The mitofusin 1 and 2 (MFN and MFN2) proteins reside in the outer mitochondrial membrane and have been shown to regulate mitochondrial network architecture by mediating tethering and fusion of mitochondria. Mitochondria normally form a tubular and branched reticular network dynamically regulated by a balance of fusion and fission events. Absence of either Mfn1 or Mfn2 results in a fragmented mitochondrial network. Züchner et al. previously described mutations in the gene mitofusin 2 (MFN2) as the cause of the major autosomal-dominant, axonal form of Charcot-Marie-Tooth neuropathy (CMT2A). CMT type 2 (CMT2) is characterized by chronic axonal degeneration of peripheral nerves leading to the loss of functional nerve fibers. Mutations in MFN2 are the most common cause of CMT2, and in Chapter 2 we report the results from a genetic screen of MFN2 in a CMT2 patient cohort. The original finding that mutations in MFN2 cause CMT2A led to investigations focused on deficiencies of mitochondrial fusion and transport, specifically in the context of long axonal processes affected in CMT. While some experimental work supports disrupted mitochondrial transport in the etiology of CMT2A, other studies on CMT2A patient fibroblasts and cell models suggest abnormal mitochondrial fusion and dynamics do not underlie the etiology of this. In the first half of Chapter 3, we present some of our initial investigations prior to de Brito and Scorrano’s report published in 2008 regarding a novel role for Mfn2 in tethering the endoplasmic reticulum (ER) to mitochondria. In Mfn2 null mouse embryonic fibroblasts (MEFs) regions of contact between mitochondria and the endoplasmic reticulum (ER) are significantly reduced. These regions of contact are thought to form specialized subdomains of the ER, called mitochondrial associated membranes (MAM). Besides observing a fragmented ER network in Mfn2 knockout (KO) mouse embryonic (MEF) cells, de Brito and Scorrano presented several lines of evidence which suggest that the underlying pathogenic mechanism in CMT2A stems from disrupted ER-mitochondria. As this observation had not been replicated in the literature, we describe our attempts to replicate these finding in the last half of Chapter 3. The MAM represents a sub-domain of the ER in close association with the mitochondrial outer membrane. The movement of phosphatidylserine (PS) from the MAM domains of the ER to mitochondria and its subsequent decarboxylation to phosphatidylethanolamine (PE) by the enzyme PS decarboxylase (Pisd) has been well characterized and is known to depend on the existence of an outer mitochondrial membrane protein. As PE has curvature inducing and fusogenic biophysical characteristics, a deficiency in PE would be an attractive mechanism contributing to the morphological and fusion defects observed in Mfn2 null cell models. We hypothesized that loss of Mfn2 would lead to specific decreases in mitochondrial and cellular levels of PE. Chapter 4 describes experiments designed to test this hypothesis. We observed significantly lower levels of PE in Mfn2 null cells, yet observe similar changes in Mfn1 null cells. Likewise, other lipid species such as ether linked PE (ePE) are decreased. To investigate how CMT2A mutations in MFN2 influence cellular phospholipid profiles, we then profiled cellular phospholipids of CMT2A patients and control lymphoblasts. We hypothesized that mutations in MFN2 would result in decreased levels of PE. In Chapter 5, we report the results of a phospholipid screen which reveal changes in ePE in CMT2A patient lymphoblasts, without the drastic decreases in PE previously observed in Mfn2 null lines. In conclusion, our data indicates an important role for both mitofusins in the mitochondrial synthesis of PE. In the context of CMT2A mutations, ePE levels are specifically reduced. Future studies may reveal how deficiencies in ePE might have important functional consequences in the pathogenesis of CMT2A.

