Dissertations / Theses on the topic 'Cellular bioenergetics'

In this thesis, thermodynamic-based mathematical modelling is combined with experimental in vitro extracellular flux analysis in order to assess drug redox cycling and cellular bioenergetics. It is widely accepted that pharmacological activity of certain classes of drugs (e.g. anticancer, antimalarial) is related to their ability to accept one or two electrons. However, pharmacological activity via redox cycling is an understated mechanism of toxicity associated with many classes of drugs. In particular, oxidative stress as a result of redox cycling plays a pivotal role in the cause of cardiac toxicity. For example, doxorubicin is an anti-neoplastic drug used to treat cancer. It has strong links to redox cycling-induced cardiac toxicity associated directly with elevated levels of reactive oxygen species (ROS) and oxidative stress within the mitochondria. The underlying mechanisms of redox cycling is very difficult to elucidate, due to the fleeting existence of the radical species. However, assessment of such cellular bioenergetics can be ameliorated with the aid of computational assistance. In chapter 2 the development of a novel thermodynamic-based in silico model of doxorubicin redox cycling is described, which is parameterized using data from in vitro extracellular flux analysis. The model is used to simulate mitochondrial-specific ROS, with its outputs confirmed against in vitro data. Chapter 3 describes construction of a pH-dependent thermodynamic model of hepatic glycolytic flux, used to determine the role of the monocarboxylate transporter 1 flux during extracellular acidification. Finally, chapter 4 describes a thermodynamic-based in silico model of mitochondrial bioenergetics, capable of simulating oxygen consumption rates of a cohort of in vitro human primary hepatocyte data. The model is finally used to simulate perturbations in key bioenergetic variables and reaction fluxes, illustrating the resulting changes on mitochondrial pH, membrane potential and subsequent oxygen consumption rates.

Primena biomarkera ćelijske energetike, uključujući indikatore metabolizma kreatina u krvi, je relativno novijeg datuma, gde se ovi indikatori koriste kao mogući pokazatelji stanja organizma pri maksimalno intenzivnim fizičkim aktivnostima. Cilj istraživanja je obuhvatao utvrđivanje efekata pojedinačnih epizoda aerobnog i anaerobnog vežbanja maksimalnog intenziteta na biomarkere perifernog zamora i ćelijske bioenergetike kod mladih muškaraca i žena. Istraživanje je dizajnirano tako da obuhvati populaciju fizički aktivnih muškaraca i žena, kao i populaciju aktivnih sportista. U prvom eksperimentalnom tretmanu fizički aktivni ispitanici muškog (n =12) i ženskog pola (n = 11) podvrgnuti su test protokolima aerobnog i anaerobnog opterećenja maksimalnog intenzivnog i kratkog trajanja. Tokom aerobnog test protokola ispitanici su trčali do maksimalnog voljnog otkaza na tredmil traci sa progresivnim povećanjem opterećenja. Pri anaerobnom test protokolu ispitanici su izvršili testiranje snažne izdržljivosti gornjih ekstremiteta do otkaza potiskom sa ravne klupe, uz opterećenje od 25% od njihove telesne težine. Drugi eksperimentalni tretman je sačinjen iz pre-eksperimentalnog testiranja kardiorespiratorne forme i eksperimentalne protokol sesije trčanja do maksimalnog voljnog otkaza na pokretnoj traci, pri konstantnoj individualnoj brzina trčanja na anaerobnom pragu. U ovom eksperimentalnom tretmanu bila je uključena populacija aktivnih sportista     (n = 10). Pre, tokom i nakon eksperimentalnih sesija praćena je koncentracija različitih biohemijskih i hematoloških markera: guanidinosirćetna kiselina (GAA); kreatin (Cr); kreatinin (Crn); laktat (Lac); interleukin-6 (IL-6); kreatin kinaza (CK); kortizol (Cor). Rezultati prvog eksperimentalnog tretmana su utvrdili statistički značajne promene u koncentraciji GAA, Cr i Crn u ktvi pre i nakon pojedinačne epizode aerobnog i anaerobnog vežbanja maksimalnim intenzitetom. Utvrđena je i statistički značajna povezanost između vežbanjem-indukovanih promena u cirkulatornim vrednostima GAA, Cr, Crn za vreme pre, tokom i nakon drugog eksperimentalnog tretmana. Uočena je statistički značajna povezanost između promena koncentracije GAA, Cr, Crn u serumu sa tradicionalnim biomarkerima perifernog zamora (IL6, Cor, Lac, CK). Dobijeni rezultati ukazuju na mogućnost primene biomarkera metabolizma kreatina u krvi prilikom praćenja i evaluacije stanja organizma tokom maksimalnih intenzivnih fizičkih aktivnosti kod mladih muškaraca i žena.
