Cells require adenosine triphosphate (ATP) to drive the myriad processes associated with growth, replication, and homeostasis. Eukaryotic cells rely on mitochondria to produce the vast majority of their ATP. Mitochondria consist of a relatively smooth outer mitochondrial membrane (OMM) and a highly complex inner mitochondrial membrane (IMM), containing numerous invaginations, called cristae, which house the molecular machinery of oxidative phosphorylation (OXPHOS). Although mitochondrial form and function are intimately connected, limitations in the resolution of live-cell imaging have hindered the ability to directly visualize the relationship between the architecture of the IMM and its associated bioenergetic properties. Using advanced imaging technologies, including Airyscan, stimulated emission depletion (STED), and structured illumination microscopy (SIM), we developed an approach to image the IMM in living cells. Staining mitochondria with various ΔΨm-dependent dyes, we found that the fluorescence pattern along the IMM was heterogeneous, with cristae possessing a significantly greater fluorescence intensity than the contiguous inner boundary membrane (IBM). Applying the Nernst equation, we determined that the ΔΨm of cristae is approximately 12 mV stronger than that of IBM, indicating that the electrochemical gradient that drives ATP synthesis is compartmentalized in cristae membranes. Notably, deletion of key components of the mitochondrial contact site and cristae organizing system (MICOS), as well as OPA1, which regulate crista junctions (CJs), decreased ΔΨm heterogeneity. Complementing our super-resolution imaging of cristae in living cells, we also developed a machine-learning protocol to quantify IMM architecture. Tracking real-time changes in cristae density, size, and shape, we determined that cristae dynamically remodel on a scale of seconds. Furthermore, we found that cristae move away from sites of mitochondrial fission, and, prior to mitochondrial fusion, the IMM forms finger-like protrusions bridging the membranes of the fusing organelles. Lastly, we investigated the role of the motor adaptor protein, Milton1/TRAK1, in mitochondrial dynamics. Patient-derived Milton1-null fibroblasts not only had impaired mitochondrial motility but exhibited fragmentation corresponding to a roughly 40% decrease in mitochondrial aspect ratio and a 17% increase in circularity, associated with increased DRP1 activity. Conversely, we found that overexpression of Milton1 led to mitochondrial hyperfusion, decreased DRP1 activity, and aberrant clustering of mtDNA. Overall, our studies directly demonstrate that maintaining mitochondrial architecture is essential for preserving the functionality of mitochondria, the hubs of eukaryotic metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19175号 / 医博第4017号 / 新制||医||1010(附属図書館) / 32167 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 松原 和夫, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aik, aCliand aNai), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 μml-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 m m tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33±1 mV. l-alanine uptake first depolarized Vm by 2±0.2 mV and then hyperpolarized Vm by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aNaiincreased by 30% from 19±2 to 25±3 m m (P < 0.01), and aKidid not change significantly from 83±3 m m. However, with added ouabain (1 m m) l-alanine caused only a 2-mV increase in Vm, but now aKidecreased from 61±3 to 54±5 m m (P < 0.05). Hyperpolarization of Vm by l-alanine uptake also resulted in a 38% decrease of aClifrom 20±2 to 12±3 m m (P < 0.001). Changes in Vm and VCl - Vm voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes. Added Ba2+ abolished the l-alanineinduced hyperpolarization, and aCliremained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte aKiis regulated by increased Na+-K+ pump activity during l-alanine uptake in spite of cell swelling and increased Vm due to increased K+ conductance. The hyperpolarization of Vm during l-alanine uptake provides electromotive force to decrease aCli. The latter may contribute to hepatocyte volume regulation during organic solute transport.

The thermotolerance, an adaptive phenomenon that is accompanied by the phenotypic plasticity which is the adjustment of physiology, biochemistry and metabolism of every cellular function by the hidden mechanism. Mitochondrion, the powerhouse of the cell that determines the functional integrity of every cellular homeostasis and functional phycological processes should provide its association in regulating the thermotolerance as well. This study assessed the mitochondrial function in regulating and determining the limit of thermo tolerance in the Daphnia magna of different geographical regions of the world, mainly sub grouped as temperature tolerant clones (IL) and temperature sensitive clones (GB). The acclimation effects or the adjustment of the preexisting biological properties help the organism adjust its biological processes to the changing habitat to maintain the cellular functional integrity. The clonal divergence as well as the acclimation show a clear pattern in limiting the thermotolerance and the prediction is the temperature tolerant clones should show higher adjustment of the mitochondrial function than temperature sensitive ones. We hypothesize that the damage in the mitochondrial membrane integrity by different mito-toxins should decrease the heat tolerance by decreasing the membrane potential and fluidity. The integrated mitochondrial function was assessed in acclimated clones by using the molecular studies as well as observation of behavioral and phenotypic plasticity. Due to the specific effects of each mito-toxins (CCCP, NaN3 and DNP) on different complexes (I-IV and ATP synthase) in ETC, we determined the mitochondrial membrane integrity by the Rhodamine 123 alongside with the lactate assay for measuring the mitochondrial integrity. Among all these three mito-toxins, CCCP show significant effect on limiting the heat tolerance. The lower lactate accumulation was observed in the temperature-tolerant clones acclimated in cold temperatures (18°C) which indicates the higher mitochondrial adjustment than the temperature sensitive clones. The concluding remark is that thermal tolerance is determined by the adjustment of mitochondrial function which accompanied with the adjustment to the mitochondrial respiration as well as the adjustment to membrane potential and fluidity.