Les mitochondries sont la principale source d’énergie dans les neurones. Les défauts mitochondriaux participent à l’apparition de maladies neurodégénératives, cependant ils peuvent être contrés par un système de contrôle qualité. Le but de ma thèse a été de déterminer si ce système est dérégulé dans la maladie de Huntington (MH) et la sclérose latérale amyotrophique (SLA) et si sa restauration est neuroprotectrice, en utilisant principalement des modèles drosophile. La MH, caractérisée par une atteinte des neurones du striatum, est due à la protéine Huntingtin mutée (mHtt). Nous avons montré que la mHtt induit une accumulation des mitochondries dans la rétine. Ceci pourrait être dû à un défaut de la mitophagie, un mécanisme qui permet l’élimination des mitochondries défectueuses et qui est orchestré par la protéine PINK1. De manière intéressante, la surexpression de PINK1 corrige le phénotype pathologique des drosophiles exprimant la mHtt. Je me suis aussi intéressé à la SLA, chez laquelle les motoneurones dégénèrent, plus exactement au gène TDP-43 qui est un contributeur majeur à la maladie. Nous avons montré que la surexpression de TDP-43 dans les neurones de drosophiles entraîne une fragmentation des mitochondries liée à une sous-expression du gène mitofusin. Ce dernier contrôle le processus de fusion entre les mitochondries saines et endommagées et donc l’intégrité de cet organite. La surexpression de Mitofusin améliore les défauts locomoteurs et l’activité neuronale altérée chez les drosophiles exprimant TDP-43. Nos résultats montrent l’importance du contrôle qualité mitochondrial dans la pathogenèse de ces maladies, et que de le renforcer pourrait être bénéfique. / Mitochondria are the main energy source in neurons. Mitochondrial defects contribute to the development of neurodegenerative diseases, however they can be countered by a quality control system. The purpose of my thesis has been to determine if this system is dysregulated in Huntington’s disease (HD) and in amyotrophic lateral sclerosis (ALS) and if restoring it can be neuroprotective, by mainly using Drosophila models. HD, which is characterized by loss of striatal neurons, is caused by the mutant Huntingtin protein (mHtt). We showed that mHtt induces the accumulation of mitochondria in the retina. This could be due to a defect in mitophagy, a mechanism which allows the elimination of defective mitochondria and which is orchestrated by the protein PINK1. Interestingly, PINK1 overexpression ameliorates the abnormal phenotype of flies expressing mHtt. I also got interested in ALS, in which motor neurons degenerate, and mainly in the TDP-43 gene which is a major contributor to the disease. We showed that TDP-43 overexpression in Drosophila neurons leads to fragmentation of mitochondria due to decreased expression levels of the mitofusin gene. The latter controls the fusion process between healthy and damaged mitochondria and therefore the organelle integrity. We show that Mitofusin overexpression ameliorates locomotor defects and abnormal neuronal activity in flies expressing TDP-43. Our results show the importance of mitochondrial quality control in the pathogenesis of these diseases, and that reinforcing it can be beneficial.

The recent surge in Type 2 Diabetes (T2D) has renewed interest in the study of cellular metabolism – which mitochondria tightly control. Previous work has shown mitochondrial dysfunction plays a critical role in the development of metabolic diseases, such as T2D. The pancreatic β-cell synthesizes and secretes insulin in vivo in response to diverse fuel signals such as glucose, fatty acids, and amino acids; failure or loss of β-cell mass is a hallmark of T2D. Pancreatic β-cell mitochondria are dynamic organelles living a life of fusion, fission, and movement collectively called mitochondrial dynamics. Mitochondrial fusion is impaired in obesity and models of obesity, while basal secretion of insulin is elevated. Previous studies demonstrate that hyperinsulinemia alone is sufficient to induce insulin resistance, yet the relationship between mitochondrial morphology and basal insulin secretion has not yet been studied. Here, we investigated the link between loss of mitochondrial fusion and insulin secretion at basal glucose concentrations by reducing the expression of mitofusin 2 (Mfn2), which controls mitochondrial morphology and metabolism. We found that forced mitochondrial fragmentation caused increased insulin secretion at basal glucose concentrations. In addition, fragmentation of mitochondria enhanced the secretory response of islets to palmitate at nonstimulatory glucose concentrations and increased fatty acid uptake and oxidation in a cell model of pancreatic β-cells. We developed unique solutions to challenges posed by the measurement of mitochondrial dynamics via confocal microscopy by using novel image analysis techniques, including a novel method of mitochondrial segmentation. This technique also revealed novel biology of brown adipose tissue mitochondria dependent on their localization within the cell. Our findings demonstrate that changes to mitochondrial dynamics in the β-cell can lead to increased insulin secretion at basal glucose concentrations. These data support the possibility that hyperinsulinemia and the downstream outcome of insulin resistance can be initiated by altered mitochondrial function in the β-cell independently of other tissues. By uncovering a new process that governs basal insulin secretion, we provide novel targets for regulation, such as mitochondrial morphology or fatty acid induced insulin secretion that may present new approaches to treatment of diabetes.