The use of biomarkers of cellular bioenergetics in exercise science appears more prevalent in recent years, where these outcomes perhaps describe changes in creatine metabolism during strenuous exercise. The aim of this study was to determine the effects of individual episodes of strenuous aerobic and anaerobic exercise on several biomarkers of peripheral fatigue and cellular bioenergetics in young men and women. The study recruited physically active men and women, and active athletes. In the first experiment, physically active men (n = 12) and women (n = 11) were subjected to strenuous aerobic and anaerobic exercise. During the aerobic test, subjects ran to exhaustion while during the anaerobic test, subjects performed repetitive bench press exercise. The second experimental treatment consisted of a pre-experimental testing of cardiorespiratory fitness, and an experimental protocol of a strenuous running session to exhaustion at constant individual running speed at the anaerobic threshold; active athletes (n = 10) were included in this experimental treatment. The blood levels of various biochemical and hematological markers were monitored before, during and after the experimental sessions, including guanidinoacetic acid (GAA); creatine (Cr); creatinine (Crn); lactate (Lac); interleukin-6 (IL-6); creatine kinase (CK); cortisol (Cor), and plethora of other physiological outcomes. We found statistically significant changes in serum GAA, Cr and Crn before and after a single session of strenuous aerobic and anaerobic exercise. A significant correlation was found between exercise-induced changes in serum GAA, Cr and Crn before, during and after the second experimental intervention. A statistically significant association was observed between changes in serum GAA, Cr, Crn and traditional biomarkers of peripheral fatigue (IL6, Cor, Lac, CK). The results of the present study suggest that biomarkers of creatine metabolism might be used as innovative tools in monitoring strenuous exercise in young men and women.

The clinical potential of pharmacological ascorbate (P-AscH-; IV delivery achieving mM concentrations in blood) as an adjuvant in cancer therapy is being re-evaluated. At mM concentrations, P-AscH- is thought to exhibit anti-cancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer, MIA PaCa-2, PANC-1, and 339 cells, to examine the effects of P-AscH- on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH- induces both nuclear and mitochondrial DNA damage. In response to this DNA damage, we observed that poly (ADP-ribose) polymerase-1 (PARP-1) is hyperactivated, as determined by increased formation of poly (ADP-ribose) polymer. Using our unique absolute quantitation, we found that the P-AscH--mediated the overactivation of PARP-1, which results in consumption of NAD+, and subsequently depletion of ATP (potential energy crisis) leading to mitotic cell death. Time-course studies with MIA PaCa-2 cells showed that the level of NAD+ and ATP were reduced by 80% immediately after a 1-h exposure to P-AscH- (4 mM; 14 pmol cell-1); both species returned to near basal levels within 24 h. In parallel with these metabolic and energetic restorations, the lesions in nuclear DNA were removed within 3 h; however, even after 24 h, lesions in mitochondrial DNA were only partially repaired. We have also found that the Chk1 pathway has a major role in the maintenance of genomic integrity following treatment with P-AscH-. Hence, combinations of P-AscH- and Chk1 inhibitors could have the potential to improve outcomes of cancer treatment. Hyperactivation of PARP-1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 Analyzer, we observed no changes in OCR or ECAR/PPR following treatment with P-AscH-. OCR and ECAR/PPR together indicate the rate of production of intracellular ATP; therefore, the rate of production is unchanged after challenge with P-AscH-. Thus, the severe decrease in ATP is due solely to increased demand. Genetic deletion and pharmacological inhibition of PARP-1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that loss of NAD+ and ATP are secondary factors in the toxicity of P-AscH-, and damage to DNA is the primary factor. These preclinical findings can guide the best use of P-AscH- as an adjuvant in cancer therapy.

