Tesi sul tema "Mitochondria fusion"

Les mitochondries sont des organelles de quelques micromètres qui proviendraient de l'incorporation d'une alpha-protéobactérie dans le cytoplasme des cellules eucaryotes par endosymbiose. Dans les cellules eucaryotes, la mitochondrie joue un rôle central dans la production d'ATP, mais aussi dans la mort cellulaire programmée par apoptose ainsi que dans la biosynthèse de nombreuses molécules. Les mitochondries sont très polymorphes, leurs taille, forme et organisation varient considérablement selon le type cellulaire ou l'état physiologique ou pathologique de la cellule. Depuis une vingtaine d'année, l'étude des mécanismes qui contrôlent la morphogenèse, la dynamique de fission et de fusion mitochondriale et leurs rôles physiologiques est devenue un domaine majeur dans la recherche sur la mitochondrie. De plus, avec les progrès de la vidéo-microscopie, il est devenu possible de filmer des mitochondries dans le cytoplasme de cellules vivantes. Durant ma thèse, j'ai participé à la caractérisation de la fonction du gène Pantagruelian Mitochondria I (PMI), un nouveau déterminant de la morphologie des mitochondries que nous avons découvert chez la drosophile. PMI est une protéine de la membrane interne qui, en intervenant dans l'organisation de cette membrane, est indispensable à la formation de mitochondries de forme tubulaire. J'ai également contribué au développement d'outils et de méthodologies permettant la visualisation et l'étude de la dynamique mitochondriale dans des embryons de drosophiles vivants
Mitochondria are organelles which are a few micrometers long and are originated from the incorporation of an alpha-proteobacteria in the cytoplasm of eukaryotic cells through endosymbiosis. In eukaryotic cells, mitochondria play a central role in ATP production as well as in programmed cell death and in the biosynthesis of many molecules. Mitochondria are highly polymorphic in size and form. Their organization also varies considerably according to the cell type or physiological or pathological state of the cell. In the last two decades, the study of the mechanisms controlling morphogenesis, dynamic of mitochondrial fission and fusion and their physiological roles has become a major research field of mitochondria. In addition, the progress in video-microscopy enable to record mitochondrial dynamics in the cytoplasm of living cells. I participated in the research on the characterization of gene function called Pantagruelian Mitochondria I (PMI), a novel determinant of the mitochondrial morphology that we discovered in Drosophila. PMI, a protein of the inner membrane, is involved in its membrane organization and essential to form tubular mitochondria. I also contributed to the development of experimental tools and protocols to visualize and study the mitochondrial dynamics in living Drosophila embryos. Interestingly, a stereotyped process of mitochondrial remodeling during Drosophila embryogenesis has been found and it raised a question about its role in developmental processes through my work

Les mitochondries sont des organites dynamiques qui fusionnent et se divisent continuellement. Cette dynamique est requise pour la biogenèse mitochondriale, la fonction et la dégradation. Les relations entre les OXPHOS, la dynamique et les mécanismes assurant la modulation de la dynamique restent largement inconnus. Nous avons étudié grâce à un essai de fusion in vivo, les relations entre la fusion et les OxPhos dans des cellules de levure portant des mutations ponctuelles dans le gène mitochondrial ATP6 qui sont associés à des maladies chez l’homme. Nous montrons que les défauts des OxPhos provoquent des défauts de fusion de la membrane interne mitochondriale mais pas de la membrane externe. L'inhibition sélective de la fusion de la membrane interne peut être mimée par les ionophores qui dissipent le potentiel de membrane interne, mais pas par des inhibiteurs des phosphorylations oxydatives. Nous montrons une inhibition dominante de fusion qui pourrait être un mécanisme d'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial pour les adresser à la mitophagie. Ces résultats indiquent que les défauts de fusion pourraient contribuer à la pathologie des maladies provoquées par des mutations de l'ADNmt. De plus ces résultats impliquent que dans des cellules, l'inhibition de la fusion dominante pourrait permettre l'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial. La fusion mitochondriale implique de nombreuses protéines de la superfamille des dynamines. Si ces protéines ont été identifiées, les mécanismes moléculaires permettant la fusion restent indéterminés. Dans le but de comprendre ces mécanismes, nous avons choisi de caractériser les protéines Mitofusine 1 et 2, essentielles à la fusion des membranes externes mitochondriales. Ces protéines sont composées de deux domaines coiled-coil et un domaine N-terminal GTPase et des domaines hydrophobes prédits pour être des segments transmembranaires. Après la détermination des activités GTPase des mitofusines, nous avons reconstitué les mitofusines ou des fragments des mitofusines dans des liposomes afin d'étudier leur capacité à fusionner ces liposomes. Les mitofusines, permettent la fusion des liposomes contenant des cardiolipides. Étonnamment, ces événements sont indépendants de la présence du GTP mais nécessitent Mg2+ dans la solution. En utilisant la microscopie électronique, nous montrons que les mitofusines 1 et 2 induisent une déformation des liposomes. Cette capacité permettant de créer localement des régions très courbes (et donc fusogènes) ouvre un nouvel angle pour comprendre les mécanismes moléculaires de la fusion mitochondriale
Mitochondria are dynamic organelles that continuously fuse and divide. This dynamic is required for mitochondrial biogenesis, function and degradation. The cross-talk between OXPHOS and dynamics and the mechanisms ensuring modulation of dynamics remain largely unraveled. We have investigated the relationship between fusion and OXPHOS in yeast cells carrying point mutations in the mitochondrial ATP6 gene that are associated to human diseases. We show that OXPHOS defects provoke severe defects of inner membrane, but not outer membrane fusion. Selective inhibition of inner membrane fusion can be recapitulated by ionophores that dissipate the inner membrane potential, but not by inhibitors of OXPHOS. We show a dominant inhibition of fusion that further provides a mechanism for the exclusion of defective mitochondria from the functional mitochondrial network, a pre-requisite for their selective targeting to mitophagy. These results suggest that defects of fusion could contribute to the pathology of diseases caused by mtDNA mutations. Moreover, these results imply that in cells, inhibition of dominant fusion could allow the exclusion of dysfunctional mitochondria mitochondrial network. Mitochondrial fusion involves many proteins of the superfamily of dynamin. If these proteins have been identified, the molecular mechanisms of fusion remain undetermined. In order to understand these mechanisms, we choose to characterize Mitofusin 1 and 2 proteins, essential for outer mitochondrial membrane fusion. These transmembrane proteins are consisting of two coiled-coil domains and one N-terminal GTPase domain. We have characterized GTPase activity of Mitofusin and reconstituted Mitofusins or fragments of Mitofusins into liposomes to study their capacity to fuse these liposomes. Full-length mitofusins can fuse liposomes containing cardiolipins. Surprisingly, these fusion events are independent of GTP but require Mg2+ in the buffer. Using electron microscopy, we show that mitofusin 1 and 2 induce local deformation of liposomes. This capacity of mitofusins to locally create highly curved (and thus fusogenic) membrane regions opens a new angle to understand the molecular mechanisms of mitochondrial fusion

Les mitochondries sont des organites dynamiques dont la morphologie dépend de l’équilibre fusion/fission de leurs membranes. Ce processus essentiel à la survie cellulaire est nommé dynamique mitochondriale et sa dérégulation est associée à des troubles neurologiques. Cependant les mécanismes précis régissant la dynamique mitochondriale ne sont pas élucidés. Cette thèse porte sur la protéine Fzo1p, une grande GTPase de la superfamille des Dynamin-related-Protein. C’est un élément clé impliqué dans la fusion mitochondriale de la membrane externe de la levure. Sa structure et sa dynamique ont été étudiées par modélisation et simulations de dynamiques moléculaires tout-atome dans une bicouche lipidique solvatée. Le modèle structural obtenu tient compte de données expérimentales, de template structuraux, et de modèles ab initio du domaine transmembranaire de Fzo1p. Ce modèle a été validé expérimentalement par mutagenèse dirigée. Des permutations de charges ont confirmé des ponts salins à longue distance prédits dans le modèle. En outre, des mutations ont montré que les domaines coiled-coil de Fzo1p, contrairement à sa partie N-terminale, sont indispensables à sa fonction. L’ensemble des résultats expérimentaux et in silico met en évidence l’implication des domaines charnières dans le changement conformationnel de Fzo1p, ainsi que des résidus critiques affectant sa stabilité. Les précisions atomiques obtenues sur l’interaction de Fzo1p avec le GDP permet de formuler des hypothèses sur le mécanisme moléculaire de la catalyse du GTP pour la fusion membranaire; voire à la compréhension de la dynamique mitochondriale
Mitochondria are dynamic organelles whose morphology is determined by fusion and fission of their membranes. This essential process is known as mitochondrial dynamics. Defects in mitochondrial dynamics are associated with neurological disorders making the investigation of physiological relevance. However, the precise sequence of events that lead mitochondrial dynamics are still not well characterised. Fzo1p, a large GTPase of the Dynamin-Related Proteins superfamily, is a key component in mitochondrial outer membrane fusion in yeast. During this PhD project I built a model of the protein Fzo1p. The structure and dynamics of the model was investigated through molecular modelling and all-atom molecular dynamics simulation in a fully hydrated lipid bilayer environment. The Fzo1p structural model integrates information from several template structures, experimental knowledge, as well as ab initio models of the transmembrane segments. The model is validated experimentally through directed mutagenesis, for instance charge-swap mutations confirm predicted long-distance salt bridges. A series of mutants indicate that coiled-coil domains are required for protein function at variance with its N-terminal region. Overall, the experimental and in silico approaches pinpoint the hinge domains involved in the putative conformational change and identifies critical residues affecting protein stability. Finally, key Fzo1p-GDP interactions provide insights about the molecular mechanism of membrane fusion catalysis. The model provides insight on atomic level and proposes a structure that will be instructional to understanding mitochondrial membrane fusion

Les mitochondries sont des organelles de quelques micromètres qui proviendraient de l'incorporation d'une alpha-protéobactérie dans le cytoplasme des cellules eucaryotes par endosymbiose. Dans les cellules eucaryotes, la mitochondrie joue un rôle central dans la production d'ATP, mais aussi dans la mort cellulaire programmée par apoptose ainsi que dans la biosynthèse de nombreuses molécules. Les mitochondries sont très polymorphes, leurs taille, forme et organisation varient considérablement selon le type cellulaire ou l'état physiologique ou pathologique de la cellule. Depuis une vingtaine d'année, l'étude des mécanismes qui contrôlent la morphogenèse, la dynamique de fission et de fusion mitochondriale et leurs rôles physiologiques est devenue un domaine majeur dans la recherche sur la mitochondrie. De plus, avec les progrès de la vidéo-microscopie, il est devenu possible de filmer des mitochondries dans le cytoplasme de cellules vivantes. Durant ma thèse, j'ai participé à la caractérisation de la fonction du gène Pantagruelian Mitochondria I (PMI), un nouveau déterminant de la morphologie des mitochondries que nous avons découvert chez la drosophile. PMI est une protéine de la membrane interne qui, en intervenant dans l'organisation de cette membrane, est indispensable à la formation de mitochondries de forme tubulaire. J'ai également contribué au développement d'outils et de méthodologies permettant la visualisation et l'étude de la dynamique mitochondriale dans des embryons de drosophiles vivants
Mitochondria are organelles which are a few micrometers long and are originated from the incorporation of an alpha-proteobacteria in the cytoplasm of eukaryotic cells through endosymbiosis. In eukaryotic cells, mitochondria play a central role in ATP production as well as in programmed cell death and in the biosynthesis of many molecules. Mitochondria are highly polymorphic in size and form. Their organization also varies considerably according to the cell type or physiological or pathological state of the cell. In the last two decades, the study of the mechanisms controlling morphogenesis, dynamic of mitochondrial fission and fusion and their physiological roles has become a major research field of mitochondria. In addition, the progress in video-microscopy enable to record mitochondrial dynamics in the cytoplasm of living cells. I participated in the research on the characterization of gene function called Pantagruelian Mitochondria I (PMI), a novel determinant of the mitochondrial morphology that we discovered in Drosophila. PMI, a protein of the inner membrane, is involved in its membrane organization and essential to form tubular mitochondria. I also contributed to the development of experimental tools and protocols to visualize and study the mitochondrial dynamics in living Drosophila embryos. Interestingly, a stereotyped process of mitochondrial remodeling during Drosophila embryogenesis has been found and it raised a question about its role in developmental processes through my work

Mitochondria exist in a dynamic network regulated by the opposing processes of mitochondrial fusion and fission. Regulation of mitochondrial morphology is critical for metabolism, quality control and cell survival, among other cellular processes. Large GTPases are responsible for shaping the mitochondrial network. Mitofusins 1 and 2 and Opa1 regulate outer and inner mitochondrial membrane fusion, respectively. Conversely, Drp1 is recruited to mitochondria to carry out fission. Although many proteins have been implicated in these processes, there are still many unknowns. We sought to identify novel regulators of mitochondrial morphology and conducted a genome-wide RNAi screen to identify candidate genes. We identified Reactive Oxygen species Modulator 1 (ROMO1) as a novel regulator of mitochondrial fusion and cristae integrity. In the absence of ROMO1, the mitochondrial network fragments and cristae are lost. These defects lead to impaired mitochondrial respiration and sensitization to cytochrome c release and downstream apoptosis. ROMO1 is regulated by mitochondrial REDOX at 4 cysteine residues that couple REDOX signaling to mitochondrial morphology. We have characterized ROMO1 as an interactor with the MINOS complex, required for cristae junction maintenance, and the inner mitochondrial membrane fusion GTPase OPA1. Through these interactions ROMO1 couples cristae junction security to mitochondrial fusion.