Mitofusin 2 (MFN2), an outer mitochondrial membrane protein expressed in virtually all human tissues, is a multi-faceted protein known to affect mitochondrial morphology, metabolism, tethering, and movement as well as overall cell cycle progression. Most intriguing among its characteristics is its ability to bind to Ras and Raf, upstream effectors in the MAPK/ERK pathway. Conditional knockout (cKO) of renal proximal tubule MFN2 in vivo showed a post-ischemic protective effect. While the two day survival of control mice was only 28%, an unexpected 86% of the MFN2 cKO mice were alive at two days post-ischemia. This is likely explained by MFN2's ability to bind and sequester Ras at baseline. Because the MFN2 deficient mice did not sequester as much Ras, renal proximal tubule cells were able to proliferate at a greater rate and restore organ function more quickly. Immunoprecipitation studies confirm a strong interaction between Ras and MFN2 in resting cells but a weaker one immediately following ischemic insult, even in cells replete with MFN2. These results suggest that blocking the MFN2-Ras interaction may be a novel method to treat acute kidney injury. A small peptide mimicking Ras to block MFN2 could be feasible. This should grant ischemic tissue an increased propensity to regenerate healthy cells while leaving non-ischemic tissue completely unaffected. Such a therapeutic agent would be novel in the treatment of acute kidney injury and may have uses in other tissues as well due to MFN2's widespread expression profile.

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 mécanismes moléculaires impliqués dans la fusion membranaire ont été amplement étudiés au cours des trente dernières années. Notre compréhension actuelle de ce phénomène est principalement basée sur des résultats obtenus par (1) le développement de modèles physiques décrivant la fusion des membranes biologiques, (2) l’étude mécanistique et structurale des protéines de fusion membranaire des virus à enveloppe et (3) l’étude des évènements de fusion intracellulaire médiés par les protéines SNARES dans les cellules eucaryotes. La découverte du complexe SNARE fut l’aboutissement de travaux interdisciplinaires qui ont exigés un large éventail de techniques tel que la génétique de la levure, l’électrophysiologie, la biologie moléculaire, la biochimie cellulaire, la biophysique expérimentale et l’imagerie. Tirant parti des paradigmes et techniques biophysiques qui ont émergés de ces études, nous avons examiné les fonctions et mécanismes de régulation des domaines « coiled-coil » dans les processus de fusion intracellulaire impliquant des protéines de la famille des Longin-SNAREs ou des Mitofusines, deux machineries protéiques de fusion dont le mode d’action exact reste encore peu clair. La conception exacte des mécanismes moléculaires de la fusion membranaire requiert la reconstitution in vitro des protéines de fusion dans un large spectre d’environnement membranaire avec des propriétés biophysiques définies et facilement modulables. Idéalement, ces systèmes membranaires devraient permettre à l’expérimentateur de contrôler la composition lipidique et protéique, ainsi que la topologie membranaire, afin de rendre compte de l’importante variabilité observée entre les différents compartiments de fusion cellulaire. La reconstitution dans des liposomes offre une incroyable flexibilité avec la possibilité de faire varier la plupart des paramètres clefs et de créer un environnement minimal dans lequel les facteurs solubles et/ou membranaires peuvent être ajoutés, seuls ou en combinaison, pour dévoiler leur rôle avec clarté. Nous avons mis au point des systèmes in vitro de reconstitution de protéines dans des plateformes membranaires artificielles pour nos deux systèmes d’études (les deux protéines Longin-SNAREs TI-VAMP et Sec 22b, ainsi que les domaines « coiled-coil » des Mitofusines) et nous avons réalisé des expériences biochimiques pour caractériser le mode d’action de ces protéines. L’objectif à long-terme de ce projet est de comparer les mécanismes moléculaires des machineries de fusion associés aux protéines SNAREs et Mitofusines, et ainsi de dévoiler des similitudes structurelles et fonctionnelles entre (1) leur protéines de fusion principales et (2) leur facteurs régulateurs. / The molecular mechanisms involved in membrane fusion have been extensively studied for the past thirty years. Our current understanding of this phenomenon is mainly based on results obtained by (i) the development of physical models describing the fusion of membranes, (ii) structural and mechanistic investigations on fusion proteins of enveloped