases efficiently the integral permeability of lipid bilayer membranes. An increase in this permeability is connected apparently with the formation of single ion channels. The channels of lipid bilayer membranes induced by the nematode homogenate show cation selectivity. PMID:17460939

  7. Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate-stimulated lipid peroxidation.

    PubMed

    Kowaltowski, A J; Castilho, R F; Grijalba, M T; Bechara, E J; Vercesi, A E

    1996-02-01

    Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances. PMID:8621682

  8. Membrane fouling potentials and cellular properties of bacteria isolated from fouled membranes in a MBR treating municipal wastewater.

    PubMed

    Ishizaki, So; Fukushima, Toshikazu; Ishii, Satoshi; Okabe, Satoshi

    2016-09-01

    Membrane fouling remains a major challenge for wider application of membrane bioreactors (MBRs) to wastewater treatment. Membrane fouling is mainly caused by microorganisms and their excreted microbial products. For development of more effective control strategies, it is important to identify and characterize the microorganisms that are responsible for membrane fouling. In this study, 41 bacterial strains were isolated from fouled microfiltration membranes in a pilot-scale MBR treating real municipal wastewater, and their membrane fouling potentials were directly measured using bench-scale cross-flow membrane filtration systems (CFMFSs) and related to their cellular properties. It was found that the fouling potential was highly strain dependent, suggesting that bacterial identification at the strain level is essential to identify key fouling-causing bacteria (FCB). The FCB showed some common cellular properties. The most prominent feature of FCB was that they formed convex colonies having swollen podgy shape and smooth lustrous surfaces with high water, hydrophilic organic matter and carbohydrate content. However, general and rigid biofilm formation potential as determined by microtiter plates and cell surface properties (i.e., hydrophobicity and surface charge) did not correlate with the fouling potential in this study. These results suggest that the fouling potential should be directly evaluated under filtration conditions, and the colony water content could be a useful indicator to identify the FCB. PMID:27232989

  9. Rapid incorporation of docosahexaenoic acid from dietary sources into brain microsomal, synaptosomal and mitochondrial membranes in adult mice.

    PubMed

    Suzuki, H; Manabe, S; Wada, O; Crawford, M A

    1997-01-01

    This study examined the incorporation of docosahexaenoic acid (DHA) from several dietary sources into the brain tissue and intracellular organelles in mice which had been fed a 5% palm oil (low n-3 fatty acid level) diet for 8 or 11 weeks. The percentages of DHA in the tissues of mice fed 5% representative oils for 30 days or 5% purified n-3 fatty acid diets for 6 days were analyzed using gas chromatography. The percentage of DHA in the brain was ranked in the following order: the salmon oil diet group > the sardine oil diet group > > the perilla oil diet group > > the lard and palm oil diet groups for the 30 day feeding trial; and the DHA diet group > > the eicosapentaenoic acid and alpha-linolenic acid diet groups for the 6 day feeding trial. The percentage of arachidonic acid showed a more dramatic decrease than that of docosapentaenoic acid. These results reflected the plasma fatty acid concentrations, but were not as pronounced as the changes observed in the plasma. The majority of the DHA incorporated into the brain was recovered in microsomal, synaptosomal, and mitochondrial fractions separated by density gradient centrifugation. These membrane fractions took up DHA within several days. These results suggest that the intake of DHA itself increases the DHA level of brain membranes more rapidly than intake of the precursors in animals fed a low n-3 fatty acid level diet. PMID:9285258

  10. Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.

    PubMed

    de Meis, Leopoldo; Ketzer, Luisa A; da Costa, Rodrigo Madeiro; de Andrade, Ivone Rosa; Benchimol, Marlene

    2010-01-01

    Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver. PMID:20209153

  11. Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models

    PubMed Central

    Kalanxhi, Erta; Wallace, Carmichael J. A.

    2007-01-01

    Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon. PMID:17614790

  12. Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex.

    PubMed

    Model, Kirstin; Prinz, Thorsten; Ruiz, Teresa; Radermacher, Michael; Krimmer, Thomas; Kühlbrandt, Werner; Pfanner, Nikolaus; Meisinger, Chris

    2002-02-22

    The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures. PMID:11866524

  13. Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane.