La morphologie mitochondriale résulte d’un équilibre dynamique entre les processus de fusion et de fission, impactant la physiologie cellulaire. Plusieurs données montrent une relation entre la fonction mitochondriale, des maladies cardiovasculaires et le vieillissement. OPA1 (optic atrophy 1) est une protéine qui contrôle la fusion de la membrane interne de la mitochondrie, et dont la mutation induit la maladie ADOA (autosomal dominant optic atrophy). Les travaux menés récemment indiquent que la mutation OPA1 est impliquée dans le dysfonctionnement cardiaque mais son impact sur la fonction vasculaire est encore inconnu. Notre étude a pour ambition d’examiner le rôle d’OPA1 sur la fonction vasculaire, notamment dans le développement de l’hypertension artérielle et le vieillissement vasculaire. Avec un modèle de souris hétérozygotes Opa1+/-, nous montrons dans cette étude que la protéine OPA1 joue un rôle protecteur dans le système vasculaire. En effet, les souris déficientes en OPA1 développent une hypertension-L-NAME dépendante plus grave qui est associée avec une dysfonction endothéliale plus importante et une altération de remodelage vasculaire. D’autre part, présentant une fonction vasculaire normale à 6 mois, les souris Opa1+/- commencent à développer un dysfonctionnement vasculaire à 12 mois qui pourrait induire le développement de pathologies vasculaires. En conclusion, ces résultats suggèrent pour la première fois que la dynamique mitochondriale peut jouer un rôle important sur la fonction et l’adaptation des vaisseaux dans les conditions pathologiques et dans le vieillissement vasculaire. Des études complémentaires seront nécessaires afin de clarifier le rôle de la protéine OPA1 dans l’hypertension. Ces données peuvent contribuer à la recherche de nouvelles cibles thérapeutiques pour prévenir les complications de l’hypertension et les maladies vasculaires liées à l’âge
Defects in mitochondrial dynamics have been associated with various disorders, including cardiovascular diseases. OPA1 is essential for mitochondrial inner membrane fusion. Mutation in Opa1 is associated with the autosomal dominant optic atrophy (ADOA). Since then, OPA1 has been reported to be associated with cell apoptosis, cell proliferation, mitochondrial ATP synthesis, calcium homeostasis and ROS production. These data suggest that OPA1 has a potential role in vascular cells and subsequently affects vascular function. On the other hand,OPA1 is also associated with age-related changes of mitochondria and simultaneously contribute to the development of many dysfunctions in different organs. In this study, we investigated impacts of OPA1 mutation on vascular function in physiological and pathological condition like hypertension and vascular aging. By using an Opa1+/- heterozygote mouse model, we show that the OPA1 protein plays a protective role in the vascular system. Indeed, Opa1+/- mice developed a hypertension more severe than WT mice which was associated with more important endothelial dysfunction and altered vascular remodeling. In addition, although initial vascular function was normal, at 12 months, Opa1+/- mice displayed vascular dysfunction which might predict onset of vascular diseases at a later time. These results suggest for the first time that mitochondrial dynamics might play an important role in vascular function and adaptation in pathological conditions and in vascular aging. More studies are needed to clarify the role of the protein OPA1 in hypertension. These data may help to identify novel therapeutic targets to prevent complications of hypertension and vascular age-related diseases

Mitochondria are essential organelles for the life of the cells since it is the major source of ATP, key molecule for many endoergonic reaction. Recently it has been demonstrated that mitochondrial play a key role in many other cellular processes like Ca2+ signaling and programmed cell death. Following an apoptotic insult mitochondria release cytochrome c and other proteins required in the cytosol for the activation of the effector caspases required for cell demise. What is remarkable about cytochrome c release is that is fast, complete and usually is not associated with mitochondrial swelling. Thanks to the advances in 3D electron microscopy it has been demonstrated that cristae are not just invagination of the inner mitochondrial membrane (IMM) as previously depicted by Palade (Palade, 1952) but rather distinct compartments of it, separated from the inter membrane space (IMS) by tubular narrow cristae junctions. The majority of cytochrome c and the other respiratory chain components are restricted in this compartment. To reach a complete cytochrome c release in the absence of mitochondrial swelling mitochondria remodel their internal structure: individual cristae fuse and tubular narrow cristae junctions widen; this process, defined cristae remodeling is associated with the mobilization of cytochrome c towards the IMS for its subsequent release across the outer mitochondrial membrane (OMM) (Scorrano et al., 2002). The molecular mechanism beyond this dynamic process is not well understood and in the laboratory where I did my doctoral Thesis it has been hypothesized that OPA1, the only dynamin related protein of the IMM (Alexander et al., 2000; Delettre et al., 2000) could control cristae remodeling. Dynamin related proteins are regulators of mitochondrial morphology promoting mitochondrial fusion and fission. To this family belong Mitofusins (MFN) 1 and 2 in the OMM and OPA1 that resides in the IMM. OPA1 is a large GTPase anchored in the IMM, facing the IMS (Olichon et al., 2002; Satoh et al., 2003); it has been shown that in yeast, its orhologue Mgm1p is required for fusion competent mitochondria by the cooperation with a protein of the same family on the OMM called Fzo1p. In our laboratory it has been demonstrated that in mammalian cells OPA1 promotes mitochondrial fusion through one of the two mammaliam orthologue of Fzo1p called MFN1. In 2000 two distinct laboratories demonstrated that mutations in OPA1 gene are the cause of dominant optic atrophy (ADOA), the leading case of inherited blindness in human, characterized by selective death of retinal ganglion cell (RGC) (Alexander et al., 2000; Delettre et al., 2000). The fact the mutation in a mitochondrial protein involved in mitochondrial morphology caused cell death opened a new scenario that corroborates the central position of mitochondria in regulating apoptotic signaling. The aim of my thesis was to analyze the role of OPA1 in mitochondria-dependent apoptosis. We started with a brute force approach by overexpressing OPA1 in murine embryonic fibroblasts (MEFs) and measuring cells viability in response to intrinsic apoptotic stimuli that specifically trigger apoptosis through the mitochondrial pathway. Overexpression of wt OPA1 but not of mutant in the GTPase domain (OPA1K301A) or a truncated mutant in the coiled coil domain (OPAR905*) is able to prevent from apoptosis induced by hydrogen peroxide, staurosporine, etoposide and overexpression of tBID, a BH3 only protein of the Bcl-2 family that promotes cristae remodeling. To confirm that OPA1 antiapoptotic activity was exerted at the mitochondrial level we analyzed two aspects of the mitochondrial dysfunction: cytochrome c release and mitochondrial depolarization. To this aim we overexpressed a mitochondrially targeted red fluorescent protein (mtRFP) as marker of the mitochondrial network and then we immunodecorated cytochrome c with a FITC-conjugated secondary antibody. OPA1 overexpression prevented cytochrome c release in response to intrinsic stimuli while its inactive mutant OPAK301A aggravated cytochrome c release kinetic. We then analyzed another aspect of the mitochondrial dysfunction: mitochondrial depolarization, taking advantage of the potentiometric probe tetramethylrhodamine-methyl ester (TMRM) which mitochondrial fluorescence is proportional to mitochondrial potential. Overexpression of OPA1, but not of its inactive K301A mutant, prevented mitochondrial depolarization induced by intrinsic stimuli, confirming that OPA may prevent from apoptosis at the mitochondrial level by reducing cytochrome c release and mitochondrial depolarization. How can a dynamin related protein prevent from apoptosis? We asked this because when our study was ongoing an intriguing hypotesys emerged: during apoptosis mitochondrial network undergoes irreversible massive fragmentation; this event and apoptotic cristae remodeling are required for complete cytochrome c release. In principle, OPA1 could prevent apoptosis at both of these levels either counteracting mitochondrial fragmentation thanks to its pro-fusion activity or by the regulation of cristae remodeling. To understand at which of these levels OPA1 was exerting its antiapototic activity, we started a genetic approach, overexpressing OPA1 in Mfn1-/-, where OPA1 pro-fusion activity was prejudiced. Overexpression of OPA1 in these cells prevented from apoptosis induced by intrinsic stimuli; in view of the fact that a residual pro-fusion activity of OPA1 could be mediated by the presence of MFN2 we repeated the same experiments in cells in which both mitofusins were ablated (DMF). Also in this conditions OPA1 prevented from apoptosis at the mitochondrial level, slowing down cytochrome c release kinetic. OPA1 has an antiapoptotica function that is independent of its pro-fusion activity on the mitochondrial network. At this point we asked whether OPA1 may have a role on apoptotic cristae remodeling. We generated stable cell lines that stably overexpressed OPA1 and its K301A mutant both in wt and in Mfn1-/- cells and a cell line depleted of OPA1 by short hairpin RNA interference (shOPA1RNAi). We then isolated mitochondria and measured cytochrome c release induced by recombinant caspase 8 cleaved BID (cBID) using a specific ELISA immunoassay. Stable overexpression of OPA1 is able to prevent cytochrome c relase independently of MFN1 while its downregulation dramatically increases its release. Using a specific assay we observed that OPA1 is also able to prevent cytochrome c mobilization from the cristae independently of MFN. These results were confirmed by the fact that overexpression of the OPA1K301A mutant increased cytochrome c mobilization that was almost complete when OPA1 levels were depleted by RNAi. A thorough morphometric analysis of isolated mitochondria from these cell lines, associated with 3D reconstruction of electron microscopy tomography, showed that OPA1 controls cristae morphology and prevents cristae junction widening in response to cBID. To better understand the molecular mechanism through which OPA1 controls cristae remodeling and cristae junctions diameter we based our hypothesis on the possible analogy with vesciculation processes regulated by cytosolic dynamin, where GTPase activity of it mediated mechanoenzimatic constriction of the vesicle collar. Despite this analogy, we should mention that OPA1, unlike dynamin, is located on the inner side of the membrane to be constricted and not on the outside as dynamin complicating the model. First, we analyzed biochemical characteristic of OPA1: gel filtration studies showed that OPA1 is eluted at very high molecular weight fractions (>600 KDa) and in response to cBID incubation it is retrieved in low molecular weight fractions. Parallel studies in our laboratory demonstrated that OPA1 is processed by a rhomboid protease, PARL, into a short form found soluble in the IMS that is responsible for the antiapototic but not of the pro-fusion activity of OPA1. We therefore reasoned that OPA1 could organize into high molecular weight complexes made up at least by the PARL generated soluble form and the membrane bound form of OPA1. To confirm this hypothesis we crosslinked this complex and confirmed the presence of a high molecular weight immunoreactive band for OPA1 that disappear following the mechanical expansion of the cristae induced by osmotic swelling. These crosslinker-stabilized oligomers contain both the soluble and the membrane bound forms of OPA1 as demonstrated by their immunoreactivity for properly tagged and co-expressed forms. The OPA1-containing oligomers is targeted by cBID in a time dependent manner and OPA1 overexpression stabilizes these complexes. We can conclude that OPA1 controls cytochrome c mobilization and cristae remodeling that occurs during apoptosis. This function of OPA1 is independent of MFNs and is correlated to the formation of high molecular weight complexes. The data collected so far on OPA1 antiapoptotic function open a new scenario. First we need to investigate on the molecular composition of these complexes in normal and apoptotic conditions. To this aim we started a biochemical study on OPA1-containing complexes in mitochondria isolated from different genetic background in normal and apoptotic conditions. The proteomic analysis of the proteins eventually found in complex with OPA1 will allow us to comprehend the function and regulation of OPA1 oligomers before and after cell death induction. OPA1 appears as a crucial protein in the apoptotic process; as a confirmation of this, it has been found that OPA1 is highly overexpressed in some lung cancer (Dean Fennel, personal communication); we then asked whether OPA1 could be a target for the development of new drugs that enhance apoptosis in tumor cells. To this aim, we started a collaboration with Stefano Moro from the Department of Medicinal Chemistry of the University of Padova, to generate a library of candidate inhibitors of OPA1 performing a virtual screening of compounds targeted to the GTPase pocket of OPA1 obtained following an homology modeling on the Dyctiostelium Discoideum GTPase domain of Dynamin A. In conclusion, the data presented in this doctoral thesis show that mitochondrial protein OPA1 participates in the regulation of cytochrome c mobilization and cristae remodeling during apoptosis. We demonstrated that OPA1 organizes into high molecular weight complexes which disruption correlates with cristae junction widening. This function is distinct from its role in mitochondrial morphology and this suggest a bifurcation and specialization of OPA1 function during evolution.