viruses and (iii) studies of SNARE protein-mediated intracellular fusion events of eukaryotic cells. Discovery of the SNARE complex was the outcome of interdisciplinary works which involved a wide range of techniques including yeast genetics, electrophysiology, molecular biology, cell-free biochemistry, adhesion/fusion biophysics and imaging. Taking advantage of the paradigms and biophysical techniques that emerged from these studies, we investigated the function and regulation of coiled-coil domains in intracellular fusion processes involving Longin-SNAREs or Mitofusins, two fusion protein machineries whose exact mode of action still remains unclear. A comprehensive understanding of the molecular mechanisms of membrane fusion requires the in vitro reconstitution of fusion proteins into a wide variety of membrane environments with defined and tunable biophysical properties. Ideally, these membrane systems should allow the experimentalists to control the lipid and protein composition as well as the membrane topology, to account for the variability observed across cellular fusing compartments. Reconstitution into liposomes offers amazing flexibility with the capacity to vary most of these relevant parameters, and to create a minimal environment in which membrane and/or soluble factors can be added, one at a time or in combination, to reveal their role with clarity. We have set up the in vitro reconstitution of proteins into various artificial membrane platforms for both systems (the Longin-SNAREs TI-VAMP and Sec22b and the coiled-coil domains of Mitofusins) and performed biochemical assays to gain insight into how these proteins execute their functions. The long-term goal of this project is to compare the molecular mechanisms of SNARE and Mitofusin fusion machineries and thus reveal structural and functional similitudes between (i) their core fusion proteins, and (ii) their regulatory factors.

Les mécanismes moléculaires impliqués dans la fusion membranaire ont été amplement étudiés au cours des trente dernières années. Notre compréhension actuelle de ce phénomène est principalement basée sur des résultats obtenus par (1) le développement de modèles physiques décrivant la fusion des membranes biologiques, (2) l’étude mécanistique et structurale des protéines de fusion membranaire des virus à enveloppe et (3) l’étude des évènements de fusion intracellulaire médiés par les protéines SNARES dans les cellules eucaryotes. La découverte du complexe SNARE fut l’aboutissement de travaux interdisciplinaires qui ont exigés un large éventail de techniques tel que la génétique de la levure, l’électrophysiologie, la biologie moléculaire, la biochimie cellulaire, la biophysique expérimentale et l’imagerie. Tirant parti des paradigmes et techniques biophysiques qui ont émergés de ces études, nous avons examiné les fonctions et mécanismes de régulation des domaines « coiled-coil » dans les processus de fusion intracellulaire impliquant des protéines de la famille des Longin-SNAREs ou des Mitofusines, deux machineries protéiques de fusion dont le mode d’action exact reste encore peu clair. La conception exacte des mécanismes moléculaires de la fusion membranaire requiert la reconstitution in vitro des protéines de fusion dans un large spectre d’environnement membranaire avec des propriétés biophysiques définies et facilement modulables. Idéalement, ces systèmes membranaires devraient permettre à l’expérimentateur de contrôler la composition lipidique et protéique, ainsi que la topologie membranaire, afin de rendre compte de l’importante variabilité observée entre les différents compartiments de fusion cellulaire. La reconstitution dans des liposomes offre une incroyable flexibilité avec la possibilité de faire varier la plupart des paramètres clefs et de créer un environnement minimal dans lequel les facteurs solubles et/ou membranaires peuvent être ajoutés, seuls ou en combinaison, pour dévoiler leur rôle avec clarté. Nous avons mis au point des systèmes in vitro de reconstitution de protéines dans des plateformes membranaires artificielles pour nos deux systèmes d’études (les deux protéines Longin-SNAREs TI-VAMP et Sec 22b, ainsi que les domaines « coiled-coil » des Mitofusines) et nous avons réalisé des expériences biochimiques pour caractériser le mode d’action de ces protéines. L’objectif à long-terme de ce projet est de comparer les mécanismes moléculaires des machineries de fusion associés aux protéines SNAREs et Mitofusines, et ainsi de dévoiler des similitudes structurelles et fonctionnelles entre (1) leur protéines de fusion principales et (2) leur facteurs régulateurs. / The