Biochemistry
Ph.D.
Mitochondrial calcium (Ca2+) uptake has been studied for over five decades, with crucial insights into its underlying mechanisms enabled by development of the chemi-osmotic hypothesis and appreciation of the considerable voltage present across the inner mitochondrial membrane (ΔΨm) generated by proton pumping by the respiratory chain (Carafoli, 1987; Nicholls, 2005). However, the molecules that regulate mitochondrial Ca2+ uptake have only recently been identified (Jiang et. al., 2009; Perocchi et. al., 2010) and further work was needed to clarify how these molecules regulate mitochondrial Ca2+ uptake. Leucine Zipper EF hand containing Transmembrane Protein 1 (LETM1) acts as a regulator of mitochondrial Ca2+ uptake distinct from the mitochondrial Ca2+ uniporter (MCU) pathway (Jiang et. al., 2009). However, a controversy exists regarding the function of LETM1 (Nowikovsky et. al., 2004). Therefore, I asked if LETM1 played a role in mitochondrial Ca2+ uptake and if LETM1 regulated cellular bioenergetics and basal autophagy. To further characterize mitochondrial calcium uptake, we asked how Mitochondrial Calcium Uptake 1 (MICU1) regulates MCU activity by quantifying basal mitochondrial Ca2+ and MCU uptake rates in MICU1 ablated cells. The following work characterizes the molecules that regulate mitochondrial Ca2+ uptake and their mechanistic function on decoding calcium signals. Since LETM1 is the Ca2+/H+ antiporter, I hypothesize that alterations in LETM1 expression and activity will decrease mitochondrial Ca2+ uptake and will result in impaired mitochondrial bioenergetics. As a regulator of free intracellular Ca2+, mitochondrial Ca2+ uptake and the orchestra of its regulatory molecules have been implicated in many human diseases. Mitochondria act both upstream by regulating cytosolic Ca2+ concentration and as downstream effectors that respond to Ca2+ signals. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter (Jiang et. al., 2009); however characterization of the functional role of LETM1-mediated Ca2+ transfer remained unstudied. Therefore the specific aims of this project were to determine how LETM1 regulates Ca2+ homeostasis and bioenergetics under physiological settings. Secondly, this project aimed to characterize how LETM1-dependent Ca2+ signaling regulates ROS production and autophagy. The data presented here confirmed that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake. Furthermore, in-depth approaches including either deletion of EF-hand or mutation of critical EF-hand residues (D676A D688KLETM1) impaired histamine (GPCR agonist)-induced mitochondrial Ca2+ uptake. Knockdown of LETM1 resulted in bioenergetic collapse and promoted LC3-positive multilamellar vesicle formation, indicative of autophagy induction. Interestingly, knockdown of LETM1 significantly reduced complex IV but not complex I and complex II-mediated oxygen consumption rate (OCR). In contrast, cellular NADH and mitochondrial membrane potential (ΔΨm) were unaltered in both control and LETM1 knockdown cells. LETM1 has been implicated in formation of the supercomplexes of the electron transport chain (Tamai et. al., 2008). In support, these studies show that LETM1 knockdown results in increased reactive oxygen species (ROS) production. These results for the first time demonstrate that LETM1 controls cellular bioenergetics through regulation of mitochondrial Ca2+ and ROS. MICU1 was identified as an essential regulator of the mitochondrial Ca2+ uniporter (Perocchi et. al., 2010). Therefore, this project specifically aimed to determine how MICU1 regulates the mitochondrial Ca2+ uniporter. Interestingly, the data presented here suggest that MICU1 is not necessary for uniporter activity. Instead, loss of MICU1 caused mitochondria to constitutively load Ca2+ at rest which resulted in a host of cellular phenotypes. This result led to further questions on how MICU1 knockdown affects cellular bioenergetics and if MICU1 is essential for cell survival under stress. MICU1 ablation influenced pyruvate dehydrogenase activity and ROS production. Subsequent investigations demonstrated that increased basal ROS left cells poised to ceramide-induced cell death thereby suggesting the role of MICU1 in cell survival. Collectively, the data presented here show that MICU1 is necessary to control constitutive mitochondrial Ca2+ uptake during rest. This work demonstrates that LETM1 regulates a distinct mode of mitochondrial Ca2+ uptake pathway whereas MICU1 controls mitochondrial Ca2+ uniporter activity. Further studies are required to uncover the potential role of these two mitochondrial-resident Ca2+ regulators in health and disease.