    PubMed

    Johnston, Amelia J; Hoogenraad, Joan; Dougan, David A; Truscott, Kaye N; Yano, Masato; Mori, Masataka; Hoogenraad, Nicholas J; Ryan, Michael T

    2002-11-01

    Tom7 is a component of the translocase of the outer mitochondrial membrane (TOM) and assembles into a general import pore complex that translocates preproteins into mitochondria. We have identified the human Tom7 homolog and characterized its import and assembly into the mammalian TOM complex. Tom7 is imported into mitochondria in a nucleotide-independent manner and is anchored to the outer membrane with its C terminus facing the intermembrane space. Unlike studies in fungi, we found that human Tom7 assembles into an approximately 120-kDa import intermediate in HeLa cell mitochondria. To detect subunits within this complex, we employed a novel supershift analysis whereby mitochondria containing newly imported Tom7 were incubated with antibodies specific for individual TOM components prior to separation by blue native electrophoresis. We found that the 120-kDa complex contains Tom40 and lacks receptor components. This intermediate can be chased to the stable approximately 380-kDa mammalian TOM complex that additionally contains Tom22. Overexpression of Tom22 in HeLa cells results in the rapid assembly of Tom7 into the 380-kDa complex indicating that Tom22 is rate-limiting for TOM complex formation. These results indicate that the levels of Tom22 within mitochondria dictate the assembly of TOM complexes and hence may regulate its biogenesis. PMID:12198123

  14. Etiology of the membrane potential of rat white fat adipocytes.

    PubMed

    Bentley, Donna C; Pulbutr, Pawitra; Chan, Sue; Smith, Paul A

    2014-07-15

    The plasma membrane potential (Vm) is key to many physiological processes; however, its ionic etiology in white fat adipocytes is poorly characterized. To address this question, we employed the perforated patch current clamp and cell-attached patch clamp methods in isolated primary white fat adipocytes and their cellular model 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were -32.1 ± 1.2 mV (n = 95) and -28.8 ± 1.2 mV (n = 87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular K(+) to 50 mM by equimolar substitution of bath Na(+) did not affect Vm, whereas substitution of bath Na(+) with the membrane-impermeant cation N-methyl-D-glucamine(+)-hyperpolarized Vm by 16 mV, data indicative of a nonselective cation permeability. Substitution of 133 mM extracellular Cl(-) with gluconate-depolarized Vm by 25 mV, whereas Cl(-) substitution with I(-) caused a -9 mV hyperpolarization. Isoprenaline (10 μM), but not insulin (100 nM), significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl(-) with an inward slope conductance of 16 ± 1.3 pS (n = 11) and a reversal potential close to the Cl(-) equilibrium potential, -29 ± 1.6 mV. Although the reduction of extracellular Cl(-) elevated the intracellular Ca(2+) of adipocytes, this was not as large as that produced by elevation of extracellular K(+). In conclusion, the Vm of white fat adipocytes is well described by the Goldman-Hodgkin-Katz equation with a predominant permeability to Cl(-), where its biophysical and single-channel properties suggest a volume-sensitive anion channel identity. Consequently, changes in serum Cl(-) homeostasis or the adipocyte's permeability to this anion via drugs will affect its Vm, intracellular Ca(2+), and ultimately its function and its role in metabolic control. PMID:24865982

  15. Influence of low-power laser radiation on the activity of some membraneous and mitochondrial enzymes of hepatocytes in rats

    NASA Astrophysics Data System (ADS)

    Cieslar, Grzegorz; Adamek, Mariusz; Sieron, Aleksander; Kaminski, Marcin

    1995-01-01

    It was observed in some experiments that visible laser radiation activates the enzymatic function of mitochondria, while infrared laser radiation affects the enzymatic activity of cellular membranes. The aim of the study was to estimate the activity of some membranous as well as mitochondrial enzymes of hepatocytes in rats irradiated with infrared laser. Experimental material consisted of 38 Wistar rats divided into 2 groups -- a studied group exposed to infrared laser radiation and a control group, in which no irradiation was made. A semiconductive infrared laser (wavelength -- 904 nm, mean power -- 8.9 mW) was used. The clean-shaven skin of the right infracostal region of animals was irradiated 5 minutes daily for 15 consecutive days. After finishing the experiment in the preparations from obtained segments of the left liver lobe, the enzymatic activity of succinate dehydrogenase (SDH, EC 1.3.99.1), lactic dehydrogenase (LDH, EC 1.1.1.27), Mg2+ dependent ATP-ase (ATP-ase Mg2+, EC 3.1.3.2.) and acid phosphatase (AcP, EC 3.6.1.8.) was estimated with the use of histochemical methods. In the case of SDH and LDH the increase of enzymatic activity was observed in all 3 zones of liver cluster, especially in male rats. In the case of ATP-ase Mg2+ and AcP the increase of enzymatic activity in biliary canaliculi of hepatocytes in all zones of the liver cluster was observed. On the basis of the obtained results it was proved that infrared laser radiation activates significantly the enzymatic activity of most of the analyzed enzymes, which means that it affects not only properties of biological membranes but also activates the oxidoreductive processes of organism, as it has been observed for visible laser radiation. On the basis of the spectrum of energetic levels in macromolecules (Jablonski's diagram) the mechanisms of availed results are discussed both for enzymes possessing and not possessing chromatophores.

  16. Triiodothyronine facilitates weaning from extracorporeal membrane oxygenation by improved mitochondrial substrate utilization

    SciTech Connect

    Files, Matthew D.; Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena R.; Xu, Chun; Des Rosiers, Christine; Isern, Nancy G.; Portman, Michael A.