molecular mechanisms involved in membrane fusion have been extensively studied for the past thirty years. Our current understanding of this phenomenon is mainly based on results obtained by (i) the development of physical models describing the fusion of membranes, (ii) structural and mechanistic investigations on fusion proteins of enveloped viruses and (iii) studies of SNARE protein-mediated intracellular fusion events of eukaryotic cells. Discovery of the SNARE complex was the outcome of interdisciplinary works which involved a wide range of techniques including yeast genetics, electrophysiology, molecular biology, cell-free biochemistry, adhesion/fusion biophysics and imaging. Taking advantage of the paradigms and biophysical techniques that emerged from these studies, we investigated the function and regulation of coiled-coil domains in intracellular fusion processes involving Longin-SNAREs or Mitofusins, two fusion protein machineries whose exact mode of action still remains unclear. A comprehensive understanding of the molecular mechanisms of membrane fusion requires the in vitro reconstitution of fusion proteins into a wide variety of membrane environments with defined and tunable biophysical properties. Ideally, these membrane systems should allow the experimentalists to control the lipid and protein composition as well as the membrane topology, to account for the variability observed across cellular fusing compartments. Reconstitution into liposomes offers amazing flexibility with the capacity to vary most of these relevant parameters, and to create a minimal environment in which membrane and/or soluble factors can be added, one at a time or in combination, to reveal their role with clarity. We have set up the in vitro reconstitution of proteins into various artificial membrane platforms for both systems (the Longin-SNAREs TI-VAMP and Sec22b and the coiled-coil domains of Mitofusins) and performed biochemical assays to gain insight into how these proteins execute their functions. The long-term goal of this project is to compare the molecular mechanisms of SNARE and Mitofusin fusion machineries and thus reveal structural and functional similitudes between (i) their core fusion proteins, and (ii) their regulatory factors.

Les mécanismes moléculaires impliqués dans la fusion membranaire ont été amplement étudiés au cours des trente dernières années. Notre compréhension actuelle de ce phénomène est principalement basée sur des résultats obtenus par (1) le développement de modèles physiques décrivant la fusion des membranes biologiques, (2) l’étude mécanistique et structurale des protéines de fusion membranaire des virus à enveloppe et (3) l’étude des évènements de fusion intracellulaire médiés par les protéines SNARES dans les cellules eucaryotes. La découverte du complexe SNARE fut l’aboutissement de travaux interdisciplinaires qui ont exigés un large éventail de techniques tel que la génétique de la levure, l’électrophysiologie, la biologie moléculaire, la biochimie cellulaire, la biophysique expérimentale et l’imagerie. Tirant parti des paradigmes et techniques biophysiques qui ont émergés de ces études, nous avons examiné les fonctions et mécanismes de régulation des domaines « coiled-coil » dans les processus de fusion intracellulaire impliquant des protéines de la famille des Longin-SNAREs ou des Mitofusines, deux machineries protéiques de fusion dont le mode d’action exact reste encore peu clair. La conception exacte des mécanismes moléculaires de la fusion membranaire requiert la reconstitution in vitro des protéines de fusion dans un large spectre d’environnement membranaire avec des propriétés biophysiques définies et facilement modulables. Idéalement, ces systèmes membranaires devraient permettre à l’expérimentateur de contrôler la composition lipidique et protéique, ainsi que la topologie membranaire, afin de rendre compte de l’importante variabilité observée entre les différents compartiments de fusion cellulaire. La reconstitution dans des liposomes offre une incroyable flexibilité avec la possibilité de faire varier la plupart des paramètres clefs et de créer un environnement minimal dans lequel les facteurs solubles et/ou membranaires peuvent être ajoutés, seuls ou en combinaison, pour dévoiler leur rôle avec clarté. Nous avons mis au point des systèmes in vitro de reconstitution de protéines dans des plateformes membranaires artificielles pour nos deux systèmes d’études (les deux protéines Longin-SNAREs TI-VAMP et Sec 22b, ainsi que les domaines « coiled-coil » des Mitofusines) et nous avons réalisé des expériences biochimiques pour caractériser le mode d’action