Temple University--Theses

L'inflammation est associée à des modifications du métabolisme cellulaire avec une glycolyse (libération de lactate) accrue accompagnée d'une baisse de la phosphorylation oxydative mitochondriale. L'inflammation cause l'hyperosmolarité du milieu extracellulaire. Cette thèse examine les effets de l'hyperosmolarité sur le métabolisme énergétique cellulaire. Nous avons mesuré la consommation d'oxygène cellulaire (OCR) et la production d'acide (PPR) c'est à dire de lactate libéré dans le milieu extérieur avec deux approches expérimentales : l'oxygraphie haute résolution (O2k Oroboros instrument) pour l'OCR et l'analyseur de flux extracellulaires (Seahorse Agilent) pour l'OCR et le PPR. L'exposition de cellules à des conditions hyperosmolaires (600 mOsmoles au lieu de la valeur normale 300) cause une répression de la consommation d'oxygène qui s'établit en quelques minutes et dure des heures (indéfiniment?) et à la longue affecte la viabilité cellulaire. Cet effet a été retrouvé sur plusieurs types cellulaires: CHO (épithélium ovarien), HT29 (colonocytes), HEK293 (rein embryonnaire), SH-SY5Y (neuroblastome). Il est reproduit avec trois osmolytes différents: le mannitol, le polyéthylène glycol et le chlorure de sodium. Un stress osmotique plus modéré (450 mOsm) cause une même chute de la respiration mais de durée limitée (une-deux heures). Une recherche des mécanismes à l'origine de cette inhibition montre que l'hyperosmolarité altère la fonction mitochondriale de différentes manières. Un premier effet est une inhibition du système enzymatique de production d'ATP. En présence de glucose cette inhibition s'accompagne d'une importante augmentation de la glycolyse qui cause une inhibition mitochondriale supplémentaire qui repose sur l'amplification de l'effet Crabtree (inhibition de la respiration par le glucose) dont la cible sont les complexes respiratoires. En l'absence de glucose le turnover cellulaire e l'ATP est sérieusement diminué mais de façon inattendue la survie cellulaire est plutôt meilleure. Ces résultats posent la question de la contribution des conditions hyperosmotiques liées à l'inflammation dans l'établissement d'un profil métabolique de type inflammatoire
Metabolic alterations associated with inflammation include increased recruitment of glycolysis (lactate release) and repression of mitochondrial oxidative phosphorylation. Inflammation causes hyperosmolar conditions in the extracellular medium. This thesis examines the consequences of hyperosmolarity on cellular bioenergetics. For this purpose we measured the cellular oxygen consumption rate (OCR) and proton production rate (PPR) for lactate release in the external medium. Two methodologies were used the high-resolution respirometer (O2k Oroboros Instruments) for OCR and the extracellular flux analyzer (Seahorse, Agilent) for OCR and PPR. The exposure cells to hypertonic conditions (600 milliOsmoles while normal value is 300) causes within few minutes a decrease in OCR (cellular respiration) that lasts for hours (indefinitely) and in the long term impact on cellular viability. This effect was observed with four different cell lines CHO (ovarian epithelial), HT29 (colonocytes), HEK293 (Embryonic kidney) and SH-SY5Y (Neuroblastoma). It was shown to be caused by three different osmolytes: Mannitol, polyethylene glycol, sodium chloride. A milder osmotic challenge (450 mOsm) caused a similar initial decrease but with restoration of initial OCR within few hours. The mechanisms underlying this effect have been investigated, hyperosmolarity impacts on mitochondrial respiration at different steps. A first effect is the inhibition of the mitochondrial ATP production step. In presence of glucose this is accompanied by a large increase in glycolysis (lactate release) that causes further mitochondrial inhibition by a second mechanism, which is likely to represent an enhancement of the Crabtree effect (inhibition of respiration by glycolysis) that impacts on respiratory complexes. In absence of glucose the cellular ATP turnover is seriously repressed surprisingly cellular survival is rather improved. These results raise therefore the question of the possible contribution of the hyperosmotic conditions caused by inflammation in the acquisition of the inflammatory metabolic profile