Les mitochondries sont des organelles très dynamiques qui subissent des phénomènes de fission et de fusion constants de leurs membranes extérieures et intérieures. Ces processus sont essentiels pour le maintien des fonctions mitochondriales essentielles telles que la phosphorylation oxydative ou la signalisation du calcium. D’un point de vue moléculaire, la fusion et la fission mitochondriale dépendent tous les deux des grandes GTPases de la famille des protéines de type dynamine. Les dynamines qui favorisent l’attachement et la fusion des membranes mitochondriales extérieures sont appelés les mitofusines.La mitofusine de la levure Fzo1 est une GTPase transmembranaire située dans la membrane externe de la mitochondrie. Son oligomérisation favorise l’attachement suivi de la fusion de la membrane externe mitochondriale. Fzo1 a été proposé récemment comme une protéine d’attachement potentielle entre les peroxysomes et les mitochondries lorsqu’elle est surexprimée. Cependant, on ignore si Fzo1 est présent sur les membranes peroxysomales dans les cellules sauvages ou si cette localisation extra-mitochondriale est une conséquence de la surexpression. De plus, nous ne savons toujours pas comment le Fzo1 peroxysomal et le Fzo1 mitochondrial interagissent dans ces contacts et quel est leur rôle dans la cellule. Durant ma thèse, j’ai pu prouver que Fzo1 se trouve réellement aux peroxysomes dans des conditions physiologiques et oligomérise avec le Fzo1 mitochondrial créant ainsi des contacts Fzo1-Fzo1 entre les peroxysomes et les mitochondries que nous appellerons maintenant des contacts « Permit Fzo1-dépendants ». On a découvert que ces contacts sont modulés par les niveaux de Fzo1 qui sont étroitement régulés par la ligase ubiquitine appelée Mdm30 mais aussi en fonction des niveaux de désaturation des acides gras dans la cellule. D’un point de vue fonctionnel et après avoir écarté plusieurs possibilités, nous avons trouvé que le rôle des contacts Permit Fzo1-dépendants est de réguler la fusion mitochondriale à travers le cycle glyoxylate, un processus qui permet aux cellules de convertir des composés unitaires de C2 en précurseurs de C4 pour la biosynthèse des acides aminés et des glucides. Nous avons découvert que les contacts Permit Fzo1-dépendants permettent le transfert mitochondrial des produits intermédiaires du cycle de glyoxylate pour stimuler la fusion mitochondriale. Ces résultats révèlent ainsi une réponse des organelles aux changements de désaturation des acides gras et aux besoins métaboliques de la cellule pour réguler la fusion mitochondriale.Enfin, les résultats obtenus au cours de ma thèse ont enrichi nos connaissances sur les contacts entre organelles et nous ont permis de prouver que Fzo1 est localisé sur les membranes mitochondriales et peroxysomales dans les cellules de type sauvage de levure. Nos études montrent également que les contacts Permit Fzo1-dépendants sont modulés en fonction des besoins de la cellule car ils jouent un rôle crucial dans l’entretien de la fusion mitochondriale en créant un raccourci possible pour les produits intermédiaires du cycle du glyoxylate pour atteindre les mitochondries lorsque cela est nécessaire
Mitochondria are highly dynamic organelles that undergo constant fission and fusion of their outer and inner membranes. These processes are critical to maintain essential mitochondrial functions such as oxidative phosphorylation or calcium signaling. On a molecular basis, mitochondrial fusion and fission both depend on large GTPases of the Dynamin-Related Protein (DRP) family. The DRPs that mediate attachment and fusion of mitochondrial outer membranes are called the Mitofusins. The yeast mitofusin Fzo1 is located in the mitochondrial outer membrane. Its oligomerization promotes mitochondrial tethering followed by mitochondrial outer membrane fusion. Fzo1 has recently been proposed as a potential tether between peroxisomes and mitochondria when overexpressed. However, whether Fzo1 is present on peroxisomal membranes in WT cells or whether this extra-mitochondrial localization is a consequence of overexpression is unknown. In addition, we still don’t know how peroxisomal and mitochondrial Fzo1 mediate these contacts and their purpose in the cell. In my thesis, we were able to prove that Fzo1 naturally localizes to peroxisomes and oligomerizes with the mitochondrial Fzo1 thus creating Fzo1-Fzo1 contacts between peroxisomes and mitochondria which we will now call “Fzo1-mediated permit” contacts. We found that these contacts are modulated by Fzo1 levels which are tightly regulated by an SCF ubiquitin ligase called Mdm30 but also depending on fatty acid desaturation levels in the cell. From a functional standpoint, we found that the role of Fzo1-mediated permit contacts is to regulate mitochondrial fusion through the glyoxylate cycle, a process which allows cells to convert C2 unit compounds to C4 precursors for amino acid and carbohydrate biosynthesis. We discovered that Fzo1-mediated permit contacts allow the mitochondrial transfer of early byproducts of the glyoxylate cycle to stimulate mitochondrial fusion. In fine, the results obtained during my thesis enriched our knowledge on organelle contacts and allowed us to prove that Fzo1 is localized on both mitochondrial and peroxisomal membranes in wild type cells. Our studies also show that Fzo1-mediated permit contacts are modulated according to the cell’s needs as they play a crucial role in upkeeping mitochondrial fusion by providing a possible shortcut for byproducts of the glyoxylate cycle to reach mitochondria when direly needed

Mitochondrial Ca2+ buffering is an important physiological modulator of neuronal signaling and bioenergetics, but this propensity toward Ca2+ regulation proves pathological during excitotoxic insult. Specifically, excessive mitochondrial Ca2+ uptake is a key component of glutamate toxicity within the penumbra surrounding the ischemic core following stroke. This mitochondrial toxicity and Ca2+ dyshomeostasis may be visualized in real time as delayed calcium deregulation (DCD). DCD is a predictor of neuronal, excitoxic death, and is composed of three phases: 1) an initial response; 2) a latent period of elevated, but stable cytosolic Ca2+; and 3) failure of mitochondrial Ca2+ retention, termed deregulation. The duration of the latent period is an index of neuronal resistance. Mitochondria are dynamic organelles that rapidly and reversibly undergo fission and fusion (MFF). MFF is tightly regulated by the phosphoregulation of fission inducing Drp1 at serine 656. Drp1-S656 phosphorelation is mediated by PKA/AKAP1, and it is dephosphorylated by PP2A/Bβ2. Phosphorylation of Drp1-S656 inactivates this contractile GTPase resulting in inhibition of mitochondrial fission and a shift toward elongated mitochondria. This PKA/AKAP1 dependent Drp1-S656 phosphorylation has proven to be neuroprotective. Likewise, attenuation of PP2A/Bβ2 signaling enhances neuronal survival during ischemia and excitotoxic insult. Based on the mitochondrial buffering role in excitotoxicity and MFF modulation of neuronal survival, we began investigating the role of Ca2+ buffering as a function of MFF during glutamate toxicity. Noted above, resistance to excitoticity is visualized by the duration of the DCD latent period. Overexpression of AKAP1 in cultured hippocampal neurons greatly prolonged DCD latency in a PKA dependent manner, while Bβ2 ablation prolonged DCD latency by hours. Pharmacological modulation of PKA required PDE4 inhibition to reproduce the AKAP1 observations. Preliminary experiments studying the effect of Bβ2 overexpression on matrix Ca2+ load suggests possible mechanism of MFF regulated of matrix Ca2+ accumulation. Using mtPericam DRG neurons as a model system for individual mitochondrial Ca2+ recording, we discovered impaired extrusion kinetics in mitochondria fragmented by both Drp1 and Bβ2 overexpression. Ca2+ uptake was comparable to that of control. Extreme elongation of mitochondria via dominant negative Drp1-K38A enhanced recovery. Understanding these observations, however, requires knowledge of the mitochondrial Ca2+ buffering mechanism. Mitochondrial uptake candidates include MCU and ccdc109b. Our neuronal characterization of MCU confirms a role in mitochondrial Ca2+ buffering, but not a requirement; other components must be involved. Ccdc109b remains an inconclusive candidate, but may be an important regulator of MCU. Mitochondrial efflux transporters include Letm1 and NCLX. Though Letm1 observations are hindered by control artifact, preliminary evidence supports a role in extrusion. The role of NCLX is complicated by possible tissue specificity. Functional expression experiments utilizing Na+ free Li+ external solution suggests absence of NCLX in hippocampal neurons; DRG neurons were capable of Li+ exchange. The above observations confirm the significance of mitochondrial Ca2+ extrusion in neuronal survival. Understanding the mechanisms and regulation of mitochondrial Ca2+ transport has the potential to provide novel therapeutic targets in pathologies of excitotoxic etiology.

La thérapie cellulaire régénératrice offre des perspectives d'applications dans de nombreuses pathologies entraînant une perte cellulaire. Cependant, suite à un infarctus du myocarde et donc une diminution importante du nombre de cardiomyocytes, l'injection de cellules souches n'a permis de mettre en évidence qu'une amélioration légère et transitoire de la fonction cardiaque. Ces résultats suggèrent qu'il est nécessaire d'améliorer l'efficacité des protocoles de thérapie cellulaire cardiaque. Cette amélioration passe par une meilleure compréhension des mécanismes mis en jeu par les cellules souches dans la régénération myocardique. Parmi les hypothèses soulevées, la fusion entre les cellules souches et les cardiomyocytes a été décrite dans plusieurs études mais le rôle physiologique de ce phénomène reste inconnu. Mon travail de thèse a consisté à étudier ce mécanisme in vitro au sein de cocultures entres des cellules souches adultes humaines (cellules hMADS pour human multipotent adipose derived stem cells) et des cardiomyocytes murins adultes. Nous avons pu mettre en évidence un processus de fusion hétérologue entre ces deux types cellulaires, aboutissant à la reprogrammation du cardiomyocyte vers un stade de progéniteur. Les cellules hybrides résultant de cette fusion ont exprimé des marqueurs cardiomyogéniques précoces et de prolifération et ont été montrées comme ayant un génotype exclusivement murin. Ces cellules hybrides ou progéniteurs cardiaques se sont formés préférentiellement par un mécanisme de fusion partielle par l'intermédiaire de structures intercellulaires appelées nanotubes composés de f-actine et de microtubules. En outre, nous avons montré que le transfert de mitochondries des cellules souches vers les cardiomyocytes était indispensable pour la reprogrammation des cardiomyocytes. En conclusion, nos résultats apportent de nouveaux éléments dans la compréhension des mécanismes de régénération médiés par les cellules souches qui est un pré-requis pour optimiser les protocoles de thérapie cellulaire cardiaque
Regenerative cell therapy offers potential applications in many diseases involving cell loss. However, following myocardial infarction and the dramatic decrease in the number of cardiomyocytes, the injection of stem cells led to a poor and transient improvement of cardiac function. Therefore stem cell-based therapy to treat myocardial infarction requires a better understanding of the mechanisms brought into play by stem cells in heart regeneration. Among the different hypothesis raised, cell fusion between stem cells and cardiomyocytes has been described in several studies. However, the respective physiological impact of cell fusion remains unknown. During my thesis, I investigated this cell fusion mechanism in vitro in a coculture model between human multipotent adipose-derived stem cells (hMADS) and murine fully differentiated cardiomyocytes. We showed intercellular exchanges of cytoplasmic and nuclear material between both cell types, followed by a heterologous cell fusion process promoting cardiomyocyte reprogramming back to a progenitor-like state. The resulting hybrid cells expressed early cardiac commitment and proliferation markers and exhibited a mouse genotype. We provided evidence that cardiac hybrid cells were preferentially generated through partial cell fusion mediated by intercellular structures composed of f-actin and microtubule filaments. Furthermore, we showed that stem cell mitochondria were transferred into cardiomyocytes and were required for somatic cell reprogramming. In conclusion, by providing new insights into previously reported cell fusion processes, our results might contribute to a better understanding of stem cell-mediated regenerative mechanisms and thus, the development of more efficient stem cell-based heart therapies

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.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008.
Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