    2014-03-20

    Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia-reperfusion and / or by ECMO.

  17. Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester.

    PubMed

    Antonenko, Yuri N; Rokitskaya, Tatyana I; Huczyński, Adam

    2015-04-01

    Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczyński et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108-2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin. PMID:25600660

  18. Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines.

    PubMed

    Jiang, Jhih-Hang; Davies, John K; Lithgow, Trevor; Strugnell, Richard A; Gabriel, Kipros

    2011-11-01

    Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic β-barrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions. We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved 'bacterial like' mechanism to infiltrate the host mitochondrial outer membrane. PMID:22032638

  19. Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of alpha-helical outer membrane proteins.

    PubMed

    Thornton, Nicolas; Stroud, David A; Milenkovic, Dusanka; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2010-02-26

    The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes. PMID:20026336

  20. [Computer modeling the hydrostatic pressure characteristics of the membrane potential for polymeric membrane, separated non-homogeneous electrolyte solutions].

    PubMed

    Slezak, Izabella H; Jasik-Slezak, Jolanta; Rogal, Mirosława; Slezak, Andrzej

    2006-01-01

    On the basis of model equation depending the membrane potential deltapsis, on mechanical pressure difference (deltaP), concentration polarization coefficient (zetas), concentration Rayleigh number (RC) and ratio concentration of solutions separated by membrane (Ch/Cl), the characteristics deltapsis = f(deltaP)zetas,RC,Ch/Cl for steady values of zetas, RC and Ch/Cl in single-membrane system were calculated. In this system neutral and isotropic polymeric membrane oriented in horizontal plane, the non-homogeneous binary electrolytic solutions of various concentrations were separated. Nonhomogeneity of solutions is results from creations of the concentration boundary layers on both sides of the membrane. Calculations were made for the case where on a one side of the membrane aqueous solution of NaCl at steady concentration 10(-3) mol x l(-1) (Cl) was placed and on the other aqueous solutions of NaCl at concentrations from 10(-3) mol x l(-1) to 2 x 10(-2) mol x l(-1) (Ch). Their densities were greater than NaCl solution's at 10(-3) mol x l(-1). It was shown that membrane potential depends on hydrodynamic state of a complex concentration boundary layer-membrane-concentration boundary layer, what is controlled by deltaP, Ch/Cl, RC and zetas. PMID:17022155

  1. Quantum squeezed light for probing mitochondrial membranes and study of neuroprotectants.

    SciTech Connect

    Gourley, Paul Lee; Copeland, Robert Guild; McDonald, Anthony Eugene; Hendricks, Judy K.; Naviaux, Robert K.

    2005-01-01

    We report a new nanolaser technique for measuring characteristics of human mitochondria. Because mitochondria are so small, it has been difficult to study large populations using standard light microscope or flow cytometry techniques. We recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles that is well suited to mitochondrial studies. This ultrasensitive detection technique uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a semiconductor biocavity laser. In this paper, we use the method to study the lasing spectra of normal and diseased mitochondria. We find that the diseased mitochondria exhibit larger physical diameter and standard deviation. This morphological differences are also revealed in the lasing spectra. The diseased specimens have a larger spectral linewidth than the normal, and have more variability in their statistical distributions.

  2. Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

    PubMed Central

    Barbi de Moura, Michelle; Uppala, Radha; Zhang, Yuxun; Van Houten, Bennett; Goetzman, Eric S.

    2014-01-01

    SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak. PMID:25165814

  3. Cigarette smoke extract affects mitochondrial function in alveolar epithelial cells.

    PubMed

    Ballweg, Korbinian; Mutze, Kathrin; Königshoff, Melanie; Eickelberg, Oliver; Meiners, Silke

    2014-12-01

    Cigarette smoke is the main risk factor for chronic obstructive pulmonary disease (COPD). Exposure of cells to cigarette smoke induces an initial adaptive cellular stress response involving increased oxidative stress and induction of inflammatory signaling pathways. Exposure of mitochondria to cellular stress alters their fusion/fission dynamics. Whereas mild stress induces a prosurvival response termed stress-induced mitochondrial hyperfusion, severe stress results in mitochondrial fragmentation and mitophagy. In the present study, we analyzed the mitochondrial response to mild and nontoxic doses of cigarette smoke extract (CSE) in alveolar epithelial cells. We characterized mitochondrial morphology, expression of mitochondrial fusion and fission genes, markers of mitochondrial proteostasis, as well as mitochondrial functions such as membrane potential and oxygen consumption. Murine lung epithelial (MLE)12 and primary mouse alveolar epithelial cells revealed pronounced mitochondrial hyperfusion upon treatment with CSE, accompanied by increased expression of the mitochondrial fusion protein mitofusin 2 and increased metabolic activity. We did not observe any alterations in mitochondrial proteostasis, i.e., induction of the mitochondrial unfolded protein response or mitophagy. Therefore, our data indicate an adaptive prosurvival response of mitochondria of alveolar epithelial cells to nontoxic concentrations of CSE. A hyperfused mitochondrial network, however, renders the cell more vulnerable to additional stress, such as sustained cigarette smoke exposure. As such, cigarette smoke-induced mitochondrial hyperfusion, although part of a beneficial adaptive stress response in the first place, may contribute to the pathogenesis of COPD. PMID:25326581