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

Le développement tumoral tel qu'il est récemment modélisé selon le concept des cellules souches cancéreuses (CSC) est un modèle statique dans lequel les CSC seraient les seules responsables de l'émergence, de la résistance aux traitements ainsi que de la récurrence tumorale. Cependant, la biologie du cancer est bien plus complexe et la plasticité des CSC suggère l'existence d'une conversion bidirectionnelle entre les CSC et les non-CSC. Cette thèse vise à élucider les mécanismes par le biais desquels l'autophagie, un processus d'auto-digestion, régit le destin des CSC mammaires et apporte une meilleure compréhension au processus de plasticité. Nos résultats soulignent l'implication de l'autophagie dans le remodelage métabolique en augmentant la glycolyse aux dépens de la phosphorylation oxydative et ceci est accompagné par l'émergence des CSC. En effet, nous montrons que l'Oncostatine M (OSM), une cytokine pro-inflammatoire de la famille de l'IL-6, régule l'autophagie et l'expression de l'hexokinase II (HK II). Cette enzyme, la première de la voie du métabolisme du glucose, est décrite pour jouer un rôle clé dans l'effet "Warburg". Nous montrons que l'invalidation de l'expression de HK II et PI3K/AKT prévient l'induction de la population CSC. De manière originale, nos résultats mettent en évidence un nouveau rôle pour l'autophagie qui confère, par acétylation, une protection à l'HK II contre la dégradation par le protéasome, permettant ainsi de maintenir une glycolyse accrue nécessaire pour l'émergence et le maintien des CSC
Tumor development as recently modelized according to the concept of cancer stem cells (CSCs) is a static model in which CSCs are the only ones responsible for emergence, resistance to treatment and tumor recurrence. However, the cancer biology is complex and the plasticity of CSCs suggests the existence of a bidirectional conversion between CSCs and non-CSCs. This thesis aims to elucidate the mechanisms by which autophagy, a process of self-digestion, governs the fate of breast CSCs and provides a better understanding of the process of plasticity. Our results highlight the involvement of autophagy in metabolic remodeling by increasing glycolysis at the expense of oxidative phosphorylation and this is accompanied by the emergence of CSCs. Indeed, we show that Oncostatin M (OSM), a pro-inflammatory cytokine of the IL-6 family, regulates autophagy and the expression of hexokinase II (HK II). This enzyme, the first of the glucose metabolism pathway, is described to play a key role in the 'Warburg' effect. Here we report that inhibition of HK II and PI3K / AKT prevent the induction of CSC population. Notably, the results presented in this thesis attribute to autophagy a new role which confers, by acetylation, a protection to HK II against the degradation by the proteasome, making it possible to maintain an increased glycolysis required for the emergence and maintenance of CSCs

Cette thèse étudie le remodelage métabolique des cellules cancéreuses. Trois modèles sont analysés par de nombreuses approches biochimiques et génétiques : (i) des cellules de poumon transduites avec une forme oncogénique de HRASG12V, (ii) des cellules HeLa soumises à une privation de glucose et (iii) des pièces chirurgicales de cancer du poumon. Sur chaque modèle, le remodelage métabolique observé met en jeu de nombreuses voies du catabolisme et de l’anabolisme, notamment la glutaminolyse et la biosynthèse de sérine. Ce travail révèle un rôle important des mitochondries dans ce remodelage, à la fois pour l’apport d’énergie et pour la