La mitochondrie forme un réseau dynamique de tubules, dont la morphologie et la distribution sont étroitement régulées. Les mitochondries sont des organelles à double membrane dont l’architecture interne est complexe. Les crêtes mitochondriales forment des invaginations de la membrane interne. Elles sont le lieu des phosphorylations oxydatives, réactions par lesquelles l’ATP synthase produit l’ATP. L’ATP synthase est également connue pour son rôle clé dans la morphogenèse des crêtes. Dans cette étude j’ai mis en évidence in vivo la localisation en cluster de l’ATP synthase au sein du réseau mitochondrial de S. cerevisiae se développant sur substrat fermentescible. Mes résultats suggèrent que ces clusters correspondent aux crêtes mitochondriales, ce qui ouvre de nouvelles perspectives pour l’étude du remaniement de la membrane interne.La morphologie du réseau mitochondrial est maintenue par un équilibre entre les processus de fusion et de fission des tubules mitochondriaux. Dans la deuxième partie de ma thèse, j’ai mis en évidence une fragmentation progressive du réseau mitochondrial lors de l’entrée des cellules en quiescence, un état cellulaire non prolifératif réversible. En quiescence, le réseau mitochondrial est constitué de petites vésicules sous corticales immobiles au contenu enzymatique variable. Lors d’un retour à l’état prolifératif ces vésicules fusionnent rapidement pour reformer un réseau tubulaire, et ce, avant l’émergence de la cellule fille. De façon surprenante j’ai mis en évidence que ni les machineries canoniques de fusion ou de fission, ni le cytosquelette d’actine ne sont requis lors du remaniement du réseau mitochondrial dans les transitions entre prolifération et quiescence. / Mitochondria form a dynamic tubular network which organization and distribution is highly regulated.Mitochondria are double membrane organites with a complex internal architecture. Cristae, which areinner membrane invaginations, are the site of oxidative phosphorylation, reactions by which ATP synthaseproduces ATP. ATP synthase also play a key role in cristae morphogenesis. In this study, I have shown thatATP synthase localized as discrete clusters along the mitochondrial network in living S. cerevisiae cellsgrown on a fermentable carbon source. Overall our data suggest that ATP synthase clusers correspond tomitochondrial cristae, opening new avenues to explore the mechanisms involved in inner membraneremodelling.Mitochondrial network morphology is regulated by a dynamic equilibrium between the fusion and fissionof mitochondrial tubules. In the second part of my thesis, I highlight a progressive mitochondrialfragmentation during quiescence establishment, a state defined as a reversible absence of proliferation.Quiescent cells mitochondrial network is composed of immobile small cortical mitochondrial vesicles witha variable enzymatic content. Upon quiescence exit, cortical mitochondrial vesicles rapidly fuse and atubular network is reconstituted prior to bud emergence. Astonishingly, neither the canonical fusion orfission machineries nor the actin cytoskeleton are required for the mitochondrial network modificationduring quiescence / proliferation transition.

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.

<p dir="ltr">Adipose tissue is characterized by the dominant presence of adipocytes, specialized cells adept at lipid metabolism. These adipocytes act as critical nodes, coordinating the complex processes of energy storage and mobilization according to the body's metabolic requirements. Within the adipocyte population of mammals, there are three main subtypes: white, beige, and brown adipocytes. White adipocytes are primarily dedicated to the sequestration of energy in the form of triglycerides. Conversely, beige and brown adipocytes are distinguished by their capacity for thermogenesis, the process of dissipating nutritional energy as heat. The contemporary challenge of chronic overnutrition has precipitated a global surge in obesity and cardiometabolic diseases. Addressing this issue necessitates the maintenance of white adipocyte homeostasis and the enhancement of the quantity and function of thermogenic adipocytes, which are imperative for mitigating the global obesity epidemics.</p><p dir="ltr">Mitochondrion, a multifunctional organelle, is integral to a broad spectrum of cellular processes, including anabolic and catabolic metabolism, bioenergetics, and signal transduction, all of which are essential for maintaining cellular functions and homeostasis. The efficacy of mitochondrial operations is intrinsically linked to their membrane dynamics. In this study, transmission electron microscopy and mass spectrometry were employed to investigate the proteins implicated in the cold-induced mitochondrial membrane remodeling in brown adipocytes. Through this approach, a poorly characterized protein, Family with Sequence Similarity 210 Member A (FAM210A), was identified as a mitochondrial inner membrane protein that is induced by cold stimulation. Subsequent loss-of-function experiments were conducted to elucidate the role of FAM210A in adipocytes. Mice with adipose-specific deletion of <i>Fam210a</i> (<i>Fam210a</i>^<em>AKO</em>) exhibited compromised mitochondrial cristae structure and a reduced thermogenic capacity in brown adipose tissue (BAT), resulting in an increased susceptibility to lethal hypothermia during acute cold challenge. Moreover, in mice with inducible ablation of <i>Fam210a</i> in adipocytes (<i>Fam210</i>^{<em>iAKO</em>}), mitochondrial alterations in BAT were negligible at thermoneutral conditions; however, they exhibited defective cold-induced mitochondrial cristae remodeling, culminating in a progressive loss of cristae and diminished mitochondrial density. Mechanistically, it was determined that FAM210A interacts with mitochondrial protease YME1L and modulates its activity toward OMA1 and OPA1 cleavage, thus compromising cold-induced mitochondrial remodeling in BAT.</p><p dir="ltr">Additionally, this research delved into the role of FAM210A in adipocytes in response to dietary stress by feeding mice with high-fat diet (HFD). The study found a consistent correlation between FAM210A expression and OPA1 cleavage in adipocytes under HFD challenge. Mice lacking FAM210A in all adipocytes and subjected to HFD exhibited lipoatrophy in white adipose tissue (WAT) and a downregulation of genes associated with adipogenesis and lipid metabolism. In contrast, mice with a brown adipocyte-specific ablation of <i>Fam210a </i>(<i>Fam210a</i>^<em>UKO</em>) displayed no significant change in WAT mass but had enlarged livers. Crucially, both <i>Fam210a</i>^<em>AKO</em> and <i>Fam210a</i>^<em>UKO</em> mice presented increased WAT inflammation, deteriorated glucose tolerance, and exacerbated insulin resistance. These findings underscore the pivotal role of FAM210A in brown adipose tissue (BAT) in the preservation of WAT homeostasis and the regulation of systemic glucose clearance in diet-induced obesity.</p><p dir="ltr">In summary, these studies characterize the mitochondrial dynamics in brown adipocytes in response to cold stress, identify a new cold-induced mitochondrial protein, FAM210A, and uncover its functions in adipocytes under cold and dietary stresses. These findings highlight the importance of mitochondrial remodeling in the adaptive response of adipocytes to evolving metabolic demands. This work establishes FAM210A as a key regulator of mitochondrial cristae remodeling, shedding light on the mechanisms that govern mitochondrial plasticity in adipocytes.</p>