  4. Experimental Investigation into the Transmembrane Electrical Potential of the Forward Osmosis Membrane Process in Electrolyte Solutions

    PubMed Central

    Bian, Lixia; Fang, Yanyan; Wang, Xiaolin

    2014-01-01

    The transmembrane electrical potential (TMEP) in a forward osmosis membrane process with a single electrolyte solution as the draw and feed solutions was investigated by experiments. The effects of membrane orientation, the electrolyte species (KCl, NaCl, MgCl2, and CaCl2), concentration and concentration ratio of solutions at both sides of membrane on water flux and TMEP were investigated. The results showed that the TMEPs at different membrane orientation cannot completely coincide, which confirmed the effect of membrane asymmetry. The ion diffusion coefficients significantly affected the TMEP across the membrane, with different patterns for different electrolytes and concentrations. PMID:24957177

  5. Photogeneration of membrane potential hyperpolarization and depolarization in non-excitable cells.

    PubMed

    Ando, Jun; Smith, Nicholas I; Fujita, Katsumasa; Kawata, Satoshi

    2009-02-01

    We monitored femtosecond laser induced membrane potential changes in non-excitable cells using patchclamp analysis. Membrane potential hyperpolarization of HeLa cells was evoked by 780 nm, 80 fs laser pulses focused in the cellular cytoplasm at average powers of 30-60 mW. Simultaneous detection of intracellular Ca2+ concentration and membrane potential revealed coincident photogeneration of Ca2+ waves and membrane potential hyperpolarization. By using non-excitable cells, the cell dynamics are slow enough that we can calculate the membrane potential using the steady-state approximation for ion gradients and permeabilities, as formulated in the GHK equations. The calculations predict hyperpolarization that matches the experimental measurements and indicates that the cellular response to laser irradiation is biological, and occurs via laser triggered Ca2+ which acts on Ca2+ activated K+ channels, causing hyperpolarization. Furthermore, by irradiating the cellular plasma membrane, we observed membrane potential depolarization in combination with a drop in membrane resistance that was consistent with a transient laser-induced membrane perforation. These results entail the first quantitative analysis of location-dependent laser-induced membrane potential modification and will help to clarify cellular biological responses under exposure to high intensity ultrashort laser pulses. PMID:19137284

  6. TOM22, a core component of the mitochondria outer membrane protein translocation pore, is a mitochondrial receptor for the proapoptotic protein Bax.

    PubMed

    Bellot, G; Cartron, P-F; Er, E; Oliver, L; Juin, P; Armstrong, L C; Bornstein, P; Mihara, K; Manon, S; Vallette, F M

    2007-04-01

    The association of Bax with mitochondria is an essential step in the implementation of apoptosis. By using a bacterial two-hybrid assay and crosslinking strategies, we have identified TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), as a mitochondrial receptor of Bax. Peptide mapping showed that the interaction of Bax with TOM22 involved the first alpha helix of Bax and possibly two central alpha helices, which are homologous to the pore forming domains of some toxins. Antibodies directed against TOM22 or an antisense knockdown of the expression of TOM22 specifically inhibited the association of Bax with mitochondria and prevented Bax-dependent apoptosis. In yeast, a haploid strain for TOM22 exhibited a decreased expression of TOM22 and mitochondrial association of ectopically expressed human Bax. Our data provide a new perspective on the mechanism of association of Bax with mitochondria as it involves a classical import pathway. PMID:17096026

  7. [Effect of ethylmaleimide on the transport of Ca+ and K+ ions across mitochondrial membranes].

    PubMed

    Lofrumento, N E; Zanotti, F; Pavone, A

    1979-04-30

    As already reported, it has been found that the gradient of protons, set up across the inner membrane during the Ca2+ uptake by rat liver mitochondria, can be completely reversed by the addition of NEM. Identical results have been obtained by following the energy dependent K+ uptake. In these last conditions, the rate of H+ efflux supported by succinate oxidation is greatly enhanced only when NEM is added after rotenone. It is proposed that the increased rate other than to the inhibition of Pi uptake, as suggested by Reynafarje and Lehninger, could also be ascribed to a further decrease in the energetic level of the membrane as well as to an increased rate of succinate-Pi exchange diffusion reaction induced by NEM. A possible direct effect of NEM on succinate oxidation has been also considered to account for the inhibition observed when it is added before rotenone. PMID:554640

  8. Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

    PubMed

    Hahn, Alexander; Parey, Kristian; Bublitz, Maike; Mills, Deryck J; Zickermann, Volker; Vonck, Janet; Kühlbrandt, Werner; Meier, Thomas

    2016-08-01

    We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology. PMID:27373333

  9. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

    PubMed Central

    Vyssokikh, M Y; Katz, A; Rueck, A; Wuensch, C; Dörner, A; Zorov, D B; Brdiczka, D

    2001-01-01

    Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein. PMID:11513733

  10. Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei

    PubMed Central

    Guler, Jennifer L.; Kriegova, Eva; Smith, Terry K.; Lukeš, Julius; Englund, Paul T.