synthèse d’antioxydants et d’acides aminés, mais aussi de phospholipides. J’ai montré l’impact étendu d’une simple mutation HRASG12V sur un très grand nombre de processus, révélant ainsi l’importance de la génétique dans le remodelage métabolique des cellules cancéreuses. Toutefois, la privation de glucose induit elle aussi un remarquable remodelage à de très nombreux niveaux, depuis l’épissage des ARN messagers jusqu’à la synthèse de sérine. Enfin, cette thèse identifie deux classes bioénergétiques de tumeurs du poumon, ouvrant de nombreuses perspectives pour le diagnostic et la compréhension de ce type de tumeurs, mais aussi pour proposer des stratégies thérapeutiques adaptées. Les résultats identifient des biomarqueurs et des cibles validées sur nos modèles in vitro. Les perspectives de cette thèse vont consister à la transposition de ces approches à la clinique
This thesis investigates the metabolic remodeling of cancer cells. Three models are analyzed by different biochemical and genetic approaches: (i) lung cells transduced with oncogenic HRASG12V, (ii) HeLa cells challenged with glucose deprivation and (iii) surgical pieces of lung tumors. On each model the observed metabolic remodeling involves numerous catabolic and anabolic pathways, including glutaminolysis and serine biosynthesis. Our work revealed an important role of mitochondria in metabolic remodeling, both for the supply of energy and for the synthesis of antioxidants and amino acids, but also phospholipids. We show the extent of a single mutation HRASG12V on a very large number of metabolic processes, revealing the importance of genetics in the metabolic remodeling of cancer cells. However, glucose deprivation also induced a remarkable remodeling at many levels of cell metabolism, from the splicing of messenger RNAs to serine biosynthesis. In the third part, this thesis identified two bioenergetic classes of lung tumors, opening interesting opportunities for the diagnosis and understanding of this type of tumor, but also to propose appropriate therapeutic strategies. The results identify biomarkers and targets validated in our in vitro models. The outlook of this thesis will be to the implementation of these approaches in the clinic

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.

Mitochondria play a central role in lipid metabolism and pathology in obesity and type 2 diabetes mellitus. Mitochondria have been shown to associate with lipid droplets (LDs) in multiple tissues but the functional role of these peridroplet mitochondria (PDM) is unknown. This work reveals that PDM have unique protein composition and cristae structure, and remain adherent to the LD in the tissue homogenate. We developed an approach to isolate PDM based on their adherence to LDs. Comparison of purified PDM to cytoplasmic mitochondria reveals that (1) PDM have increased pyruvate oxidation, electron transport, and ATP synthesis capacities. (2) PDM have reduced beta oxidation capacity and depart from LDs upon activation of brown adipose tissue thermogenesis and beta oxidation. (3) PDM support LD expansion as Perilipin 5-induced recruitment of mitochondria to LDs increases ATP-dependent triacylglyceride synthesis. (4) PDM maintain a distinct protein composition due to uniquely low fusion-fission dynamics. We conclude that PDM represent a segregated mitochondrial population with unique structure and function that supports triacylglyceride synthesis. We suggest that increased mitochondrial recruitment to LDs may be part of a generalized adaptive response in physiological conditions that require LD expansion, such as post-prandial lipid synthesis and storage. Furthermore, PDM-mediated LD expansion may play a role in muscle and liver injury from lipotoxicity in conditions of nutrient excess, such as obesity and hyperlipidemia. A better understanding of PDM and LD biology may therefore lead to new therapies for lipotoxic tissue injury and insulin resistance.
2020-10-24T00:00:00Z