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.

Plusieurs études ont découvert et renforcé l'implication de la dynamique mitochondriale dans le cancer. J'ai découvert un rôle inattendu des protéines kinases de la famille PKD dans la fission mitochondriale. La perte de l'activité PKD a conduit à un blocage de la fission et a entraîné une élongation significative des mitochondries par fusion continue. D'un point de vue mécanique, nous avons montré que les protéines PKD régulent la dynamique mitochondriale en activant le facteur de fission mitochondrial (MFF) par phosphorylation de plusieurs sites. MFF agit comme un récepteur principal de la GTPase DRP1, qui resserre les mitochondries, et il est essentiel à une bonne division mitochondriale. Les trois membres de la famille PKD peuvent phosphoryler MFF. La phosphorylation de MFF est médiée par PKD et la fragmentation mitochondriale se produit pendant la mitose. Comme démontré dans études sur les phosphoprotéomes, la phosphorylation du MFF est augmentée dans les cancers très mitotiques. Ainsi, l'axe de signalisation PKD-MFF régulant la dynamique mitochondriale en mitose pourrait devenir une voie thérapeutique attrayante pour le traitement du cancer. / Over the last two decades, multiple studies have uncovered and strengthen the implication of mitochondrial dynamics in cancer. During my thesis, I discovered an unanticipated role for the PKD kinase family in mitochondrial fission. Loss of PKD activity led to blockade of mitochondrial fission and resulted in a significant elongation of mitochondria by unopposed fusion. Mechanistically, we showed that PKDs regulated mitochondrial dynamics by activating the mitochondrial fission factor (MFF) through phosphorylation of multiple sites. MFF acts as a main receptor for the large GTPase DRP1, which constricts mitochondria, and it is critical for proper mitochondrial division. All three PKD family members could phosphorylate MFF. PKD-mediated MFF phosphorylation and mitochondrial fragmentation occurred specifically during mitosis. As MFF phosphorylation was found to be significantly upregulated in highly mitotic cancers, which was evidenced in several global phosphoproteome studies, the discovered PKD-MFF signaling axis regulating mitochondrial dynamics in mitosis could become an attractive therapeutic avenue for cancer treatment.

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.

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.