    2013-01-01

    Summary Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function. PMID:18221265

  11. Drug-induced mitochondrial dysfunction and cardiotoxicity.

    PubMed

    Varga, Zoltán V; Ferdinandy, Peter; Liaudet, Lucas; Pacher, Pál

    2015-11-01

    Mitochondria has an essential role in myocardial tissue homeostasis; thus deterioration in mitochondrial function eventually leads to cardiomyocyte and endothelial cell death and consequent cardiovascular dysfunction. Several chemical compounds and drugs have been known to directly or indirectly modulate cardiac mitochondrial function, which can account both for the toxicological and pharmacological properties of these substances. In many cases, toxicity problems appear only in the presence of additional cardiovascular disease conditions or develop months/years following the exposure, making the diagnosis difficult. Cardiotoxic agents affecting mitochondria include several widely used anticancer drugs [anthracyclines (Doxorubicin/Adriamycin), cisplatin, trastuzumab (Herceptin), arsenic trioxide (Trisenox), mitoxantrone (Novantrone), imatinib (Gleevec), bevacizumab (Avastin), sunitinib (Sutent), and sorafenib (Nevaxar)], antiviral compound azidothymidine (AZT, Zidovudine) and several oral antidiabetics [e.g., rosiglitazone (Avandia)]. Illicit drugs such as alcohol, cocaine, methamphetamine, ecstasy, and synthetic cannabinoids (spice, K2) may also induce mitochondria-related cardiotoxicity. Mitochondrial toxicity develops due to various mechanisms involving interference with the mitochondrial respiratory chain (e.g., uncoupling) or inhibition of the important mitochondrial enzymes (oxidative phosphorylation, Szent-Györgyi-Krebs cycle, mitochondrial DNA replication, ADP/ATP translocator). The final phase of mitochondrial dysfunction induces loss of mitochondrial membrane potential and an increase in mitochondrial oxidative/nitrative stress, eventually culminating into cell death. This review aims to discuss the mechanisms of mitochondrion-mediated cardiotoxicity of commonly used drugs and some potential cardioprotective strategies to prevent these toxicities. PMID:26386112

  12. Mitochondrial dynamism and cardiac fate--a personal perspective.

    PubMed

    Dorn, Gerald W

    2013-01-01

    Defects in mitochondrial biogenesis are well known to contribute to cardiac dysfunction. By contrast, mechanistic details of essential homeostatic mechanisms that maintain mitochondrial health in the heart are only recently being uncovered, and the pathological potential of these processes is largely hypothetical. I will review the role of mitochondrial dynamics, focusing on cyclic organelle fission and fusion, in normal and diseased hearts. Special attention is given to recent insights into the non-canonical functioning of the mitofusin 2 (Mfn2) outer mitochondrial membrane fusion protein as a regulator of sarcoplasmic-reticular calcium crosstalk and a critical determinant of mitophagic culling of damaged mitochondria. Because mitochondrial fusion in normal adult cardiomyocytes occurs so slowly and infrequently, I postulate that the major function of Mfn2 in the heart may not be to redundantly promote mitochondrial fusion with Mfn1, but to centrally orchestrate mitochondrial quality control.   PMID:23615052

  13. Membrane-Protein Crystallography and Potentiality for Drug Design

    NASA Astrophysics Data System (ADS)

    Yamashita, Atsuko

    Structure-based drug design for membrane proteins is far behind that for soluble proteins due to difficulty in crystallographic structure determination, despite the fact that about 60% of FDA-approved drugs target membrane proteins located at the cell surface. Stable homologs for a membrane protein of interest, such as prokaryotic neurotransmitter transporter homolog LeuT, might enable cooperative analyses by crystallography and functional assays, provide useful information for functional mechanisms, and thus serve as important probes for drug design based on mechanisms as well as structures.

