7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...

AMPK is a master regulator of cellular energy homeostasis. The gain-of-function AMPKg3 R225W mutation in human skeletal muscle increases resistance to fatigue during exercise, mitochondrial content, and glycogen storage. We demonstrate that primary myotubes exhibit increased OCR, decreased ECAR, increased FAO, and increased activities of several mitochondrial complexes. To examine whether functional effects are attributable to mitochondrial content, we inhibited AMPK; differences between R225W and control were diminished. Glycogen phosphorylase inhibition demonstrated normal respiration independent of glycogen. We examined markers of quality/quantity control of mitochondria. In R225W muscle, fusion markers increased, biogenesis markers remained unchanged, mTOR pathway was inhibited, and there was greater capacity for autophagic flux and mitophagy. We thus determine that bioenergetic effects of R225W are in part due to active AMPK, but also due to capacity for more robust mitochondria. Overall, R225W provides a model for evaluating effects of AMPK, and new avenues toward treatment of metabolic disease.

The gram-negative endotoxin, lipopolysaccharide (LPS), has been extensively researched in high doses (10-200ng/ml) and is well-documented in the literature for its ability to result in devastating effects such as multi-organ failure, sepsis, and septic shock. In high doses, LPS signals through Toll-like-receptor 4 (TLR4) and triggers a cascade of events culminating in the release of pro- and anti-inflammatory cytokines and the activation of NF-κB. In contrast, super-low doses of LPS (1-100pg/ml) are able to trigger the persistent release of pro-inflammatory mediators while evading the compensatory activation of NF-κB. This mild yet persistent induction of inflammation may lie at the heart of numerous inflammatory diseases and disorders and warrants studies such as this to elucidate the novel mechanisms. In this study, we explored the novel mechanisms utilized by super-low dose LPS in cellular stress and low-grade inflammation. In the first study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and necroptosis were examined. We show that in the presence of super-low dose LPS (50pg/ml), the key regulators of mitochondrial fission and fusion, Drp1 and Mfn1 respectively, are inversely regulated. An increase in mitochondrial fragmentation and cell death which was not dependent on caspase activation was observed. In addition, super-low dose LPS was able to activate RIP3, a kinase responsible for inducing the inflammatory cell death, necroptosis. These mechanisms were regulated in an Interleukin-1 receptor-associated kinase 1 (IRAK-1) dependent manner. In the second study, the molecular mechanisms governing the role of super-low dose LPS on cellular stress and endosome/lysosome fusion were examined. In the presence of low-dose LPS (50pg/ml), endosomal-lysosomal fusion is inhibited and a loss of endosomal acidification required for the successful clearance of cellular debris and resolution of inflammation was observed. Additionally, super-low dose LPS induced the accumulation of p62 indicative of the suppression of autophagy. Tollip and Interleukin-1 receptor-associated kinase 3 (IRAK-M) appear to be critical regulators in this process. Collectively, these studies show that low-dose endotoxemia is capable of causing persistent cellular stress, not observed in the presence of high-dose LPS (10-200ng/ml), and that it promotes necroptotic cell death while suppressing mechanisms necessary for the resolution of inflammation such as endosome-lysosome fusion. This research reveals novel mechanisms utilized by low-dose endotoxemia which could aid future efforts to develop prevention and treatment for various debilitating inflammatory diseases. / Ph. D.

No / Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.