ABSTRACT Mitochondria are the energy producing organelles in eukaryotic cells providing ATP through oxidative phosphorylation (OXPHOS). Mitochondria are highly dynamic and undergo fission, fusion and move into the cell along the microtubules to generate the mitochondrial network. Mitochondrial dynamics play a critical role in the control of organelle shape, size, number, function and quality control of mitochondria. It is regulated by several GTPases that play an important role in fusion and fission processes. In mammals, mitochondrial fusion is controlled by Mitofusin 1 (Mfn1), Mitofusin 2 (Mfn2) and Optic atrophy protein 1 (Opa1), while mitochondrial fission is regulated by Dynamin related protein 1 (Drp1). The aim of this study is to understand how mitochondrial distribution in neuronal cells is affected by mitochondria function and/or morphology. We use Drosophila melanogaster , whose genome contains homologs for all mitochondrial fusion and fission proteins, as a modelorganism to study how loss of fusion and fission protein modify the axonal distribution and motility of mitochondria. We demonstrate that loss of Marf (Mitochondrial associated regulatory factor, homologous to human mitofusins) or Opa1 causes an accumulation of mitochondria in the soma, a defect in the axonal distribution of mitochondria, a severe depletion of mitochondria in neuromuscular junctions (NMJs) and reduced mitochondrial motility. Simultaneous loss of Drp1 rescues the Opa1 phenotype very robustly while loss of Marf essentially does not. Viability data however show the opposite trend. The expression of Marf RNAi or Opa1 RNAi cause lethality, and so does the double down regulation of Opa1 and Drp1. Conversely individuals expressing Marf RNAi and Drp1 RNAi simultaneously survive and are comparable to the controls. We then examined possible alterations of mitochondrial function by analyzing the mitochondrial respiratory capacity, the activity of the respiratory chain complexes and ATP production capacity. The data show that individuals where Marf, Opa1 or simultaneously Opa1 and Drp1 are down-regulated display severe alterations in mitochondrial function, while there are no obvious energy defects in individuals in which the expression of Marf and Drp1 is simultaneously reduced. Collectively our results obtained suggest that mitochondrial morphology is important for a homogeneous distribution of mitochondria along the axon and their transport to synapses and that these mechanisms are independent of mitochondria function.
RIASSUNTO I mitocondri sono organelli essenziali per la cellula e la loro funzione primaria è di produrre energia sottoforma di ATP. I mitocondri sono organelli altamente dinamici:processi di fusione e fissione delle membrane mitocondriali ne controllano la forma, la lunghezza e il numero e un equilibrio tra i due meccanismi è fondamentale per una corretta morfologia mitocondriale. Numerose proteine sono coinvolte nei processi di fusione e fissione mitocondriale: Mitofusina 1 e Mitofusina 2 (Mfn1 e Mfn2) e Optic atrophy 1 (Opa1) regolano i processi di fusione mitocondriale, mentre Dynamin-related protein 1 (Drp1)mediala fissione. Drosophila possiede il gene mitochondrial assembly regulatory factor (MARF), espresso in modo ubiquitario ed omologo al gene MFN2. Nel tessuto muscolare la riduzione di espressione di Marf induce frammentazione e alterazione della morfologia del mitocondrio. Inoltre, mutanti di Marf mostrano una severa deplezione dei mitocondri nelle giunzioni neuromuscolari (NMJs) ed un’alterazione della morfologia della giunzione caratterizzata dall’aumento nel numero e da una riduzione nella dimensione dei bottoni sinaptici. Un altro aspetto della dinamica mitocondriale, oltre ai processi di fusione e fissione, è la motilità dei mitocondri, che deve essere altamente regolata soprattutto in cellule come i neuroni. Il trasporto mitocondriale e la continua ridistribuzione dei mitocondri lungo l’assone è essenziale per il mantenimento dell’integrità assonale e delle normali funzioni della cellula. Studi hanno messo in evidenza come la mancanza di mitocondri a livello delle giunzioni neuromuscolari in Drosophila comprometta la trasmissione sinaptica e come difetti nel trasporto mitocondriale assonale siano implicati nello sviluppo di disordini neurologici e malattie neurodegenerative (Chan, 2006). Lo scopo di questo lavoro è quello di capire il ruolo della morfologia e della funzione mitocondriale nella distribuzione intracellulare dei mitocondri nei neuroni. Per fare questo abbiamo utilizzato Drosophila melanogaster, organismo modello efficace per l’analisi della funzione genica, inclusa quella di geni responsabili di patologie umane. L’analisi della morfologia mitocondriale è stata effettuata utilizzando linee di Drosophilache esprimono in vivo un transgene per RNA interference e che permette di ridurre l’espressione di geni endogeni coinvolti nei processi di fusione e fissione mitocondriale, quali Marf, Opa1 e Drp1. Abbiamo inoltre creato linee che esprimono contemporaneamente i trangeni per RNAi di Marf e Drp1 o Opa1 e Drp1, con lo scopo di bilanciare i meccanismi di fusione e/o fissione. Ci siamo soffermati in particolare sullo studio di due aspetti principali, la morfologia e la funzionalità mitocondriale, per capire se difetti nella morfologia e nella funzionalità mitocondriale siano collegate e concorrano insieme allo sviluppo di patologie.Numerose patologie neurodegenerative sono infatti caratterizzate da alterazioni del trasporto mitocondriale e spesso questo è associato a difetti nella morfologia e nella funzionalità mitocondriale. Per studiare la morfologia mitocondriale, le linee UAS-RNAi sono state incrociate con una linea che contiene il promotore ELAV per l’espressione tessuto-specifica nei neuroni ed esprime una GFP mitocondriale. Abbiamo analizzato la morfologia dei mitocondri, sia nel corpo cellulare sia negli assoni e la distribuzione mitocondriale in assoni lunghi come i motoneuroni e assoni corti come quelli del nervo ottico e la distribuzione mitocondriale nella giunzione neuromuscolare.I risultati ottenuti mostrano che frammentazione dei mitocondri e alterazione della distribuzione mitocondriale assonale in individui in cui sia ridotta l’espressione di proteine di fusione. Inoltre si osserva una diminuzione della percentuale dei mitocondri mobili e del numero assoluto dei mitocondri anterogradi e retrogradi. Questi dati dimostrano che vi è una stretta correlazione tra morfologia mitocondriale e distribuzione dei mitocondri, in particolare in assoni lunghi. Inoltre analizzando le linee Marf RNAi Drp1 RNAi e Opa1 RNAi Drp1 RNAi, nelle quali gli eventi di fusione e fissione ridotti ma sono in equilibrio tra loro, si osserva un miglioramento la morfologia, la distribuzione e il trasporto mitocondriale assonale in modo particolare nel caso di Opa1 e non nel caso di Marf. Abbiamo cercato di capire quindi se in questi individui vi fossero alterazioni delle funzionalità mitocondriali attraverso l’analisi della capacità respiratoria mitocondriale, dell’attività dei complessi della catena respiratoria e della capacità di produzione di ATP. I risultati ottenuti dimostrano che morfologia e funzionalità mitocondriale non sempre sono collegate tra loro hanno effetti diversi nella modulazione della distribuzione mitocondriale assonale. In conclusione possiamo affermare che solamente la morfologia e la dimensione del mitocondrio sembrano essere essenziali per la corretta distribuzione mitocondriale assonale.

Intracytoplasmic protein aggregates called Lewy Bodies (LB) characterize the neurodegeneration in Parkinson’s Disease (PD). However, whether aggregates are linked to cell death or they represent a protective mechanism is debated. Recent studies suggest that autophagy participates in the degradation of cytoplasmic protein inclusions. Moreover, growing evidence shows that mitochondrial impairments and altered mitochondrial fission and fusion play a role in the pathogenesis of PD. Synphilin-1 is a protein that is present in LB and that interacts with key elements of PD pathogenesis, such as α-synuclein. Overexpression of synphilin-1 in various cellular models leads to cytoplasmatic inclusions that fulfil the criteria of aggresomes, dynamic structures formed under proteolytic stress. HtrA2/Omi is a mitochondrial protease that is thought to be involved in protection of mitochondria against stress in physiological conditions, besides its pro-apoptotic function under apoptosis induction. Loss of HtrA2/Omi function leads to neurodegeneration in mouse models. Mutations in both synphilin-1 and HtrA2/Omi have been found in PD patients. We used HEK293 cells overexpressing wild type (WT) or R621C mutant synphilin-1 to evaluate if autophagy activation may influence cell viability by modulating synphilin-1 inclusions. Moreover, we studied also the effects synphilin-1-overexpression and aggregation on mitochondria. In addition, we evaluated the consequences of loss of HtrA2/Omi on mitochondrial function and morphology in fibroblasts from knockout mice, as well as in HeLa cells and in Drosophila melanogaster S2R+ cells. We observed co-localization of synphilin-1 inclusions with autophagic structures and the pharmacological activation of autophagy was effective in reducing the percentage of cells bearing synphilin-1 inclusions. However, this treatment couldn’t protect the cells from apoptosis induced by proteasome inhibition. Synphilin-1-overexpressing cells revealed higher levels of mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential (MMP) compared to empty vector controls. HtrA2/Omi deficiency caused accumulation of ROS within mitochondria and reduced MMP as well, together with morphological alterations of the mitochondria. Interestingly, we observed a selective up-regulation of the fusion factor OPA1 in cells lacking HtrA2/Omi and co-immunoprecipitation experiments showed that these two proteins can physically interact. To conclude, our findings support the view that aggresomes are actively built to remove excesses of noxious proteins; however, the enhancement of their clearance via autophagy is not sufficient to protect against proteasome inhibition and mitochondrial impairments in the presence of high levels of aggregate-prone proteins. Concerning HtrA2/Omi, we confirmed previous reports showing an involvement of this protease in maintaining mitochondrial homeostasis and we reported for the first time a direct effect of loss of HtrA2/Omi on mitochondrial morphology. Finally, we showed a novel role of HtrA2/Omi in the modulation of OPA1.

VAP-A est un récepteur ancré à la surface du réticulum endoplasmique (RE) pour des centaines de protéines contenant un motif FFAT. Ses partenaires possèdent des structures et des fonctions très variées. VAP-A participe à la formation des sites de contact membranaire (MCSs) entre le RE et les autres organelles et ceci permet notamment le trafic non vésiculaire des lipides entre les membranes. Par exemple, la protéine de transfert de lipide OSBP interagit avec VAP aux MCSs RE/Golgi afin de transporter le cholestérol contre son gradient de concentration par contre-échange et hydrolyse de phosphatidylinositol-4-phoshate (PI4P). L'interaction entre le domaine Major-Sperm-Protein (MSP) de VAP-A et le motif FFAT de ses partenaires était déjà caractérisé. Cependant, la façon dont ce récepteur universel peut s'adapter à toutes ses cibles dans tous les MCSs, qui sont très différents en terme de géométrie et de stabilité était inconnue.Dans cette étude nous avons utilisé une approche pluridisciplinaire afin de démontrer que VAP-A contient deux régions intrinsèquement désordonnées (IDRs) qui fournissent à la protéine une flexibilité indispensable à son organisation fonctionnelle dans les MCSs. Nous avons montré qu'un mutant de VAP-A sans ses linkers flexibles possède une localisation subcellulaire restreinte aux MCSs RE/mitochondrie. Ce mutant ne peut donc pas soutenir l'activité d'OSBP et CERT aux MCSs RE/Golgi. En revanche, il interagit avec VPS13A et PTPIP51 à la mitochondrie et permet ainsi le transport de lipides qui contribuent au métabolisme des cardiolipines et à la fusion mitochondriale.Ces résultats indiquent que la flexibilité de VAP-A fournie par ses IDRs, joue un rôle clé pour assurer son adaptabilité à différents contextes et plus précisément aux MCSs à durée de vie courte comme les MCSs RE/Golgi, cette étude démontre également l'implication de VAP-A dans la fusion mitochondriale
VAP-A is a receptor at the surface of the endoplasmic reticulum (ER) for hundreds of proteins containing a FFAT motif and having a wide range of structures and functions. VAP-A is also required for creating multiple membrane contact sites (MCSs) between the ER and other compartments, which notably enable non-vesicular lipid exchanges between membranes. For example, the lipid-transfer protein (LTP) OSBP interacts with VAP at ER/Golgi MCS to transport cholesterol through coupled counter-exchange and hydrolysis of PI4P. It is well known that VAP-A partners contain a FFAT motif specifically recognized by the Major-Sperm-Protein (MSP) domain of VAP, however, how this receptor adapts to its different targets in MCSs that are so different in geometry and lifetime is not understood.In this study, we used a multidisciplinary approach to demonstrate that VAP-A contains two intrinsically disordered linkers that provide it with a high degree of flexibility to enable functional organization of different MCSs. A VAP-A mutant without flexible linkers is restricted in its subcellular localization, and does not support lipid transport by OSBP and CERT at ER/Golgi MCS. However, this mutant is present at ER/mitochondria MCS by interacting with VPS13A and PTPIP51, and thus facilitates lipid transport contributing to cardiolipin metabolism and mitochondrial fusion.In conclusion, this work indicates that VAP-A conformational flexibility mediated by its intrinsically disordered regions is key to ensure membrane tethering especially at short-lived MCSs; it also demonstrates the implication of VAP-A in mitochondrial fusion