  14. Potential Indexing of the Invasiveness of Breast Cancer Cells by Mitochondrial Redox Ratios.

    PubMed

    Sun, Nannan; Xu, He N; Luo, Qingming; Li, Lin Z

    2016-01-01

    The invasive/metastatic potential of cancer cells is an important factor in tumor progression. The redox ratios obtained from ratios of the endogenous fluorescent signals of NADH and FAD, can effectively respond to the alteration of cancer cells in its mitochondrial energy metabolism. It has been shown previously that the redox ratios may predict the metastatic potential of cancer mouse xenografts. In this report, we aimed to investigate the metabolic state represented by the redox ratios of cancer cells in vitro. Fluorescence microscopic imaging technology was used to observe the changes of the endogenous fluorescence signals of NADH and FAD in the energy metabolism pathways. We measured the redox ratios (FAD/NADH) of breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and SKBR3. We found that the more invasive cancer cells have higher FAD/NADH ratios, largely consistent with previous studies on breast cancer xenografts. Furthermore, by comparing the fluorescence signals of the breast cancer cells under different nutritional environments including starvation and addition of glutamine, pyruvate and lactate, we found that the redox ratios still effectively distinguished the highly invasive MDA-MB-231 cells from less invasive MCF-7 cells. These preliminary data suggest that the redox ratio may potentially provide a new index to stratefy breast cancer with different degrees of aggressiveness, which could have significance for the diagnosis and treatment of breast cancer. PMID:27526133

  15. Mitochondrial membrane peroxidizability index is inversely related to maximum life span in mammals.

    PubMed

    Pamplona, R; Portero-Otín, M; Riba, D; Ruiz, C; Prat, J; Bellmunt, M J; Barja, G

    1998-10-01

    The oxidative stress theory of aging predicts a low degree of fatty acid unsaturation in tissues of longevous animals, because membrane lipids increase their sensitivity to oxidative damage as a function of their unsaturation. Accordingly, the fatty acids analyses of liver mitochondria from eight mammals, ranging in maximum life span from 3.5 to 46 years, show that the total number of double bonds and the peroxidizability index are negatively correlated with maximum life span (r = -0. 88, P < 0.003; r = -0.87, P < 0.004, respectively). This is not due to a low content of unsaturated fatty acids in longevous animals, but mainly to a redistribution between kinds of the polyunsaturated n-3 fatty acids series, shifting from the highly unsaturated docosahexaenoic acid (r = -0.89, P < 0.003) to the less unsaturated linolenic acid (r = 0.97, P < 0.0001). This redistribution pattern strongly suggests the presence of a constitutively low delta6-desaturase activity in longevous animals (r = -0.96, P < 0.0001). Thus, it may be proposed that, during evolution, a low degree of fatty acid unsaturation in liver mitochondria may have been selected in longevous mammals in order to protect the tissues against oxidative damage, while maintaining an appropriate environment for membrane function. PMID:9788245

  16. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles.

    PubMed

    Torrano, Adriano A; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ∼50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ∼150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine. PMID:27341699

  17. Betaine is a positive regulator of mitochondrial respiration

    SciTech Connect

    Lee, Icksoo

    2015-01-09

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

  18. K+ transport and membrane potentials in isolated rat parotid acini

    SciTech Connect

    Nauntofte, B.; Dissing, S.

    1988-10-01

    42K+ transport properties of isolated rat parotid acini were characterized concomitant with measurements of membrane potentials (Em) by means of the fluorescent dye diSC3-(5). In unstimulated acini suspended in a 5 mM K+ buffer, Em was governed by the K+ and Cl- gradients and amounted to about -59 mV, a value that remained unaffected on cholinergic stimulation. In unstimulated acini, 42K+ influx was largely mediated by the Na+-K+ pump, and the residual influxes were mediated by a bumetanide-sensitive component (cotransport system) and by K+ channels. Efflux of 42K+ was largely mediated by a bumetanide-sensitive component and by K+ channels. In the unstimulated state, the cotransport system was mediating K+-K+ exchange without contributing to the net uptake of K+. Within 10 s after stimulation, a approximately 10-fold increase in the acinar K+ conductance (gK) occurred, resulting in a rapid net efflux of K+ that amounted to approximately 3.8 mmol.l cells-1.s-1. Measurements of 42K+ fluxes as a function of the external K+ concentration revealed that in the stimulated state gK increases when external K+ is raised from 0.7 to 10 mM, consistent with an activation of acinar gK by the binding of external K+ to the channel. 42K+ flux ratios as well as the effect of the K+ channel inhibitor from scorpion venom (LQV) suggest that approximately 90% of K+ transport in the stimulated state is mediated by ''maxi'' K+ channels.

  19. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    SciTech Connect

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  20. Tetrahydrocannabinol induces brain mitochondrial respiratory chain dysfunction and increases oxidative stress: a potential mechanism involved in cannabis-related stroke.