de ces protéines. L’objectif à long-terme de ce projet est de comparer les mécanismes moléculaires des machineries de fusion associés aux protéines SNAREs et Mitofusines, et ainsi de dévoiler des similitudes structurelles et fonctionnelles entre (1) leur protéines de fusion principales et (2) leur facteurs régulateurs. / The molecular mechanisms involved in membrane fusion have been extensively studied for the past thirty years. Our current understanding of this phenomenon is mainly based on results obtained by (i) the development of physical models describing the fusion of membranes, (ii) structural and mechanistic investigations on fusion proteins of enveloped viruses and (iii) studies of SNARE protein-mediated intracellular fusion events of eukaryotic cells. Discovery of the SNARE complex was the outcome of interdisciplinary works which involved a wide range of techniques including yeast genetics, electrophysiology, molecular biology, cell-free biochemistry, adhesion/fusion biophysics and imaging. Taking advantage of the paradigms and biophysical techniques that emerged from these studies, we investigated the function and regulation of coiled-coil domains in intracellular fusion processes involving Longin-SNAREs or Mitofusins, two fusion protein machineries whose exact mode of action still remains unclear. A comprehensive understanding of the molecular mechanisms of membrane fusion requires the in vitro reconstitution of fusion proteins into a wide variety of membrane environments with defined and tunable biophysical properties. Ideally, these membrane systems should allow the experimentalists to control the lipid and protein composition as well as the membrane topology, to account for the variability observed across cellular fusing compartments. Reconstitution into liposomes offers amazing flexibility with the capacity to vary most of these relevant parameters, and to create a minimal environment in which membrane and/or soluble factors can be added, one at a time or in combination, to reveal their role with clarity. We have set up the in vitro reconstitution of proteins into various artificial membrane platforms for both systems (the Longin-SNAREs TI-VAMP and Sec22b and the coiled-coil domains of Mitofusins) and performed biochemical assays to gain insight into how these proteins execute their functions. The long-term goal of this project is to compare the molecular mechanisms of SNARE and Mitofusin fusion machineries and thus reveal structural and functional similitudes between (i) their core fusion proteins, and (ii) their regulatory factors.

No description available.

<p>  </p> <p>Zebrafish (<em>Danio rerio</em>) is a well-established model to study neutrophil biology. However, a lack of standard tissue-specific knockdown or knockout technique in the zebrafish field has limited the power of this model organism when studying developmental essential genes, such as those related to mitochondrial function. We have developed a robust and flexible neutrophil-restricted knockout in zebrafish based on CRISPR/Cas9 system, with which we gained insights into the role of Rac2 in regulating the actin cytoskeleton and the subcellular location of Rac activation in zebrafish neutrophils.</p> <p>Previous study in our lab using another neutrophil-specific knockout system addressed multiple mitochondrial proteins regulate neutrophil motility in zebrafish. Interestingly, we observed <em>Mfn2</em>-deficient neutrophils trapped in the vasculature in zebrafish embryos. Here we further characterized the function of MFN2 in regulating cell migration with neutrophil-like HL-60 cells and mice embryonic fibroblasts (MEFs). We found significant changes in actin organization in both <em>MFN2</em>-deficient neutrophil-like cells and MEFs and mechanistically, disrupted mitochondria-ER interaction, increased intracellular Ca2+ levels. We also investigated the cytoskeleton proteins and observed hyperactivation of RhoA and Myosin light chain kinase, along with accumulation of phosphorylated myosin light chain at the cell boundary in <em>MFN2</em>-deficient MEFs. These altered MFN2-Ca2+-RhoA/MLCK-myosin signaling finally affects the peripheral actin bundle architecture and forms the “Peripheral Actin Myosin Belt (PAMB)” structure. The formation of PAMB hampered cell adhesive migration in <em>Mfn2</em>-null MEFs. </p> <p>Altogether, our research gained new insights into the essential role of MFN2 in cytoskeleton regulation and the underlying molecular mechanisms, which may provide a new direction to understand the relevance of this gene in immune cell dysfunction and other MFN2-associated diseases.</p>