Leydig-ove  ćelije  testisa  su  primarno  mesto  sinteze muških polnih hormona. Ovi hormoni su neophodani za reproduktivno,  ali  i  za  opšte  zdravlje  budući  da  suozbiljni zdravstveni problemi često povezani sa njihovom smanjenom produkcijom.  Insulin i insulinu sličan faktor rasta  1,  IGF1  (engl.  insulin  like  growth  factor  1),  isignalizacija koju pokreću preko svojih receptora  (INSR i IGF1R),  su  jedan  od  ključnih  faktora  koji  regulišu specifični razvoj tkiva, pa i samih gonada. Ipak,  uloga  imehanizmi  delovanja  ovih  receptora  u  steroidogenim tkivima nisu  u potpunosti  poznati.  Stoga je  istraživanje  uokviru ove  doktorske  disertacije  koncipirano sa ciljem da se,  na  modelu  prepubertalnih  (P21)  i  adultnih  (P80) mužjaka miševa sa kondicionalnom delecijom Insr i Igf1r gena  u  steroidogenim  ćelijama  (Insr/Igf1r-DKO), definiše uloga INSR i IGF1R u regulisanju diferencijacije i  steroidogene  funkcije  Leydig-ovih  ćelija.  Pored  toga, mužjaci  i  ženke  P21  miševa  sa  istom  delecijom  su korišćeni  za  praćenje  ekspresije  glavnih  markera mitohondrijalne  biogeneze  i  fuzije/arhitekture  u  Leydigovim  ćelijama,  ovarijumima  i   nadbubrežnim  žlezdama. Rezultati  su  potvrdili  da  delecija  Insr  i  Igf1r  usteroidogenim  tkivima  utiče  na  diferencijaciju  i funkcionalne karakteristike Leydig-ovih ćelija P21 i P80 miševa,  upućujući  na  pojavu  tzv.  „feminizacije“.  BrojLeydig-ovih  ćelija  izolovanih  iz  P21  i  P80  Insr/Igf1rDKO  miševa  bio  je  smanjen,  a  morfologija  i ultrastruktura  ovih  ćelija  izmenjene  kod  P21  Insr/Igf1rDKO  miševa.  Steroidogeni  kapacitet  i  aktivnost,  kao  i ekspresija  glavnih  elemenata  steroidogene  mašinerije (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1  i  6, Hsd17b3,Sf1)  bili su  smanjeni  u Leydig-ovim ćelijama P21 i P80 Insr/Igf1r-DKO miševa,  dok je ekspresija transkripcionih represora  steroidogeneze  (Arr19  i  Dax1)  bila  povećana specifično  u  istim  ćelijama,  ali  ne  i  u  ostatku  testisa.Transkripcioni  profil  markera  muškog  pola  (Sry,  Sox9, Amh)  bio  je  izmenjen  u Leydig-ovim ćelijama P21 i P80 Insr/Igf1r-DKO  miševa.  Transkripcija  markera  ženskog pola (Rspo1, Wnt4) u testisima,  kao i ekspresija  Cyp19a1 i  produkcija estradiola (E2) u Leydig-ovim ćelijama,  P21 i  P80  Insr/Igf1r-DKO  miševa  bile  su  povećane. Transkripcija  markera  mitohondrijalne  biogenze (Ppargc1a,  Tfam,  Mtnd1)  bila  je  smanjena  u  Leydigovim  ćelijama  P21  Insr/Igf1r-DKO  miševa,  dok  supromene  ekspresije  izostale  u  ovarijumima  ženki  istog  genotipa.  Isti  markeri  su  bili  povećani  u  nabdubrežnim  žlezdama  oba  pola.  Markeri  mitohondrijalne fuzije/arhitekture  (Mfn1  i  Mfn2)  bili  su  povećani  u Leydig-ovim ćelijama P21 Insr/Igf1r-DKO miševa, što je  praćeno  i  narušenom  mitohondrijalnom  fazom steroidogeneze (produkcija progesterona), kao i brojem i  morfologijom ovim organela.  Ekspresija istih markera u ovarijumima  bila  je  nepromenjena.  Sumirano,  rezultati ovog istraživanja  su  pokazali  da su  INSR i IGF1R  važni za  diferencijaciju  i  steroidogenu  funkciju  Leydig-ovih  ćelija  P21  i  P80  miševa.  Takođe,  ovi  receptori  su  važni regulatori  markera  mitohondrijalne  biogeneze  i fuzije/arhiteture u steroidogenim ćelijama muških gonada  P21 miševa, ali ne i u steroidogenim ćelijama ovarijuma. 
Leydig cells of testes are the primary site of the male sex hormones  synthesis.  These  hormones  are  indispensable for  both  reproductive  and  general  health  since  serious health  problems  are  often  associated  with  their  reduced production.  Insulin  and  insulin-like  growth  factor  1, IGF1  (insulin  like  growth  factor  1),  and  signaling triggered through  their receptors (INSR and IGF1R), are  one of the key  factors  that regulate specific development of  tissue  including  gonads.  However,  the  role  and mechanisms  of  these  receptors  action  in  steroidogenic tissues are not known enough. This study was designed to  observe   the role of INSR and IGF1R in regulating the differentiation and steroidogenic function of Leydig cells by using the model of prepubertal (P21) and adult (P80) male mice with the conditional deletion of the  Insr  and Igf1r  genes  in  steroidogenic  cells  (Insr/Igf1r-DKO).  In addition,  male  and  female  P21  mice  with  the  samedeletion were used to monitor the expression of the main markers  of  mitochondrial  biogenesis  and fusion/architecture  in  Leydig  cells,  ovaries  and  adrenal glands.  The  results  confirmed  that  deletion  of  Insr  and Igf1r  in  steroidogenic  tissues  influences  differentiation and  functional  characteristics  of  Leydig  cells  isolated from  P21  and  P80  mice,  suggesting  an  appearance  of "feminization".  The  number  of  Leydig  cells  isolated from  both  P21  and  P80  Insr/Igf1r-DKO  mice  was reduced.  Morphology  and  ultrastructure  of  Leydig  cells were  disturbed  in  P21  Insr/Igf1r-DKO  mice. Steroidogenic capacity and activity, as well as expression of the main elements of  steroidogenic machinery (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1  and  6, Hsd17b3, Sf1) were  decreased  in  Leydig  cells  from  P21  and  P80 Insr/Igf1r-DKO  mice,  while  the  expression  of transcriptional  repressors  of  steroidogenesis  (Arr19  and Dax1) was increased  in the same cells, but not in the rest of  the  testes.  Transcription  profile  of  the  male  sex markers  (Sry,  Sox9,  Amh)  was  altered  in  Leydig  cells from  P21  and  P80  Insr/Igf1r-DKO  mice.  Transcription of the female sex markers (Rspo1, Wnt4) in the testes, as well  as  Cyp19a1  expression  and  estradiol  (E2) production in Leydig cells,  from P21 and P80  Insr/Igf1rDKO  mice  were  increased.  Transcription  of mitochondrial  biogenesis  markers  (Ppargc1a,  Tfam, Mtnd1)  was  declined  in  Leydig  cells  from  P21 Insr/Igf1r-DKO mice, while changes were absent in  the ovaries of the same genotype.  Transcription of the  same markers  was  increased  in  the  adrenal  glands  of  both sexes.  The  mitochondrial  fusion/architecture  markers (Mfn1  and  Mfn2)  were  increased  in  Leydig  cells  from Insr/Igf1r-DKO  mice  and  followed  by  disturbedmitochondrial  phase  of  steroidogenesis  (progesterone production), as well as  decreased  number and  disturbed morphology  of  mitochondria.   Expression  of  the  same markers  in  the  ovaries  was  unchanged.  In  summary, results  of  this  study  showed  that  INSR  and  IGF1R  are important in differentiation and steroidogenic function of Leydig  cells  from  P21  and  P80  mice.  Also,  these receptors  are  important  regulators  of  mitochondrial biogenesis  and   fusion/architecture  markers  in steroidogenic  cells  of  P21  male  mice,  but  not  in steroidogenic cells of ovaries.

Depuis quelques années, de nombreux travaux suggèrent que des perturbations des fonctions mitochondriales contribuent aux maladies neurodégénératives. Les mitochondries sont particulièrement importantes pour les neurones en raison à leur rôle dans la régulation calcique, la signalisation redox, la plasticité synaptique et, in fine, la survie cellulaire. La dynamique mitochondriale contrôle la morphologie de l'organelle via un équilibre délicat entre deux forces opposées : la fusion et la fission régulées par des dynamines de la grande famille des GTPases. Notre équipe a montré que la perte ou des mutations de la protéine de fusion OPA1 entraînent des dysfonctionnements de la membrane interne mitochondriale pouvant mener à l'apoptose, qui revêtent une importance particulière dans l'atrophie optique autosomale dominante (ADOA-1). Pour comprendre les mécanismes par lesquels des altérations de la dynamique mitochondriale pourraient contribuer à des dysfonctionnements mitochondriaux et éventuellement à l'origine de la neurodégénérescence, nous avons étudié les effets de la perte d'OPA1 dans des neurones corticaux ex vivo. La perte de fonction à l'interférence à l'ARN mène à la fragmentation mitochondriale sans perturbation de la distribution mitochondriale, ni mort neuronale. Si l'arborescence dendritique est inchangée, la quantité de plusieurs protéines synaptiques est réduite, suggérant une déficience synaptique. De plus, dans ces conditions, l'état redox est altéré et la quantité protéique de complexes respiratoires spécifiques est réduite. Enfin, l'enregistrement des propriétés électrophysiologiques montrent des changements dans la transmission synaptique, notamment par une diminution de la fréquence des courants excitateurs et une augmentation de la fréquence des courants inhibiteurs. De façon intéressante, un traitement à la forskoline permet de restaurer un fonctionnement électrophysiologique normal. Pour conclure, nos données offrent de nouvelles pistes non seulement dans la compréhension de maladies neurodégénératives liées directement à la dynamique mitochondriale comme l'ADOA1, mais aussi d'autres pathologies neurodégénératives liées à un défaut du métabolisme oxydatif comme les maladies d'Alzheimer, Parkinson ou Huntington
In the past few years, multiple findings have suggested that disruptions of mitochondrial functions and dynamics contribute to neurodegenerative diseases. Mitochondrial functions in neurons include regulation of calcium and redox signaling, developmental and synaptic plasticity as well as the arbitration of cell survival and death. Mitochondrial dynamics controls the organelle's morphology via a delicate balance of two opposing forces: mitochondrial fusion and fission that are regulated by large dynamin-related GTPases evolutionary conserved from yeast to human. We have previously demonstrated that the fusion protein OPA1 loss or mutations led to mitochondrial inner membrane dysfunctions and apoptosis of particular importance in optic nerve pathologies like ADOA1 (autosomal dominant optic atrophy). While links emerge between defects in mitochondrial fusion and neurodegeneration, the processes involved are still largely unknown. To understand the mechanisms by which alterations of mitochondrial dynamics could contribute to mitochondria dysfunction, eventually leading to neurodegeneration, we studied the effects of OPA1 loss of function in neurons ex vivo. In cortical neurons, RNA interference of the fusion protein OPA1 led to mitochondrial fragmentation without altering neither mitochondrial distribution nor neuronal death rate. While there was no incidence on dendrites and axon size and numbers, the quantity of several synaptic proteins was reduced, suggesting synaptic impairment. In these conditions, the redox state of OPA1 depleted-neurons was impaired and specific respiratory complex proteins quantities were decreased. Finally, electrophysiological recordings showed that OPA1 depletion induced changes in synaptic transmission, particularly in decreasing of EPSC frequency and by increasing IPSC frequency. Interestingly, forskolin treatment rescue these electrophysiological defaults. In conclusion, our data may offer new insights not only into mitochondrial dynamics-linked neurodegenerative diseases like ADOA1 but to other neurodegenerative pathologies correlated with oxidative metabolism such as Huntington's, Parkinson's and Alzheimer's diseases

Le projet de doctorat porte sur l'étude structurale des mitofusines (Mfn1/2 chez l'homme et Fzo1 chez la levure) en utilisant principalement des méthodes basées sur la modélisation telles que la dynamique moléculaire ou les méthodes de prédiction de structure basées sur l'intelligence artificielle (principalement AlphaFold). Les mitochondries forment un réseau complexe à l'intérieur des cellules, subissant des événements continus de fusion et de fission. Ces processus façonnent la dynamique mitochondriale et sont essentiels pour l'entretien, la fonction, la distribution et l'héritage des mitochondries. La morphologie de ces dernières répond donc aux changements physiologiques constants de la cellule. Les larges GTPase impliquées dans l'ancrage et la fusion des membranes externes de mitochondrie sont des protéines transmembranaires appelées mitofusines. Les mitofusines Mfn1 et Mfn2 se trouvent chez les mammifères. Fzo1 (Fuzzy Onion 1) est l'homologue unique de Mfn1/2 chez la levure Saccharomyces cerevisiae. La fusion de la membrane interne mitochondriale et l'organisation des crêtes sont médiées par l'OPA1 humaine (Atrophie Optique 1) et la Mgm1 de la levure (Maintenance du Génome Mitochondrial 1). La dysfonction de la fusion mitochondriale est liée à plusieurs troubles neurodégénératifs, tels que Parkinson, Alzheimer et la maladie de Huntington. En effet, il a été montré que les mutations dans Mfn2 induisent le développement et la progression de dystrophies musculaires, telles que la maladie de Charcot-Marie-Tooth de type 2A, la forme la plus courante de la maladie CMT axonale. Le mécanisme exact par lequel les mitofusines contribuent à la dysfonction mitochondriale, ainsi que le mécanisme moléculaire exact de la fusion, ne sont pas encore entièrement compris. Dans l'ensemble, la fusion mitochondriale joue un rôle important dans la CMT2A, il est donc d'une importance capitale de comprendre pleinement le processus au niveau moléculaire. Les structures de Mfn1 et Mfn2 ont étés partiellement résolue, le domaine transmembranaire étant exclu, mais aucune structure résolue n'est disponible pour Fzo1. Fzo1 est intégré à membrane externe de mitochondrie avec ses deux domaines transmembranaires, exposant les parties N- et C-terminales vers le cytosol et une boucle vers l'espace intermembranaire. Du côté N-terminal, on trouve deux domaines de répétitions en heptad (HRs), HRN (présent uniquement chez la levure) et HR1, flanquant un domaine GTPase. Un troisième domaine HR, HR2, se trouve dans la partie C-terminale. Certains modèles de Fzo1 ont été construits avec comme template la protéine bactérienne de type dynamin-like (BDLP). BDLP est impliquée dans le remodelage des membranes et existe sous deux états conformationnels, une version compacte fermée qui passe à une structure étendue ouverte lors de la liaison au GTP, sur laquelle les modèles construits étaient basés. L'objectif du doctorat est de mettre à jour le modèle de Fzo1 construit en 2017, en travaillant dans un premier temps le domaine transmembranaire à l'aide de dynamiques moléculaires à plusieurs échelles. Un autre projet a consisté à étudier l'hélice amphipathique du domaine HR1 de Mfn1 (MfnA-AH), à tester ses capacités de liaison à la membrane. Initialement, nous avons utilisé des simulations gros grains, établissant ainsi une base solide pour évaluer la capacité prédictive de la famille de champs de force MARTINI. En utilisant d'autres simulations réalisées avec la pénétratine, nous avons pu fournir une analyse comparative des interactions AH-membranes dans les champs de force MARTINI. Mfn1-AH a ensuite été caractérisé plus en détail à l'aide de simulations tout-atomiques
The Phd project is the structural study of mitofusins (Mfn1/2 in humans and Fzo1 in yeasts) using mainly modeling-based methods such as molecular dynamics or structure prediction methods based on artificial intelligence (mainly AlphaFold). This project is a part of an ANR (MITOFUSION) shared between different partners (Laboratoire de Biochimie Théorique: Antoine Taly, Marc Baaden, Laboratoire des Biomolécules: Patrick Fuchs, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes: Mickaël Cohen, Institut de Psychiatrie et Neurosciences de Paris: David Tareste) whose goal is to understand the structure-function relationships of the mitofusin. Mitochondria form a complex network inside the cells, undergoing continuous fusion and fission events. These processes shape mitochondrial dynamics and are essential for the maintenance, function, distribution and inheritance of mitochondria. The morphology of the latter therefore respond to the ever-changing physiological changes of the cell. The large GTPase involved in the tethering and fusion of the mitochondrial outer membranes (OM) are transmembrane proteins called mitofusins. The mitofusins Mfn1 and Mfn2 can be found in mammals. Fzo1 (Fuzzy Onion 1) is the unique mitofusin homologue in Saccharomyces cerevisiae. The mitochondrial inner membrane fusion and cristea organisation is mediated by human OPA1 (Optic Atrophy 1) and yeast Mgm1 (Mitochondrial Genome Maintenance 1). Mitochondrial fusion dysfonction is related to several neurodegenerative disorders, such as Parkinson, Alzheimer and Huntingtion diseases. As a matter of fact, research has shown that mutations in Mfn2 induce the development and progression of muscular dystrophies, such as Charcot-Marie-Tooth Type 2A, the most common form of axonal CMT disease. The exact mechanism by which the mitofusins contributes to mitochondria dysfunction as well as the exact molecular fusion mechanism is not fully understood yet. Overall, mitochondrial fusion plays an important role in CMT2A, it is thus of paramount importance to get a full understanding of the process at the molecular level. The structure of both Mfn1 and Mfn2 was partially solved, the transmembrane domain being excluded, and no solved structure are available for Fzo1. With our ANR partners, we decided to work on the yeast version of Mitofusin (named Fzo1) as it is a good model (of homology with human Mfn1 and Mfn2) as yeast are convenient hosts for testing how other protein partners are involved in the process (e.g. Ugo1). Fzo1 is embedded in the mitochondrial OM as it possesses two transmembrane domains, exposing N- and C- terminal portions towards the cytosol and a loop towards the intermembrane space. On the N-terminal side can be found two coiled-coil heptad repeats (HRs) domains, HRN (in yeast only) and HR1, flanking a GTPase domain. A third coiled-coil heptad repeats domain HR2 is on the C-terminal portion. Some models of Fzo1 were built based on the mitofusin related bacterial dynamin-like protein (BDLP). BDLP is involved in membrane remodelling and exists in two conformational states, a closed compact version which changes to an opened extended structure, upon GTP-binding, on which the built models were based. The goal of the PhD is to update the model of Fzo1 built in 2017, by working on the transmembrane domains using multiscale molecular dynamics, and then update the overall structure using artificial intelligence methods. An other project consisted in studying the amphipathic helix of HR1 domain of Mfn1 (MfnA-AH), test its membrane binding capabilities. Initially, we employed coarse-grained simulations, establishing a robust foundation for evaluating the predictive capacity of the MARTINI family of force fields. Using other simulations ran with the penetratin, we were able to provide a comparative analysis for the AH-membranes interactions in the MARTINI force-fields. The Mfn1-AH was then further characterized using all-atom simulations