    PubMed

    Wolff, Valérie; Schlagowski, Anna-Isabel; Rouyer, Olivier; Charles, Anne-Laure; Singh, François; Auger, Cyril; Schini-Kerth, Valérie; Marescaux, Christian; Raul, Jean-Sébastien; Zoll, Joffrey; Geny, Bernard

    2015-01-01

    Cannabis has potential therapeutic use but tetrahydrocannabinol (THC), its main psychoactive component, appears as a risk factor for ischemic stroke in young adults. We therefore evaluate the effects of THC on brain mitochondrial function and oxidative stress, key factors involved in stroke. Maximal oxidative capacities V max (complexes I, III, and IV activities), V succ (complexes II, III, and IV activities), V tmpd (complex IV activity), together with mitochondrial coupling (V max/V 0), were determined in control conditions and after exposure to THC in isolated mitochondria extracted from rat brain, using differential centrifugations. Oxidative stress was also assessed through hydrogen peroxide (H2O2) production, measured with Amplex Red. THC significantly decreased V max (-71%; P < 0.0001), V succ (-65%; P < 0.0001), and V tmpd (-3.5%; P < 0.001). Mitochondrial coupling (V max/V 0) was also significantly decreased after THC exposure (1.8±0.2 versus 6.3±0.7; P < 0.001). Furthermore, THC significantly enhanced H2O2 production by cerebral mitochondria (+171%; P < 0.05) and mitochondrial free radical leak was increased from 0.01±0.01 to 0.10±0.01% (P < 0.001). Thus, THC increases oxidative stress and induces cerebral mitochondrial dysfunction. This mechanism may be involved in young cannabis users who develop ischemic stroke since THC might increase patient's vulnerability to stroke. PMID:25654095

  1. Tetrahydrocannabinol Induces Brain Mitochondrial Respiratory Chain Dysfunction and Increases Oxidative Stress: A Potential Mechanism Involved in Cannabis-Related Stroke

    PubMed Central

    Wolff, Valérie; Schlagowski, Anna-Isabel; Rouyer, Olivier; Charles, Anne-Laure; Singh, François; Auger, Cyril; Schini-Kerth, Valérie; Marescaux, Christian; Raul, Jean-Sébastien; Zoll, Joffrey; Geny, Bernard

    2015-01-01

    Cannabis has potential therapeutic use but tetrahydrocannabinol (THC), its main psychoactive component, appears as a risk factor for ischemic stroke in young adults. We therefore evaluate the effects of THC on brain mitochondrial function and oxidative stress, key factors involved in stroke. Maximal oxidative capacities Vmax (complexes I, III, and IV activities), Vsucc (complexes II, III, and IV activities), Vtmpd (complex IV activity), together with mitochondrial coupling (Vmax/V0), were determined in control conditions and after exposure to THC in isolated mitochondria extracted from rat brain, using differential centrifugations. Oxidative stress was also assessed through hydrogen peroxide (H2O2) production, measured with Amplex Red. THC significantly decreased Vmax (−71%; P < 0.0001), Vsucc (−65%; P < 0.0001), and Vtmpd (−3.5%; P < 0.001). Mitochondrial coupling (Vmax/V0) was also significantly decreased after THC exposure (1.8±0.2 versus 6.3±0.7; P < 0.001). Furthermore, THC significantly enhanced H2O2 production by cerebral mitochondria (+171%; P < 0.05) and mitochondrial free radical leak was increased from 0.01±0.01 to 0.10±0.01% (P < 0.001). Thus, THC increases oxidative stress and induces cerebral mitochondrial dysfunction. This mechanism may be involved in young cannabis users who develop ischemic stroke since THC might increase patient's vulnerability to stroke. PMID:25654095

  2. The mitochondrial genome of Euphausia superba (Prydz Bay) (Crustacea: Malacostraca: Euphausiacea) reveals a novel gene arrangement and potential molecular markers.

    PubMed

    Shen, Xin; Wang, Haiqing; Ren, Jianfeng; Tian, Mei; Wang, Minxiao

    2010-02-01

    Euphausiid krill are dominant organisms in the zooplankton population and play a central role in marine ecosystems. In this paper, we described the gene organization, gene rearrangement and codon usage in the mitochondrial genome of Euphausia superba Dana 1852 (sampling from Prydz Bay, PB). The mitochondrial genome of E. superba is more than 15,498 bp in length (partial non-coding region was not determined). Translocation of four tRNAs (trnL ( 1 ), trnL ( 2 ), trnW and trnI) and duplication of one tRNA (trnN) were founded in the mitochondrial genome of E. superba when comparing its genome with the pancrustacean ground pattern. To investigate the phylogenetic relationship within Malacostraca, phylogenetic trees based on currently available malacostracan mitochondrial genomes were built with the maximum likelihood and the Bayesian models. All analyses based on nucleotide and amino acid data strongly support the monophyly of Stomatopoda, Penaeidae, Caridea, and Brachyura, which is consistent with previous research. However, the taxonomic position of Euphausiacea within Malacostraca is unstable. From comparing the mitochondrial genome between E. superba (PB) and E. superba (sampling from Weddell Sea, WS), we found that nad2 gene contains maximal variation with 61 segregating sites, following by nad5 gene which has 12 segregating sites. Thus, nad2 and nad5 genes may be used as potential molecular markers to study the inherit diversity among different E. superba groups, which would be helpful to the exploitation and management of E. superba resources. PMID:19578978

  3. Three-dimensional organization of the endoplasmic reticulum membrane around the mitochondrial constriction site in mammalian cells revealed by using focused-ion beam tomography.