Le virus Epstein-Barr (EBV), un membre de la famille des Herpesviridae, est associé à la mononucléose infectieuse et à différents types de cancers comme le lymphome de Burkitt, les lymphomes post-transplantation ou encore le carcinome du nasopharynx. Ce virus est capable de persister à vie dans l’organisme en combinant des phases de latence et des phases de multiplication active. L’autophagie est un processus cellulaire primordial qui conduit à la dégradation et au recyclage de protéines à longue durée de vie et d’organites endommagés ou vieillissants. Elle contribue non seulement à maintenir l’homéostasie cellulaire mais aussi à s’adapter aux conditions environnementales. Souvent décrite comme un mécanisme antiviral, l’autophagie est contrecarrée par de nombreux virus. Elle peut également être détournée à leur profit. Il a été démontré que l’EBV est capable de stimuler l’autophagie durant le cycle lytique et d’échapper à la dégradation dans les autolysosomes en bloquant la maturation des autophagosomes. Le but de cette étude était d’identifier des protéines virales impliquées dans la modulation du processus autophagique par l’EBV. Nous avons démontré que l’expression ectopique de BHRF1, une protéine transmembranaire de 17kDa orthologue de la protéine cellulaire Bcl-2, module l’autophagie.Alors que Bcl-2 est une protéine anti-autophagique, nous avons établi par différentes approches que l’expression de BHRF1 conduit à l’accumulation d’autophagosomes. De plus, en utilisant une sonde tandem bifluorescente LC3 (mRFP-GFP-LC3) pour étudier le flux autophagique, nous avons montré que BHRF1 stimule l’autophagie. BHRF1 est engagée dans un complexe avec Beclin1, une protéine de la machinerie autophagique. Nous avons établi que BHRF1 est localisée au niveau des membranes mitochondriales et du réticulum endoplasmique (RE). L’expression de BHRF1 est associée à une réorganisation du réseau mitochondrial conduisant à la formation d’agrégats mitochondriaux juxta-nucléaires. Considérant l’importance des microtubules dans l’autophagie et le transport des mitochondries, nous avons exploré la dynamique des microtubules et les modifications post-traductionnelles de la tubuline après expression de BHRF1. Nous avons observé un recrutement d’acétyl-tubuline autour des mito-aggresomes associé à un réseau intact de microtubules. Nos résultats ont montré que le réseau de microtubules et l’hyper-acétylation de l’alpha-tubuline sont nécessaires pour former les mito-aggrésomes induits par BHRF1. Par différentes approches, nous avons démontré le rôle de BHRF1 dans l’induction de la mitophagie, un processus qui entraine la clairance des mitochondries endommagées par autophagie. Considérant le rôle des mitochondries endommagées dans l’induction de l’apoptose, nous suggérons que le rôle anti-apoptotique de BHRF1 pourrait être associé à l’induction de la mitophagie. / Epstein-Barr virus (EBV), a member of the Herpesviridae family, is associated with infectious mononucleosis and with several types of cancers including Burkitt’s lymphoma, post-transplant B-cell lymphoma disease and nasopharyngeal carcinoma. This virus is able to establish persistent infection and to undergo lytic cycle after reactivation. Autophagy is a critical cellular process leading to degradation of long lasting proteins and damaged or aging organelles. It contributes not only to maintain cell homeostasis but also to the adaptation to environmental stresses. Sometimes, autophagy is described as an antiviral mechanism, and viruses have evolved multiple strategies to subvert it or to hijack it to their own profit. It has been reported that EBV is able to stimulate autophagy during lytic cycle and then to escape degradation within autolysosomes by blocking autophagosomes maturation. The aim of my study was to identify EBV viral proteins involved in this modulation. Among the numerous viral proteins encoded by EBV, we have identified BHRF1, a transmembrane protein homolog of cellular protein Bcl-2, which was able to modulate autophagy by ectopic expression.Whereas Bcl-2 is an anti-autophagic protein, we demonstrated by different approaches that BHRF1 expression leads to accumulation of autophagosomes. Moreover, using tandem-fluorescent-tagged LC3 (mRFP-GFP-LC3), which is based on different pH stability of GFP and mRFP fluorescent proteins, for monitoring autophagic flux, we clearly confirmed that BHRF1 stimulates autophagy. By co-immunoprecipitation we demonstrated that BHRF1 is part of acomplex including Beclin1, a protein of the autophagic machinery. We characterized the subcellular localization of BHRF1, and report that BHRF1 is localized in mitochondria and ER membranes. Expression of BHRF1 leads to a complete reorganization of the mitochondria network to form juxtanuclear mitochondrial aggregates. Based on the importance of microtubules on both autophagy and mitochondria transport, we explored microtubule dynamics and tubulin post-translational modifications after BHRF1 expression. We observed a clustering of acetyl-tubulin around the mito-aggresomes associated with an intact microtubules network. Our results showed that the microtubules network and the hyperacetylation of alpha-tubulin were both required to form BHRF1-induced mito-aggresomes.By different approaches, we demonstrated the role of BHRF1 in the induction of mitophagy, a process which promotes the clearance of impaired mitochondria by autophagy. We hypothesized that the role of BHRF1 to protect against apoptosis and to promote cell survival is related to the induction of selective autophagy.

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

Autophagy is the delivery of cytoplasmic cargo to lysosomes for degradation. Defects in autophagy are responsible for various diseases, including neurodegenerative diseases and cancer. While studies in yeast have largely characterized autophagy in response to nutrient starvation, these elegant studies do not account for autophagy in other contexts, including selective autophagy of organelles. A previous screen identified Vps13D as a gene required for the autophagic removal of mitochondria, mitophagy. Vps13D is highly conserved and essential in animals, and Vps13d loss-of-function mutants have enlarged mitochondria and mitophagy deficiencies in both cell and animal models. However, the mechanism by which Vps13D regulates these processes has not been defined. Here, I use mitochondrial clearance in the developing Drosophila intestine and fibroblasts from VPS13D mutant patients as experimental models to investigate the function of Vps13D. I discover that Vps13D is a regulator of ubiquitin and Atg8a/LC3/GABARAP localization around mitochondria. These functions are dependent on Pink1, a ubiquitin kinase, and the core autophagy machinery, respectively. Furthermore, Vps13D regulates mitochondria and endoplasmic reticulum (ER) contact sites downstream of Vmp1, a repressor of mitochondria and ER contact sites. I find that Marf, a mitochondria and ER tether and regulator of mitochondrial fusion, acts downstream of both Vmp1 and Vps13D. These findings explain the phenotypes in Vps13d mutants, as dysregulation of ubiquitin, Atg8a, and mitochondria and ER membrane contact sites impair regulation of both autophagy and mitochondria morphology.

Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation. ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte coculture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A -induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis. iv HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD. In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.

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