Les jeunes oiseaux exposés au froid assurent leur homéothermie en stimulant les oxydations mitochondriales dans les muscles squelettiques. L’exposition prolongée au froid accroit les capacités de thermogenèse musculaire grâce à une plasticité bioénergétique mitochondriale dont le contrôle reste hypothétique. Chez les mammifères, des protéines de fusion (les mitofusines (Mfns) et OPA1(OPtic Atrophy 1)) participent au remaniement des réseaux dynamiques mitochondriaux dans de multiples types cellulaires. Le but de ce travail de thèse était de caractériser l’expression d’homologues aviaires des protéines de fusion mammaliennes et d’étudier leurs variations d’expression lors de la mise en place des processus bioénergétiques chez l’oiseau en croissance, lors d’une exposition aiguë ou prolongée au froid ou lors de challenges nutritionnels ou endocrines.Sur le plan méthodologique, une approche intégrative a été utilisée de l’animal entier (calorimétrie indirecte) à l’expression protéique (western blot) ou transcriptionnelle (RT-PCR) en passant par des mesures de la fonctionnalité bioénergétique sur des fibres musculaires perméabilisées et mitochondries isolées. Deux modèles animaux ont été utilisés, une espèce naturellement adaptée aux conditions extrêmes de l’Antarctique, le manchot Adélie (Pygoscelisadeliae), et un modèle de laboratoire, le canard de Barbarie (Cairina moschata). Nos résultats ont permis de caractériser chez l’oiseau l’expression de protéines de fusion (Mfn2, OPA1) immunoréactives homologues à celles des mammifères. Le séquençage d’une partie de la séquence codante des gènes codant les Mfns a montré une bonne similitude entre les espècesd’oiseaux et les mammifères. Chez le manchot, l’abondance relative de ces protéines dans lesmitochondries musculaires variait avec la croissance et l’exposition thermique en corrélation positiveavec les capacités bioénergétiques musculaires. Chez le canard, l’activité respiratoire et l’abondance relative de ces protéines étaient également corrélées suite à un jeûne de 60h ou, bien que dans une moindre mesure, après altération pharmacologique du statut thyroïdien.Ces résultats montrent pour la première fois chez l’oiseau l’expression de protéines homologues aux protéines de fusion des mammifères. L’association entre les variations d’expression de ces protéines et les modifications bioénergétiques du muscle squelettique indiquent qu’elles pourraient contribuer à la plasticité bioénergétique observée chez l’oiseau en croissance. Ces résultats suggèrent que des modifications potentielles de l’organisation des réseaux mitochondriaux musculaires pourraient contribuer aux réponses adaptatives des organismes face aux contraintes environnementales
Cold-exposed young birds maintain their homeothermy by stimulating mitochondrial oxidations in skeletal muscle. Prolonged cold exposure enhances muscle thermogenic capacities through mitochondrial bioenergetics plasticity which control still remains hypothetical. In mammals, fusion proteins (mitofusins (Mfns) and OPA1 (Optic Atrophy 1)) contribute to the permanent and dynamic changes in mitochondrial networks in multiple cell types. The aim of our work was to characterize the expression of avian homologues of mammalian fusion proteins and to study the variations of their expression during the establishment of bioenergetics processes in growing birds, during an acute or a prolonged cold exposure and finally during nutritional or endocrine challenges. Methodologically, an integrative approach has been used from whole animal (indirect calorimetry) to protein (western-blot) or gene (RT-PCR) expression through measurements of the bioenergetics functionality of permeabilized muscle fibers and isolated mitochondria. Two animal models were used, a species naturally adapted to Antarctica harsh conditions, the Adélie penguin (Pygoscelis adeliae), and a laboratory model, the Muscovy duck (Cairina moschata).Our results allowed us to characterize, in birds, the expression of immunoreactive fusion proteins (Mfn2, OPA1) which were homologous to those of mammals. The sequencing of a part of the coding sequence of Mfns genes showed a great similitude between avian and mammalian species. In penguins, the relative abundance of these proteins in muscle mitochondria was modified by growth in the cold and was positively correlated with muscle bioenergetics capacities. In ducks, the respiratory activity and the relative abundance of these proteins were also correlated after a 60h fasting period or,though a lesser extent, after a pharmacological alteration of thyroid status. Our results show, for the first time in birds, the expression of proteins homologous to mammalian fusion proteins. The association between the changes in expression of these proteins and the bioenergetics modifications in skeletal muscle indicates that these proteins could contribute to thebioenergetics plasticity observed in growing chicks. These results suggest that potential modifications of the muscle mitochondrial network organization could play a role in the adaptive responses of organisms to the environmental constraints

During aging there is a decline in (MuSCs) and muscle regeneration, though the underlying reason is unknown. Interestingly, mitochondrial fragmentation is a common feature in aging, however, how this impacts MuSC function and maintenance has not been investigated. To address the effect of mitochondrial fragmentation in MuSCs, we generated a knockout mouse model using the Pax7CreERT2 inducible system to target deletion of the mitochondrial fusion protein Opa1 specifically within MuSCs (Opa1-KO). Analysis of MuSC function following muscle injury revealed a defect in the regenerative potential of Opa1-KO MuSCs. Moreover, following injury there was a substantial decrease in the number of MuSC in Opa1-KO animals with a concomitant increase in the number of committing cells, illustrating that loss of Opa1 drives MuSC towards commitment at the expense of self-renewal. Furthermore, loss of Opa1 in MuSCs alters the quiescence state, priming MuSCs for activation, as indicated by a reduction in quiescence-related genes, increased EdU incorporation, and enhanced cell cycle kinetics. To address the impact of mitochondrial dysfunction on muscle stem cell capacity, we generated a model of chronic Opa1 loss. Analysis of muscle stem cell function 3 months after Opa1 ablation revealed mitochondrial dysfunction and a defect in proliferation upon activation, leading to failed muscle regeneration. These data are the first to demonstrate a novel role for mitochondrial structure in the regulation of MuSC maintenance and regenerative capacity.

In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by dOpa1 knockdown not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distribution and morphology differentially. We found that changes to the spatial distribution of axonal mitochondria under dOpa1 knockdown could not be fully accounted for by changes to their motility but, instead, resulted from the disruption of inner membrane fusion. To understand the complex dynamic behavior of axonal mitochondria observed in our experimental studies quantitatively and at the mechanistic level, we built experimental data driven computational models. We found that the stationary mitochondria were composed of two morphologically different populations, which were generated by fusion/fission and long pause, respectively. Furthermore, computational modeling confirmed our experimental findings that motility and morphological dynamics of mitochondria synergistically regulated their spatial distribution in the axon. Together, our data revealed that stationary mitochondria within the axon interconnected with moving mitochondria through fusion and fission and that fusion between individual mitochondria mediated their global distribution.

L'identification des genomes nucleaires, chloroplastiques et mitochondriaux de plantes issues de fusion de protoplastes entre differentes especes de medicago a ete realisee par analyse du polymorphisme des fragments de restriction (rflp). Les trois plantes h1, h2 et h3, issues d'une experience de fusion entre medicago falcata (p1) et medicato sativa (p2) possedent un genome nucleaire hybride constitue de l'addition des deux genomes parentaux; leur genome chloroplastique est identique a celui du parent p1 et leur genome mitochondrial presente des rearrangements par rapport aux genomes parentaux. Dans une deuxieme partie, nous avons etudie ces remaniements de adn mitochondrial (mt). Nous avons donc construit une banque de l'adnmt de p2 et de h3 dans le site sall du cosmide phc79. Par hybridation moleculaire et cartographie, nous avons mis en evidence, une recombinaison intergenomique entre les deux adnmt parentaux. Cette recombinaison a eu lieu au niveau d'une sequence homologue, localisee chez les deux parents en amont d'une copie du gene alpha-atpase. Chacun des genomes mitochondrial parental contient 2 copies de ce gene, l'hybride h3 en contient trois. Des hybridations northern ont montre que la sequence homologue, ayant donne lieu a la recombinaison, est transcrite et vraisemblablement co-transcrite avec le gene alpha-atpase chez p1, p2 et h3. La transcription du gene alpha-atpase ne semble pas modifiee par la recombinaison. Par contre le profil de transcription de la sequence homologue est modifie chez l'hybride h3

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.

MtDNA mutations in mammalian cells are implicated in cellular ageing and encephalomyopathies, although mechanisms involved are not completely understood. The mitochondrial genetic bottleneck has puzzled biologists for a long time. Approximate models of genetic bottleneck proposed in the literature do not accurately model underlying biology. Recent studies indicate mitochondrial morphology changes during cellular aging in culture. In particular, the rates of mitochondrial fission and fusion are shown to be in tight balance, though this rate decreases with age. Some proteins involved in mitochondrial morphology maintenance are implicated in apoptosis. Hence, mitochondrial genetic and morphologic dynamics are critical to the life and death of cells. By working closely with experimental collaborators and by utilizing data derived from literature, we have developed stochastic simulation models of mitochondrial genetic and morphologic dynamics. Hypotheses from the mitochondrial genetic dynamics model include: (1) the decay of mtDNA heteroplasmy in blood is exponential and not linear as reported in literature. (2) Blood heteroplasmy measurements are a good proxy for the blood stem cell heteroplasmy. (3) By analyzing our simulation results in tandem with published longitudinal clinical data, we propose for the first time, a way to correct for the patient's age in the analysis of heteroplasmy data. (4) We develop a direct model of the genetic bottleneck process during mouse embryogenesis. (5) Partitioning of mtDNA into daughter cells during blastocyst formation and relaxed replication of mtDNA during the exponential growth phase of primordial germ cells leads to the variation in heteroplasmy inherited by offspring from the same mother. (6) We develop a “simulation control” for experimental studies on mtDNA heteroplasmy variation in cell cultures. Hypothesis from the mitochondrial morphologic dynamics model: (7) A cell adjusts the mitochondrial fusion rate to compensate for the fluctuations in the fission rate, but not vice versa. A deterministic model for this control is proposed. Contributions: extensible simulation models of mitochondrial genetic and morphologic dynamics to aide in the powerful analysis of published and new experimental data. Our results have direct relevance to cell biology and clinical diagnosis. The work also illustrates scientific success by tight integration of theory with practice.
Ph. D.

