Implication des sites de contact entre le réticulum endoplasmique et les endosomes dans la régulation de l'autophagie en situation normale et cancéreuse

Autophagy is a conserved process in evolution actively involved in maintaining cellular homeostasis. Each cell in the human body can degrade a portion of its intracellular material by incorporating it into a double-membrane vesicle called an autophagosome, which fuses with the lysosome to facilitate degradation and recycling of the incorporated components. Many pathologies, such as cancer, are associated with dysregulations of the autophagic process, making the understanding of the molecular mechanisms associated with this pathway essential for the development of new therapies. The formation of the autophagosome, the first step in the autophagic pathway, is initiated when the cell is under stress, such as nutrient deprivation, and occurs at the surface of the endoplasmic reticulum (ER). The ER membrane can form dynamic contacts with other organelles, notably to enable molecule exchange. Endosomes are membranous structures essential for intracellular trafficking, facilitating communication between the internal and external milieu, thus are mobilized in response to environmental stress. During my PhD, I focused on the role of endosomes in the early stages of the autophagic pathway by studying their ability to form membrane contact sites with the ER in response to nutritional stress. Initially, I investigated the morphological adaptations of the endosomal system following nutrient deprivation. I demonstrated that in response to autophagy induction, a population of endosomes tubulated through SNX1 and SNX2 proteins. These tubular endosomes can then form contacts with the ER to participate in autophagosome biogenesis. Next, through the use and optimization of several techniques (expansion microscopy, biochemical purification of organelles, split-TurboID, video microscopy, FRAP, proximity ligation, electron microscopy), I documented and characterized the dynamics and role of ER-endosome contact sites (EERCS) necessary for autophagosome formation. Thus, I discovered that EERCS form at specific regions, thereby blocking the trafficking of newly synthesized proteins. The morphology of these contacts suggests that endosomes adopt a particular shape to isolate the site of autophagosome formation from the rest of the cytoplasm. This isolation promotes local calcium accumulation and induces phase transition of proteins present in this confined space. These local changes in physicochemical properties facilitate the formation and fusion of nano-vesicles, giving rise to the primary membrane required for autophagosome formation. Interestingly, the artificial stabilization of these contacts induces autophagy inhibition, highlighting the importance of dynamics in this system. This study is the first to report a role of membrane contact sites in creating sub-compartments with isolated chemical properties from the cytoplasm. Furthermore, these results reconcile many hypotheses regarding the origin of autophagosomes, including the nature of the required membranes and the intracellular localization. Finally, I highlighted that cancer cells from acute myeloid leukemia form more EERCS to achieve an effective autophagic response. This observation sheds light on a new avenue of therapeutic investigation by encouraging medical research to focus on membrane contact sites in a pathological context.