La cellule cardiaque requiert un apport énergétique conséquent qui exige une production et un transfert énergétiques efficaces. Si la production de l’énergie dépend essentiellement des propriétés intrinsèques des mitochondries, il semblerait que l’efficacité du transfert d’énergie du site de production vers les sites consommateurs (ATPases) pourrait être liée à l’architecture spécifique du cardiomyocyte qui conduit à une organisation spatiale singulière des structures internes (mitochondries, réticulum sarcoplasmique, myofilaments). Pour comprendre ce qui lie la cytoarchitecture, la compartimentation cellulaire et la fonction contractile, il a été entrepris d’étudier l’architecture cellulaire et la signalisation énergétique de cardiomyocytes au cours du processus de maturation de la cytoarchitecture et dans un modèle présentant une désorganisation des structures intracellulaires. La première partie de ce travail, réalisée durant le développement postnatal de la souris, a permis de démontré qu’il existe une synchronisation parfaite entre la mise en place de la cytoarchitecture et la maturation fonctionnelle du transfert d’énergie par canalisation directe des nucléotides adényliques entre les mitochondries et les ATPases. Si cette étude apporte un élément qui tendrait à démontrer l’implication de l’architecture cellulaire dans l’efficacité des transferts d’énergie, elle a également mis en avant la maturation très précoce de l’énergétique cellulaire. La mitochondrie faisant partie intégrante de cette architecture et étant modelée par des mécanismes de fusion et de fission, la deuxième étape de ce travail de thèse a consisté à étudier l’implication de la morphologie mitochondriale dans l’énergétique du cardiomyocyte. Il a ainsi été montré que, chez la souris, la diminution d’expression de la protéine OPA1, impliquée dans la fusion mitochondriale, conduit à des perturbations de la morphologie mitochondriale qui n’affectent pas la fonction intrinsèque mitochondriale mais qui altèrent le système de canalisation directe entre les mitochondries et les ATPases des myofilaments. De manière générale, ces résultats démontrent clairement une dépendance des transferts d’énergie à l’architecture cellulaire spécifique de la cellule musculaire cardiaque. / The cardiac cell function requires a large amount of energy and therefore needs a high efficiency of energetic production and energetic transfer. While the energy production depends on the intrinsic properties of the mitochondria, it appears that the efficiency of energetic transfers from the main producers (mitochondria) to consumers (ATPases) could be related to the specific architecture of the cardiomyocyte, which ensures a unique spatial organization of internal structures (mitochondria, sarcoplasmic reticulum, myofilaments). In order to reveal the role of mitochondrial network organization in cardiac energy metabolism, we studied the cellular architecture and the energetic signalling of cardiomyocytes in the process of maturation of the cytoarchitecture and in a model which exhibits a perturbation of the mitochondrial dynamics. The first part of this work, which was performed during postnatal development of the mouse, showed the perfect synchronisation between the establishment of the cytoarchitecture and the maturation of the transfer of energy by direct channelling of adenine nucleotides between mitochondria and ATPases. While this study provides an element which would demonstrate the involvement of cellular architecture in the efficiency of energy transfer, it also highlighted the very early maturation of the energetic system of the cell. Knowing that the mitochondria are an integral part of the cell architecture and that the mitochondrial network is controlled by fusion and fission mechanisms, the second step of this work consisted in investigating the involvement of mitochondrial dynamics in cardiomyocyte energetics. Our work has shown that a decrease in expression of OPA1, a protein responsible for mitochondrial fusion, leads to disruption of mitochondrial morphology which does not affect intrinsic mitochondrial function but affects the direct channelling of ATP and ADP between mitochondria and ATPases of the myofilaments. Overall, these results clearly demonstrate that energy transfer in cardiomyocytes strictly depends on specific cellular architecture.

La peau est un épithélium spécialisé vital et fragile, qui évolue avec l’âge et est influencé par l’environnement, notamment les radiations solaires. Des données sont disponibles sur la réponse du réseau mitochondrial et le devenir des mitochondries endommagées en réponse à des stress chimiques et environnementaux dans plusieurs systèmes expérimentaux, mais ces processus restent peu étudiés dans les cellules cutanées. Dans ce contexte, le projet de thèse visait à analyser l’effet (i) de l’irradiation UVB sur la dynamique mitochondriale (en particulier la fragmentation des mitochondries) dans des kératinocytes primaires humains normaux, qui constituent la première ligne de défense contre les agressions externes ; (ii) d’un traitement par des poisons mitochondriaux sur les mitochondries contenues dans des kératinocytes ou des fibroblastes primaires humains normaux. Dans un premier axe de la thèse, nous avons mis au point une méthode originale (Mitoshape) basée sur l’imagerie confocale, permettant d’estimer à la fois qualitativement et quantitativement la morphologie du réseau mitochondrial dans des cellules vivantes après irradiation UVB. Grâce à cette technologie, nous avons pu montrer que les UVB induisaient une fragmentation du réseau mitochondrial dans les kératinocytes primaires, dont nous avons étudié les acteurs biochimiques. Dans un deuxième axe, nous avons montré que les poisons mitochondriaux avaient la capacité d’endommager les mitochondries dans des kératinocytes et des fibroblastes humains primaires et induisaient une autophagie générale sans toutefois exclure la présence d’une mitophagie dépendante de la voie PINK1/PARKIN. Outre son intérêt fondamental, ce travail (réalisé en collaboration avec la société de cosmétologie SILAB dans le cadre d’un partenariat industriel CIFRE) ouvre la voie à l’identification d’actifs naturels capables de préserver et/ou restaurer les paramètres fonctionnels mitochondriaux suite à des stress. / The skin is a specialized type of epithelium, both vital and fragile, which evolves with age and is continuously exposed to environmental stresses, such as solar radiations. While data is available about the response of the mitochondrial network and the fate of damaged mitochondria after chemical or environmental stresses in numerous experimental systems, little is known about these processes in skin cells. The aim of the present thesis was to study the impact (i) of UVB irradiation on mitochondrial dynamics (especially mitochondrial fragmentation) in normal human epidermal keratinocytes, which represent the first line of defence against environmental insults; (ii) of poisoning mitochondria of keratinocytes and normal human fibroblasts with chemical drugs. In a first axis, we developed an original method (called Mitoshape) based on confocal microscopy, to estimate qualitatively and quantitatively the morphology of the mitochondrial network within live cells following UVB irradiation. Using this technology, we demonstrated that UVB irradiation induces mitochondrial fragmentation in normal human keratinocytes, and studied the biochemical actors involved in this response. In a second axis, we showed that the use of mitochondrial poisons could damage mitochondria of keratinocytes and normal human fibroblasts and induce bulk autophagy, although it is not possible to formally rule out the involvement of a PINK1/PARKIN-dependent pathway of mitophagy. In addition to its fundamental interest, this work (performed in collaboration with the cosmetic company SILAB in the context of a CIFRE PhD fellowship from ANRT) paves the way for the screening of novel bioactive agents able to protect and restore mitochondria following stresses.