Neurons depend on mitochondria to produce ATP, regulate calcium signaling/homeostasis, and control apoptosis. Motor proteins of the kinesin and dynein families actively transport mitochondria through axons. Miro and Milton are two proteins responsible for linking kinesin to mitochondria, and Miro regulates dynein-based transport. Drosophila Miro (dMiro) has three isoforms (dMiro-M, -L, and -S). Milton has four: Milton-A, -B, -C, and -D. To study the role of the dMiro isoforms, we expressed a transgene of each in a dmiro null background, and examined their phenotypic effects on mitochondrial distribution, transport, and morphology in motor neuron axons by using live time-lapse imaging of GFP-tagged mitochondria. The potential role of the Milton-A isoform was examined by chronically or acutely overexpressing (OE) the transgene in motor neurons. We found that Milton-A OE causes a massive loss of mitochondria within 20 hours. In dMiro-L and -S rescues, compared to wildtype, there was decrease the total mitochondrial density, but not the percent of motile mitochondria. To determine components of the signaling pathway controlled by Miro-L and -S, we performed a genetic screen to isolate dominant suppressor mutations of the lethal overexpression phenotypes of dMiro-L and -S. Many suppressor mutations were isolated and mapped to specific chromosomes.

La thérapie cellulaire régénératrice offre des perspectives d'applications dans de nombreuses pathologies entraînant une perte cellulaire. Cependant, suite à un infarctus du myocarde et donc une diminution importante du nombre de cardiomyocytes, l'injection de cellules souches n'a permis de mettre en évidence qu'une amélioration légère et transitoire de la fonction cardiaque. Ces résultats suggèrent qu'il est nécessaire d'améliorer l'efficacité des protocoles de thérapie cellulaire cardiaque. Cette amélioration passe par une meilleure compréhension des mécanismes mis en jeu par les cellules souches dans la régénération myocardique. Parmi les hypothèses soulevées, la fusion entre les cellules souches et les cardiomyocytes a été décrite dans plusieurs études mais le rôle physiologique de ce phénomène reste inconnu. Mon travail de thèse a consisté à étudier ce mécanisme in vitro au sein de cocultures entres des cellules souches adultes humaines (cellules hMADS pour human multipotent adipose derived stem cells) et des cardiomyocytes murins adultes. Nous avons pu mettre en évidence un processus de fusion hétérologue entre ces deux types cellulaires, aboutissant à la reprogrammation du cardiomyocyte vers un stade de progéniteur. Les cellules hybrides résultant de cette fusion ont exprimé des marqueurs cardiomyogéniques précoces et de prolifération et ont été montrées comme ayant un génotype exclusivement murin. Ces cellules hybrides ou progéniteurs cardiaques se sont formés préférentiellement par un mécanisme de fusion partielle par l'intermédiaire de structures intercellulaires appelées nanotubes composés de f-actine et de microtubules. En outre, nous avons montré que le transfert de mitochondries des cellules souches vers les cardiomyocytes était indispensable pour la reprogrammation des cardiomyocytes. En conclusion, nos résultats apportent de nouveaux éléments dans la compréhension des mécanismes de régénération médiés par les cellules souches qui est un pré-requis pour optimiser les protocoles de thérapie cellulaire cardiaque

Le transporteur mitochondrial d'adénine nucleotides est une protéine membranaire qui catalyse l'échange d'ADP cytoplasmique contre l'ATP néo-synthétisé dans la matrice. Le travail présenté dans ce mémoire porte sur la coloration du transporteur Anc2p de Saccharomyces cerevisiae pour faciliter sa cristallisation dans une phase cubique de lipide. Le premier volet présente une approche de coloration chimique basée sur l'emploi de réactifs des thiols. Le second volet expose une stratégie de coloration par fusion génique du transporteur avec le cytochrome c de S. Cerevisiae. Les protéines de fusion obtenues Anc2-Cycl (His6)p et Cycl-Anc (His6)p, sont focntionnelles et leurs caractéristiques ont été étudiées de manière détaillée. La protéine Anc2-Cycl (His6)p a été purifiée à homogénéité pour entreprndre les essais de cristallisation
The mitochondrial adenine nucleotide carrier is a membrane protein which catalyses the exchange of cytoplasmic ADP against ATP synthésized in the matrix. The present work concerns the coloration of the Anc2p carrier from Saccharomyces cerevisiae with the aim at facilitating its crystallization in lipidic cubic phase. The first part presents results from a chemical coloration approach based on the use of thiol reactive compounds. The second part reports on coloration of carrier following a gene fusion strategy. We elaborated two chimera proteins, namely Anc2-Cycl (His6)p and Cycl-Anc2 (His6)p, in which the S. Cerevisiae cytochrome c was fused to the carrier. Both fusion proteins are functional and have been characterized in detail. Anc2-Cycl (His6)p has been purified to homogeneity to undertake cristallization trials

Les mitochondries forment un réseau très dynamique remodelé par deux processus antagonistes appelés : fusion et fission mitochondriales. Chez l’homme, une altération de ces processus, sont à l’origine de nombreuses maladies qui affectent essentiellement le système nerveux. L'objectif principal des travaux de ma thèse était de caractériser l'impact d'un déséquilibre entre la fusion et la fission mitochondriale dans le contexte d'une neuropathie héréditaire : la maladie de Charcot-Marie-Tooth de type 2A (CMT2A), qui est causée par des mutations dominantes dans la mitofusine MFN2. Dans le but d’étudier les mécanismes à l’origine de cette maladie, j’ai développé le premier modèle drosophile de CMT2A en exprimant dans les neurones de mouches quatre allèles de mitofusine retrouvés fréquemment chez les patients. De manière surprenante, les différents allèles altèrent très différemment la morphologie mitochondriale. En effet, alors que les mutations associées au domaine GTPase inhibent la fusion et agrègent les mitochondries, les mutations du domaine dit HB1 induisent au contraire un excès de fusion. J’ai pu ensuite déterminer que l’agrégation des mitochondries et l’excès de fusion, conduisent de manière commune à un défaut de transport des mitochondries au niveau des synapses et à une altération du métabolisme oxydatif associée à une accumulation de mutation dans l’ADN mitochondrial. Chez les drosophiles exprimant des allèles dominants actifs de mitofusine, tous ces dysfonctionnements disparaissent lorsqu’on augmente la fission suggérant que la pathogénicité des allèles du domaine HB1 résulte d’un déséquilibre de la balance entre fusion et fission en faveur de la fusion
Mitochondria form a dynamic network remodeled by two antagonistic processes called mitochondrial fusion and fission. While mitochondrial fusion creates interconnections between mitochondria, mitochondrial fission result in fragmentation. These processes are mediated by Dynamin-related GTPases, the outer-membrane fusion protein mitofusin, and the fission factor DPR1.The main aim of my resaearch was to characterize the impact of an imbalance between mitochondrial fusion and fission in neurons in the context of a severe hereditary neuropathy called Charcot-Marie-Tooth type 2A (CMT2A). Indeed, this disease is caused by dominant mutations in the mitofusinMFN2.In order to dissect the mechanisms by which these mutations alter mitofusin properties and neuronal function, we developed four drosophila models of CMT2A expressing the two most frequent substitutions (R94Q, R364W) and two others localizing to similar domains (T105M, L76P). The four alleles resulted in mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism, increased mtDNA mutations, and impaired locomotion that were associated with aberrant mitochondrial morphology. Interestingly, while GTPase domain-associated mutations (R94Q, T105M) aggregate unfused mitochondria, mutations within helix bundle 1 (R364W, L76P) unexpectedly enhance mitochondrial fusion, as demonstrated by rescue of mitochondrial morphology and locomotion provided by the DRP1 fission factor. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A, and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients

In killer whales, fish- versus mammal-eating ecological differences are regarded as key ecological drivers of sociality, but the potential influence of specific target prey characteristics remains unclear. This thesis aimed to study the social patterns and dynamics of Icelandic killer whales feeding upon herring, a schooling prey that undergoes frequent changes in distribution and school size. I used a multi-disciplinary approach combining photo-identification and genetic data to understand the sociality, role of kinship and genetic differentiation within the population. Individuals sighted in summer-spawning and overwintering herring grounds during at least five separate days (N = 198) were considered associated if photographed within 20 seconds of each other. Photo-identified individuals were genotyped (N = 61) for 22 microsatellites and mitochondrial DNA control region (611 bp). The population had weak but non-random associations, fission-fusion dynamics at the individual level and seasonal patterns of preferred associations. The society was significantly structured but not hierarchically. Social clusters were highly diverse and, whilst kinship was correlated with association, it was not a prerequisite for social membership. Indeed, some cluster members had different mitochondrial haplotypes, representing separate maternal lineages. Individuals with different observed movement patterns were genetically distinct, but associated with each other. No sex-biased dispersal or inbreeding was detected. This study revealed that the Icelandic population has a multilevel society without clear hierarchical tiers or nested coherent social units, different from the well-studied salmon- (‘residents') and seal-eating populations in the Northeast Pacific. In the Icelandic population kinship drives social structure less strongly than in residents. These findings suggest effective foraging on schooling herring in seasonal grounds promotes the formation of flexible social groupings which can include non-kin. Killer whale sociality may be strongly influenced by local ecological context, such as the characteristics of the specific target prey (e.g., predictability, biomass, and density) and subsequent foraging strategies of the population.

Given the debilitating effect that mitochondrial dysfunction has on human health, it is important to understand mitochondrial dynamics that are vital for the maintenance of mitochondrial function, genome, morphology, and quality control. Mitochondrial dynamics result from a balance in mitochondrial fusion and fission. Although the mechanism and regulation of mitochondrial fission are largely elucidated, less is known about mitochondrial fusion. Mgm1 is a protein that mediates mitochondrial fusion in yeast. However, the molecular mechanism of Mgm1 function in mediating mitochondrial fusion is unclear. In this thesis, first, I show that Mgm1 contains a lipid-binding domain by demonstrating that purified Mgm1 has lipid-binding activity and by identifying mutations in conserved residues that abrogate these interactions. Second, I show that Mgm1 assembles into hexameric rings and undergoes nucleotide-dependent structural transitions that, I believe, initiate membrane fusion. Lastly, I demonstrate that Mgm1 exhibits membrane-remodeling activities that are crucial for the tethering and lipid-mixing steps in the membrane fusion event. Together, I propose a mechanistic model of Mgm1 function in mediating mitochondrial fusion that advances the fields of mitochondrial biology, cellular protein-membrane dynamics, and the etiology of neurodegenerative diseases.

碩士
國立清華大學
生物科技研究所
99
Mitochondria are dynamic organelles that involved in ATP synthesis, calcium concentration regulations and apoptosis. Aberrant mitochondrial morphology caused by abnormal equivalence of fusion and fission in were found in neurodegenerative patients. The aim of my thesis is to study the regulation of mitochondria dynamics. In the first part, I focused on the protein that interacts with mitochondria fission Drp1 called CAP2 (cyclase associated protein 2). CAP2 is a highly conserved protein that plays critical roles in regulating acting dynamics and Ras/cAMP signaling pathway. I manipulated CAP2 expression levels by transfecting either overexpression vectors or RNAi and applied confocal microscope to monitor mitochondrial morphology. My experiments found that overexpression of CAP2 caused mitochondria fragmentation and inhibition of CAP2 lead to elongation. These results indicated that CAP2 does involved in regulating mitochondria dynamics. The second part of my thesis is focus on the effects of nature products on mitochondria dynamics. The dehydrated citrus peels used in traditional Chinese medicine contain the most common flavones: polymethoxylated flavones (PMFs). Both nobiletin and tangeretin are abundant PMFs in citrus peels. Many researches had demonstrated that PMF and 5-OH PMFs involved in anti-inflammatory, antioxidants, antithrombotic and anticarcinogenic processes and affected apoptosis. In my experiments, the survival rates of Hela cells treated with different concentrations of nobiletin, tangeretin, 5-OH nobiletin and 5-OH tangeretin were dosage dependent. In addition, 10 μM PMFs treatments altered mitochondrial dynsmics in HeLa cells. Our results of PMFs suggested these molecules may be therapeutically useful in treating the neurodegenerative disorders caused by mitochondrial fusion and fission regulation defects.

SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...