    PubMed

    Ohta, Keisuke; Okayama, Satoko; Togo, Akinobu; Nakamura, Kei-Ichiro

    2014-11-01

    The endoplasmic reticulum (ER) and mitochondria associate at multiple contact sites to form specific domains known as mitochondria-ER associated membranes (MAMs) that play a role in the regulation of various cellular processes such as Ca2+ transfer, autophagy, and inflammation. Recently, it has been suggested that MAMs are also involved in mitochondrial dynamics, especially fission events. Cytological analysis showed that ER tubules were frequently located close to each other in mitochondrial fission sites that accumulate fission-related proteins. Three-dimensional (3D) imaging of ER-mitochondrial contacts in yeast mitochondria by using cryo-electron tomography also showed that ER tubules were attached near the constriction site, which is considered to be a fission site1). MAMs have been suggested to play a role in the initiation of mitochondrial fission, although the molecular relationships between MAMs and the mitochondrial fission process have not been established. Although an ER-mitochondrial membrane association has also been observed at the fission site in mammalian mitochondria, the detailed organization of MAMs around mammalian mitochondria remains to be established. To visualize the 3D distribution of the ER-mitochondrial contacts around the mitochondria, especially around the constriction site in mammalian cells, we attempted 3D structural analysis of the mammalian cytoplasm using high-resolution focused ion-beam scanning electron microscopy (FIB-SEM) tomography, and observed the distribution pattern of ER contacts around the mammalian mitochondrial constriction site.Rat hepatocytes and HeLa cells were used. Liver tissue was obtained from male rats (Wistar, 6W) fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) under deep anesthesia. HeLa cells were fixed with the same fixative. The specimens were then stained en bloc to enhance membrane contrast and embedded in epoxy resin2). The surface of

  4. Rapid novel test for the determination of biofouling potential on reverse osmosis membranes.

    PubMed

    Manalo, Cervinia V; Ohno, Masaki; Okuda, Tetsuji; Nakai, Satoshi; Nishijima, Wataru

    2016-01-01

    A novel method was proposed to determine biofouling potential by direct analysis of a reverse osmosis (RO) membrane through fluorescence intensity analysis of biofilm formed on the membrane surface, thereby incorporating fouling tendencies of both feedwater and membrane. Evaluation of the biofouling potential on the RO membrane was done by accelerated biofilm formation through soaking of membranes in high biofouling potential waters obtained by adding microorganisms and glucose in test waters. The biofilm formed on the soaked membrane was quantified by fluorescence intensity microplate analysis. The soaking method's capability in detecting biofilm formation was confirmed when percentage coverage obtained through fluorescence microscopy and intensity values exhibited a linear correlation (R(2) = 0.96). Continuous cross-flow experiments confirmed the ability and reliability of the soaking method in giving biofouling potential on RO membranes when a good correlation (R(2) = 0.87) between intensity values of biofilms formed on the membrane during soaking and filtration conditions was obtained. Applicability of the test developed was shown when three commercially available polyamide (PA) RO membranes were assessed for biofouling potential. This new method can also be applied for the determination of biofouling potential in water with more than 3.6 mg L(-1) easily degradable organic carbon. PMID:27332844

  5. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles

    NASA Astrophysics Data System (ADS)

    Torrano, Adriano A.; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ~50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ~150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and

  6. Two Membrane-Associated Regions within the Nodamura Virus RNA-Dependent RNA Polymerase Are Critical for both Mitochondrial Localization and RNA Replication

    PubMed Central

    Gant, Vincent U.; Moreno, Stephanie; Varela-Ramirez, Armando

    2014-01-01

    ABSTRACT Viruses with positive-strand RNA genomes amplify their genomes in replication complexes associated with cellular membranes. Little is known about the mechanism of replication complex formation in cells infected with Nodamura virus. This virus is unique in its ability to lethally infect both mammals and insects. In mice and in larvae of the greater wax moth (Galleria mellonella), Nodamura virus-infected muscle cells exhibit mitochondrial aggregation and membrane rearrangement, leading to disorganization of the muscle fibrils on the tissue level and ultimately in hind limb/segment paralysis. However, the molecular basis for this pathogenesis and the role of mitochondria in Nodamura virus infection remains unclear. Here, we tested the hypothesis that Nodamura virus establishes RNA replication complexes that associate with mitochondria in mammalian cells. Our results showed that Nodamura virus replication complexes are targeted to mitochondria, as evidenced in biochemical, molecular, and confocal microscopy studies. More specifically, we show that the Nodamura virus RNA-dependent RNA polymerase interacts with the outer mitochondrial membranes as an integral membrane protein and ultimately becomes associated with functional replication complexes. These studies will help us to understand the mechanism of replication complex formation and the pathogenesis of Nodamura virus for mammals. IMPORTANCE This study will further our understanding of Nodamura virus (NoV) genome replication and its pathogenesis for mice. NoV is unique among