La dynamique mitochondriale est impliquée dans la maintenance et la fonction des mitochondries. Dans les graines sèches tout les processus cellulaires sont arrêtés du fait de la faible teneur en eau des tissues, et la transition développementale que représente la germination requiert la réactivation de la dynamique cellulaire. Une approche de bio-imagerie sur la plante modèle Arabidopsis a été utilisée afin d’étudier la réactivation des mitochondries nécessaire à la germination. La réactivation bioénergétique des mitochondries, mesurée par la présence du potentiel membranaire, intervient dès le début de l’hydratation des tissus. Cependant les mitochondries restent statiques et la dynamique mitochondriale ne reprend que plus tardivement. La réactivation des mitochondries provoque une réorganisation du chondriome impliquant la biogenèse de membranes et une fusion massive menant à la formation de structures réticulaires et périnucléaires, qui permet le mélange des nucléoïdes d’ADNmt précédemment isolés en unités discrètes. La mitophagie, un indicateur de la qualité mitochondriale, est réactivée de manière concomitante à la dynamique, alors qu’elle est réprimée durant la biogenèse des mitochondries. La fin de la germination coïncide avec la fragmentation du chondriome tubulaire, menant au doublement du nombre de mitochondrie et à une redistribution hétérogène des nucléoïdes dans le chondriome, générant une population de mitochondrie adaptée à la croissance des plantules. Cette thèse met en évidence l’imbrication des processus de dynamique mitochondriale, de biogenèse et de contrôle qualité des mitochondries requis pour la germination et pour la transition vers l’autotrophie. / Mitochondrial dynamics underpin their function and maintenance. In dry seeds, all cellular processes are in stasis due to a low water content. Thus, the developmental switch leading to germination necessarily involves a reactivation of cellular dynamics. In order tobetter understand the role played by mitochondrial dynamics during germination we used Arabidopsis as a model for a bioimaging approach to investigate the rapid reactivation of mitochondria that is required in order to provide ATP to support germination. Bioenergetic reactivation, visualised as the presence of a mitochondrial membrane potential, is almost immediate upon rehydration. However, the reactivation of mitochondrial dynamics only occurs after several hours of rehydration. The reactivation of mitochondrialbioenergetics and dynamics lead to a dramatic reorganisation of the chondriome involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mtDNA nucleoids. Mitophagy, an indicator of mitochondrial quality, is reactivated concomitant with a reactivation of mitochondrial dynamics, but is repressed at time of mitochondrial biogenesis. The end of germination coincides with fragmentation of the tubular chondriome leading to a doubling of mitochondrialnumber and heterogeneous redistribution of the nucleoids amongst the mitochondria, generating a population of mitochondria tailored to seedling growth. This thesis provides strong evidence for the tight interweaving of mitochondrial dynamics, mitochondrialbiogenesis and mitochondrial quality control that is required to ensure effective germination and the transition to autotrophy.

<p>The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. </p><p>A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. </p><p>In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.</p>

The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.