Viele Funktionen der Mitochondrien basieren auf Prozessen, an denen sowohl mitochondriale wie auch kernkodierte Genprodukte beteiligt sind. Durch zahlreiche Interaktionen ist der Einfluss dieser Einzelkomponenten auf das zelluläre System oftmals nur schwierig erkennbar. Mit Hilfe von rho0 -Zellen, deren Mitochondrien über kein eigenes Genom mehr verfügen, kann die mitochondriale Genkomponente ausgeschlossen werden. Im Rahmen dieser Arbeit wurden zunächst die metabolischen, proliferativen und morphologischen Eigenschaften einer rho0-Zelllinie 143B.TK-K7 untersucht, welche durch die Expression einer mitochondrial zielgesteuerten Restriktionsendonuklease hergestellt wurde. Während der Kultivierung bilden sich im Zytoplasma der 143B.TK-K7-Zellen mit fortlaufender Kultivierungszeit und zunehmenden Azidifizierung des Mediums Mega-Mitochondrien. Diese entstehen sowohl durch zahlreiche Fusionsereignisse als auch einem Schwellen durch vermehrten Wassereinfluss in die Mitochondrienmatrix. Alle Mitochondrien liegen dann als große kugelförmige Strukturen in der Zelle vor und nehmen somit die geringste Oberfläche zu einem vorhandenen Volumen ein. Die Entstehung der Mega-Mitochondrien ist dabei abhängig von einer hohen Protonenkonzentration zusätzlich zu einer ausreichend großen Menge an Laktat im Medium (Milchsäure). Zudem zeigt sich, dass auch in Zellen, welche noch ein mitochondriales Genom besitzen, durch diese Bedingungen die Bildung von Mega-Mitochondrien induziert werden kann. Bei der Entstehung der Mega-Mitochondrien handelt es sich zunächst nicht um apoptotische Vorgänge, da durch den Austausch des aziden Mediums eine äußerst schnelle Rückbildung in ein, den rho0-Zellen ähnliches Mitochondriennetzwerk erfolgt. Metabolische Untersuchungen zeigen, dass für die Rückbildung der Mega-Mitochondrien zu einem Netzwerk ausschließlich die im Medium vorhandene Protonenkonzentration ausreichend gering sein muss. Durch immunzytochemische Untersuchungen wurde deutlich, dass sowohl das mitochondriale Fusionsprotein MFN2 wie auch das Fissionsprotein DNM1L während der Entstehung und auch Rückbildung der Mega-Mitochondrien in punktförmigen Bereichen an der äußeren Mitochondrienmembran lokalisieren. Um zu überprüfen, ob die Bildung der Mega-Mitochondrien durch einer Überexpression von Proteinen der Fissionsmaschinerie verhindert wird, wurden PAGFP- bzw. EGFP-Fusionsproteine mit hFis1 und DNM1L hergestellt und in die 143B.TK-K7-Zellen transfiziert. Dabei führt eine verstärkte Expression von hFis1 zu aggregierten Mitochondrien, welche zwar anschwellen, nach einem Mediumwechsel jedoch trotzdem bestehen bleiben. Eine Überexpression von DNM1L hat keinen Einfluss auf die Entstehung und Rückbildung der Mega-Mitochondrien. Durch Inhibierung des Tubulin- bzw. Aktin-Zytoskeletts, konnte gezeigt werden, dass eine Zerstörung des Tubulin-Zytoskeletts auf die Entstehung und Rückbildung der Mega-Mitochondrien keine Auswirkungen hat. Die Untersuchungen zu dem Einfluss des Aktin-Zytoskeletts zeigen, dass die Mega-Mitochondrien ringförmig von dem Aktin-Zytoskelett umgeben sind. Mit Hilfe von Fluoreszenzprotein-Markern für die äußere und innere Mitochondrienmembran wurden die Mega-Mitochondrien als Modellsystem für mitochondriale Fusions- und Fissionsstudien verwendet. Somit konnte in der vorliegenden Arbeit mitochondriale Fusion und Fission zum ersten Mal an lebenden Zellen direkt beobachtet werden und führte nachfolgend zu der Einteilung von Fusionsvorgängen der Mitochondrien in einen Modus 1, bei dem eine zeitlich gekoppelte vollständige Fusion von sowohl äußerer wie auch innerer Membran geschieht und einen Modus 2, bei dem die Fusion der äußeren Membranen ohne die Fusion der inneren Membranen erfolgt. In ähnlicher Weise kann die Fission von Mitochondrien unterteilt werden. In einem als Modus 1 bezeichneten Mechanismus beginnt die Rückbildung der Mega-Mitochondrien zunächst mit einer Tubulierung der Mitochondrien hin zu langen Mitochondrienschläuchen, die einen nur geringen Durchmesser besitzen. Erst dann treten vermehrt zeitlich sehr schnell ablaufende Fissionsvorgänge auf. Zusätzlich wurde ein Modus 2-Mechanismus der Fission beobachtet, welcher aus einer unvollständigen Fusion resultiert, bei dem die inneren Membranen noch nicht miteinander verschmolzen sind. Auf elektronenmikroskopischer Ebene finden während der Mega-Mitochondrien-Bildung drastische Veränderung von zwiebelringartigen Cristae hin zu einer Abnahme von inneren Membranstrukturen und der elektronendichte im Matrixraum statt. Somit ist im Rahmen dieser Arbeit zum ersten Mal eine optische Beobachtung sowohl dieser Bewegungen wie auch von Fusions- und Fissionsprozessen und deren zeitlich Auflösung in vivo mit Hilfe der Mega-Mitochondrien gelungen
A variety of mitochondrial features are based on processes involving mitochondrially encoded as well as nuclear encoded gene products. By means of these manifold interactions it is difficult to discern the influences of the single components. One effort to overcome these difficulties was the development of cells devoid of endogenous mtDNA (so called rho0 cells) and therefore to exclude the mitochondrial genetic component. The aim of this thesis was the investigation of the metabolic, proliferative and morphologic characteristics of a rho0 cell line (143B.TK-K7) based on a 143B.TK- background. This cell line was developed by the expression of a mitochondrially targeted restriction endonuclease. During the cultivation and proceeding acidification of the culture medium by lactic acid megamitochondria developed in the cytoplasm of the 143B.TK-K7 cells. These megamitochondria form both by multiple fusion events and an additional increase in water influx into the matrix. All mitochondria then exist as large spherical structures with diameters of up to 7 µm and therefore receive the smallest surface area to a given volume. The formation of megamitochondria is dependent on a high proton production level additional to a sufficient amount of lactate (lactic acid) in the medium. Furthermore it is possibly to induce megamitochondria in cells still possessing a mitochondrial genome by these conditions. The formation of megamitochondria is not a sign of apoptotic processes per se, because the back-formation of the megamitochondria into a rho0-like network can be induced very fast by the exchange of the acidulated medium. Initial deformations of the megamitochondria are followed by tubulation in progressive mitochondria tubules and numerous fission events. Metabolic analyses show that this backformation only depends on a sufficient low concentration of protons in the medium. When the given threshold is not being traversed the megamitochondria persist. Immunocytological investigations both of the fusion protein MFN2 and the protein of the fission machinery DNM1L demonstrate a constant mitochondrial distribution in focal regions of the outer mitochondrial membrane during formation as well as back-formation of megamitochondria. By overexpressing the fission proteins hFis1 and DNM1L respectively in 143B.TK-K7 cells, it should be tested whether or not megamitochondria develop. The enhanced expression of hFis1 led to the formation of aggregated mitochondria that indeed swell but persist after changing the medium. The overexpression of DNM1L has no influence on the formation as well as the back-formation of the megamitochondria. Incubation of the cells with inhibitors for the tubulin respectively actin cytoskeleton evidenced that the destruction of the tubulin cytoskeleton has no consequence for the formation and back-formation of megamitochondria. Unclear results were obtained with inhibitors of the actin cytoskeleton probably due to secondary effects of the inhibitors to the cells. However the findings showed that the megamitochondria are embedded into the actin cytoskeleton. Additionally the megamitochondria were used as a model system for mitochondrial fusion and fission events. For this purpose fluorescent protein markers for the inner and outer mitochondrial membrane were created. With these tools it was possible to observe directly mitochondrial fusion and fission in living cells by confocal microscopy. Furthermore this led to the classification of fusion processes of the mitochondria in a mode 1 with temporally coupled fusion of outer and inner membrane and a mode 2 where the fusion of outer membrane occurs independent of the inner membrane fusion. In a similar way the fission of mitochondria can be sub-classified: mode 1 is featured during the back-formation of megamitochondria by increasing tubulation events in long mitochondrial tubules with thin diameters. Only at this point very fast fission events could be observed. Furthermore in a fission mode 2 that results from an incomplete fusion of inner mitochondrial membranes, the outer membrane invaginates from one side along the unfused inner membranes until two separate mitochondrial units exist. During the megamitochondria formation on electron microscopic level drastic changes occur from fuzzy onion like structures to a decrease of inner membranes and electron density in the matrix. Additionally inversions and inclusions consisting of one membrane and also double membranes are evident. Comparisons with confocal microscopy images show that these inclusions apparently accomplish undirected movements with high velocity. In the present thesis it was possible to observe for the first time these movements as well as mitochondrial fusion and fission in living cells with an outstanding optical and temporal resolution

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
A doença de Parkinson é a segunda doença neurodegenerativa mais comum e a doença neurodegenerativa associada ao movimento mais comum. É causada pela perda dos neurónios dopaminérgicos na substantia nigra pars compacta levando a um défice de dopamina no estriado. Perceber a base molecular da doença de Parkinson tem se revelado um grande desafio no campo das doenças neurodegenerativas. Apesar de terem sido propostas várias hipóteses para explicar os mecanismos subjacentes a patogenia da doença de Parkinson, um crescente corpo de evidencias tem enfatizado o papel da disrupção da dinâmica mitocôndrial como um grande contribuidor para a etiopatogenia da doença de Parkinson. Tem se vindo a acumular dados que sugerem que uma dinâmica mitocondrial anormal se encontra envolvida na disfunção mitocôndrial ou medeia a morte neuronal em diferentes modelos da doença de Parkinson. Aliás, a integração da fissão, fusão e autofagia mitocondrial forma um mecanismo de manutenção de qualidade mitocôndrial da homeostase mitocôndrial na qual defeitos na função mitocôndrial têm sido associados à doença de Parkinson. A maioria dos casos surge como condição esporádica e os restantes são herdados com mutações em vários genes que tem sido ligados a essas formas genéticas da doença. Os estudos de duas das proteínas ligadas as formas familiares da doença, nomeadamente a PINK1 e a Parkin, forneceram evidências que estas duas proteínas actuam na mesma via regulando a fissão e fusão mitocôndria e a mitofagia. Uma vez que uma dinâmica mitocôndrial anormal tem sido cada vez mais implicada na patogenia da doença de Parkinson, nesta tese, investigámos a regulação da dinâmica mitocôndrial em diferentes modelos celulares da doença de Parkinson. Nos observamos que uma diminuta localização mitocôndrial da OPA1 leva a um dano da dinâmica mitocôndrial na maioria dos modelos celulares de 13 Parkinson. Para além do mais, a clivagem das isoformas longas da Opa1 parecem ser responsáveis pelo padrão de fragmentação observado nos modelos celulares de PD esporádicos derivados da mitocôndria. A sobrexpressão da alpha-synucleina induz a tubulação da rede mitocondrial com estruturas elongadas que são o oposto do observado em modelos celulares de PD. Inesperadamente, a sobrexpressao da Opa1 não resgatou o aumento de produção de espécies reactivas de oxigénio induzida pelo MPP+. Globalmente, as nossas observações sugerem que uma fusão mitocondrial dependente da Opa1 desempenha um papel crucial na mediação nas anormalidades mitocôndriais e disfunção celular induzida MPP+/mtDNA. Estes estudos sugerem que a dinâmica mitocôndrial desempenha um papel importante na patogenia da doença de Parkinson, e um melhor entendimento destes mecanismos podem levar à descoberta de novos alvos terapêuticos para esta doença.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder and the most common neurodegenerative movement disorder. It is caused by the loss of dopaminergic neurons in the substantia nigra pars compacta leading to a dopamine deficit in the striatum. Understanding the molecular basis of PD has proven to be a major challenge in the field of neurodegenerative diseases. Although several hypotheses have been proposed to explain the molecular mechanisms underlying the pathogenesis of PD, a growing body of evidence has highlighted the role of mitochondrial dynamics disruption as a major contributor to PD etiopathogenesis. Accumulating data suggests that abnormal mitochondrial dynamics is involved in mitochondrial dysfunction or mediates neuronal death in different PD models. Moreover, integration of mitochondrial fusion, fission and mitochondrial autophagy forms a quality maintenance mechanism of mitochondrial homeostasis that defects in mitochondrial function have been associated with PD. Most of the cases arise as sporadic conditions and the others are inherited with mutations in several genes being linked to these genetic forms of PD. Studies of two of the proteins linked to the familial forms of the disease, namely PINK1 and Parkin, provided evidence that these two proteins act in the same pathway regulating mitochondrial fusion, fission and mitophagy. Because abnormal mitochondrial dynamics are increasingly implicated in the pathogenesis of PD, in this thesis, we investigated the regulation mitochondrial dynamics in different PD cellular models. We observed that a decreased OPA1 mitochondrial localization-drive mitochondrial dynamics impairment in most cellular PD models. Moreover, OPA1 long isoforms cleavage seems to be responsible for mitochondrial fragmented pattern observed in sporadic mitochondrial-driven cellular PD models. Alpha-synuclein overexpression induces a tubular mitochondrial network 15 with elongated structures that is the opposite of what was observed in mitochondrial PD cellular models. Unexpectedly, OPA1 overexpression did not rescued MPP+-induced increase in reactive oxygen species (ROS). Overall, our findings suggest that OPA1-dependent mitochondrial fusion plays a crucial role in mediating MPP+/mtDNA induced mitochondria abnormalities and cellular dysfunction. These studies suggest that mitochondrial dynamics can play an important role in PD pathogenesis, and a better understanding of these mechanisms can lead to the discover of new therapeutical targets for this disease.

We have studied the mechanisms of mitochondrial fusion and fission in S. cerevisiae. Using a proteomics-based approach, we have identified the WD domain protein Caf4p as a Fis1p binding partner that, along with its paralog Mdv1p, functions as a molecular adaptor between Fis1p and Dnm1p. This work defines a role for Mdv1p and Caf4p in the recruitment of Dnm1p to mitochondrial fission complexes. In a separate study, we focus on the role of Fzo1p during mitochondrial fusion. Fzo1p and its mammalian homologs, Mfn1 and Mfn2, are conserved transmembrane GTPases that are required for mitochondrial fusion. A structure/function analysis has established an essential role for three Fzo1p heptad repeat regions during mitochondrial fusion. Furthermore, we show that Fzo1p functions as an oligomer and forms critical interactions between the HRN/GTPase and HR1/HR2 regions. Finally, we have identified Om14p as a novel regulator of mitochondrial morphology. Om14p interacts with Fzo1p and Ugo1p and may be the first inhibitor of mitochondrial fusio