Membrane transport processes underlie many purification technologies. The efficiency of a membrane separation process depends upon material throughput (flux), and the degree to which the membrane discriminates amongst species in the feed stock (selectivity). In a supported liquid membrane, flux may be enhanced by carrier molecules, which act as catalysts of translocation. Carrier molecules also confer selectivity, via differential molecular recognition of the substances in the feed stock. The effect of electrical potential on the flux and selectivity of carrier-containing supported liquid membranes is not well documented. We elected to study the effect of electrical potential on supported liquid membranes containing valinomycin, a potassium ionophore, and a calixarene ester, a sodium ionophore. In these systems, the open circuit membrane potential could be made positive or negative by the choice of anion. With both of these carriers, we observed that selectivity for potassium or sodium salts was dependent on the open circuit membrane potential. To confirm that electrical potential was responsible for the observed selectivity variance, we applied a potential across the membrane using a potentiostat. The applied potential created conditions for carrier-mediated electrodialysis, where oxidation and reduction reactions on either side of the membrane act as the driving force for transmembrane flux of charged species. In chronoamperometry experiments, we found that selectivity for potassium or sodium ion was dependent on the applied electrical potential. Subject to some constraints, selectivity and flux could be controlled by the application of positive or negative electrical potentials. Linear sweep voltammetry experiments allowed for the rapid prediction of the potential that must be applied to achieve optimal selectivity. We also found that membrane potential measurements, as well as the magnitude of current that flows in chronoamperometry experiments, could be interpreted to predict Eisenman and Hofmeister sequences. These results are novel, and await a convincing theoretical justification. The results also suggest that a separation technology could be developed around the idea of modulating selectivity with electrical potential. In this regard, carrier-mediated electrodialysis may be suitable for the sequestration of toxic or radioactive heavy metals, and a large number of carrier molecules for metal ions are currently known. The technique may also be suitable for separating organic molecules, such as high-value chiral pharmaceuticals. Supported liquid membranes are a useful research tool, but industrial applications may require a more stable membrane architecture. / Graduate

Complexity lies halfway between stochasticity and determinism, suggesting that brain activity is neither fully random nor fully predictable but lives by the rules of nonlinear high- and low-complexity dynamics. One important aspect of brain function is noise-like activity (NLA), defined as background, electrical potential fluctuations in the nervous system distinct from spiking rhythms in the foreground. The objective of this thesis was to investigate the neurodynamical complexity of NLA recorded at the cellular and local network scales in in vitro preparations of mouse and human hippocampal tissue, under healthy and epileptiform conditions. In particular, it was found that neuronal NLA arises out of the physiological contributions of gap junctions and chemical synaptic channels and is characterized by a spectrum of complexity, ranging from high- to low-complexity, that was measured using methods from nonlinear dynamical systems theory. Importantly, the complexity of background, neuronal NLA was shown to depend on the degree of cellular interconnectivity to the surrounding local network. In addition, the complexity and multifractality of NLA was further studied at the cellular and local network scales in epileptiform transitions to seizure-like events, identifying emergent low-complexity and reduced multifractality (bordering on monofractal-type dynamics) in the pathological ictal state. Finally, dual intracellular recordings of hippocampal epileptiform activity were analyzed to measure NLA synchronicity, showing evidence for increased same- and cross-frequency correlations and increased phase synchronization in the pathological ictal state. Convergence towards increased phase synchrony manifested in lower frequency regions including theta (4-10 Hz) and beta (12-30 Hz), but also in higher frequency bands (gamma, 30-80 Hz). In summary, there is evidence to suggest that background NLA captures important neurodynamical information pertinent to the classification and identification of brain state transitions in healthy and epileptiform hippocampal dynamics, using sophisticated neuroengineering analyses of these physiological signals.

La complexité des mécanismes de transport en nanofiltration (NF) nécessite ledéveloppement d’outils de modélisation fiables permettant de comprendre et d’optimiser lesopérations de séparation en NF.La présente étude comprend deux volets. Le premier porte sur la rétention d’un soluténeutre – le poly(éthylène glycol) à 600 g mol-1 (PEG) – seul et en présence d’ions, par unemembrane organique de NF (polyamide). Les résultats obtenus montrent que la rétention dusoluté neutre chute en présence d’ions et ce, d’autant plus que la concentration du sel estélevée. Ce phénomène ne peut être expliqué par le seul phénomène de déshydratation dusoluté neutre par les ions environnants. Un phénomène supplémentaire de gonflement de porea été mis en évidence et corrélé à la densité de charge membranaire. La contribution des deuxphénomènes à la chute du taux de rejet du PEG a été évaluée pour différents sels à plusieursconcentrations.Le deuxième volet du travail est consacré à (i) l’évaluation de la constante diélectrique desolutions à l’intérieur de nanopores (ep) d’une membrane organique de NF (polyamide) àpartir de mesures de potentiel de membrane et (ii) la validation de cette méthode. Les valeursobtenues s’avèrent être inférieures à celle de la solution externe (effet du confinement) etdiminuer avec l’augmentation de la proportion en ions calcium du mélange (effet structurantdes ions). L’accord entre les constantes diélectriques ep déterminées à partir de mesures depotentiel de membrane et de taux de rejet ionique (mélanges ternaires) valide la cohérence dumodèle de transport utilisé (exclusion stérique, électrique et diélectrique aux interfaces) etmontre que les mesures de potentiel de membrane peuvent être envisagées pour l’évaluationcorrecte de la constante diélectrique à l’intérieur de nanopores / The complexity of transport mechanisms in nanofiltration (NF) requires the developmentof reliable modelling tools for understanding and optimizing the separation process.This study is composed of two parts. The first one focuses on the retention of a neutralsolute – poly(ethylene glycol) 600 g mol-1 (PEG) – in single solute solutions and in thepresence of mineral salts by an organic NF membrane (polyamide). Results show that PEGrejection is significantly lower in mixed-solute solutions and that rejection rate drop increaseswith salt concentration. This phenomenon cannot be imputed to only the partial dehydrationof PEG molecules by surrounding ions (salting-out effect). The additional hypothesis whichwas considered in the present work is an increase in the effective pore size (pore swelling).This hypothesis was supported by electrokinetics charge density data. The contribution ofpore swelling and salting-out to the overall decrease in the rejection rate of PEG wasevaluated for different salts at various concentrations.The second part of this study deals with (i) the determination of the dielectric constantinside pores (ep) of an NF organic membrane from membrane potential measurements and (ii)the validation of this technique. Membrane potential data were analyzed by means of theSEDE (steric, electric and dielectric exclusion) transport model. ep values were found to besmaller than the bulk value and to decrease when sodium ions were replaced by calcium ions.The agreement between ep values obtained from membrane potential measurements and thosecalculated from ion rejection rate data (ternary mixtures) highlights the global coherence ofthe transport model used and shows that membrane potential measured with electrolytemixtures can be used to determine the dielectric constant inside pores with no requirement ofadditional rejection rate measurements.

O quadro de resistência à insulina em humanos está fortemente relacionado ao acumulo de lipídeos intracelulares, a inatividade física e ao aumento de espécies reativas de oxigênio (ERO). O objetivo deste estudo foi verificar se o aumento na oferta de nutrientes incluindo glicose e ácido graxo palmítico pode alterar o potencial de membrana mitocondrial, a respiração, a produção de espécies reativas de oxigênio e a resposta a insulina em células do tecido muscular. Nossos resultados mostram que a exposição de células musculares a elevada disponibilidade de substratos resultou em diminuição do potencial de membrana mitocondrial, e aumento da respiração no estado IV e da expressão do RNAm da proteína desacopladora mitocondrial UCP-3. Mostrando a existência de um mecanismo de desacoplamento intrínseco em células do músculo esquelético ativado em situações de elevada oferta de nutrientes. Nessas condições observamos redução do acoplamento e da eficiência termodinâmica mitocondrial. Interessantemente, essa capacidade de desacoplamento parece ser perdida cronicamente como indicado pelos nossos resultados de consumo de oxigênio no período de 48h favorecendo uma menor atividade mitocondrial, aumento de EROs e redução da razão GSH/GSSG. Imagens de microscopia eletrônica em cultura primária e expressão gênica do PGC1-, um reconhecido gene regulador da biogênese mitocondrial, não demonstraram diferença entre controle e tratamento com palmitato. O ácido palmito resultou na redução da fosforilação de Akt, bem como, na captação de glicose estimulada por insulina. Nossos achados, portanto, sugerem que uma redução do acoplamento termodinâmico mitocondrial e do sistema antioxidante, juntamente com aumento do peróxido de hidrogênio, estão fortemente relacionados a redução da resposta a insulina. Deste modo, nosso estudo sugere um papel importante da mitocôndria na resposta a insulina. / The insulin resistance in human framework is strongly related to the accumulation of intracellular lipids, physical inactivity and increased reactive oxygen species (ROS). The aim of this study was to determine whether the increase in nutrient supply including glucose and palmitic fatty acid can change the mitochondrial membrane potential, respiration, production of reactive oxygen species and the insulin response in muscle tissue cells. Our results show that exposure of muscle cells to high availability of the substrate resulted in decreased mitochondrial membrane potential, in increased respiration in the state IV and mRNA expression of mitochondrial uncoupling protein UCP-3. Showing the existence of an intrinsic uncoupling mechanism of skeletal muscle cells activated in situations of high supply of nutrients. However, under these conditions we observed a reduction of the coupling and mitochondrial thermodynamic efficiency. Interestingly, this decoupling capacity was chronically lost as indicated by our results in the 48 hours period favoring a lower mitochondrial activity, increase of ROS and reduced GSH / GSSG ratio. Images from electron microscopy and gene expression of PGC1-, a recognized regulatory gene of mitochondrial biogenesis, showed no difference between control and treatment with palmitate. The palm acid resulted in reduced phosphorylation of Akt, as well as glucose uptake stimulated by insulin. Our findings thus suggest that a reduction in mitochondrial antioxidant and thermodynamic coupling system, along with increase in the hydrogen peroxide, are closely related to reducing insulin response. Thus, our findings suggest a role of mitochondria in insulin response.

O quadro de resistência à insulina em humanos está fortemente relacionado ao acumulo de lipídeos intracelulares, a inatividade física e ao aumento de espécies reativas de oxigênio (ERO). O objetivo deste estudo foi verificar se o aumento na oferta de nutrientes incluindo glicose e ácido graxo palmítico pode alterar o potencial de membrana mitocondrial, a respiração, a produção de espécies reativas de oxigênio e a resposta a insulina em células do tecido muscular. Nossos resultados mostram que a exposição de células musculares a elevada disponibilidade de substratos resultou em diminuição do potencial de membrana mitocondrial, e aumento da respiração no estado IV e da expressão do RNAm da proteína desacopladora mitocondrial UCP-3. Mostrando a existência de um mecanismo de desacoplamento intrínseco em células do músculo esquelético ativado em situações de elevada oferta de nutrientes. Nessas condições observamos redução do acoplamento e da eficiência termodinâmica mitocondrial. Interessantemente, essa capacidade de desacoplamento parece ser perdida cronicamente como indicado pelos nossos resultados de consumo de oxigênio no período de 48h favorecendo uma menor atividade mitocondrial, aumento de EROs e redução da razão GSH/GSSG. Imagens de microscopia eletrônica em cultura primária e expressão gênica do PGC1-, um reconhecido gene regulador da biogênese mitocondrial, não demonstraram diferença entre controle e tratamento com palmitato. O ácido palmito resultou na redução da fosforilação de Akt, bem como, na captação de glicose estimulada por insulina. Nossos achados, portanto, sugerem que uma redução do acoplamento termodinâmico mitocondrial e do sistema antioxidante, juntamente com aumento do peróxido de hidrogênio, estão fortemente relacionados a redução da resposta a insulina. Deste modo, nosso estudo sugere um papel importante da mitocôndria na resposta a insulina. / The insulin resistance in human framework is strongly related to the accumulation of intracellular lipids, physical inactivity and increased reactive oxygen species (ROS). The aim of this study was to determine whether the increase in nutrient supply including glucose and palmitic fatty acid can change the mitochondrial membrane potential, respiration, production of reactive oxygen species and the insulin response in muscle tissue cells. Our results show that exposure of muscle cells to high availability of the substrate resulted in decreased mitochondrial membrane potential, in increased respiration in the state IV and mRNA expression of mitochondrial uncoupling protein UCP-3. Showing the existence of an intrinsic uncoupling mechanism of skeletal muscle cells activated in situations of high supply of nutrients. However, under these conditions we observed a reduction of the coupling and mitochondrial thermodynamic efficiency. Interestingly, this decoupling capacity was chronically lost as indicated by our results in the 48 hours period favoring a lower mitochondrial activity, increase of ROS and reduced GSH / GSSG ratio. Images from electron microscopy and gene expression of PGC1-, a recognized regulatory gene of mitochondrial biogenesis, showed no difference between control and treatment with palmitate. The palm acid resulted in reduced phosphorylation of Akt, as well as glucose uptake stimulated by insulin. Our findings thus suggest that a reduction in mitochondrial antioxidant and thermodynamic coupling system, along with increase in the hydrogen peroxide, are closely related to reducing insulin response. Thus, our findings suggest a role of mitochondria in insulin response.

<p>Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged <i>in vitro</i> culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.</p><p>In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.</p>