L'analyse en molécule unique du récepteur de l’IL-2 révèle l'importance de son oligomérisation pour l'endocytose et la signalisation dans les lymphocytes

Signaling by the interleukin-2 receptor (IL-2R) is regulated by its clathrin-independent endocytosis (CIE) and subsequent degradation, while playing a critical role in immunity. Interestingly, CIE lacks a coat protein that drives pit formation, raising the question of how the CIE vesicle is initiated. Protein clustering generates forces that can induce membrane conformational changes. Notably, IL-2R has been shown to accumulate at the base of membrane protrusions, where receptors might cluster and thereby initiate the pit.To study the relevance of IL-2R clustering in its endocytosis, we generated a CRISPR-edited T cell line expressing GFP-IL-2Rᵧ and analyzed its stoichiometry at the plasma membrane, by TIRF microscopy coupled to a single-molecule endocytic tracking method. We identified distinct IL-2Rᵧ cluster populations. IL-2Rᵧ seems to reach the cell surface as a preassembled cluster to which further molecules are added, reaching an optimal cluster size that is key for its internalization. Binding of IL-2 promotes the formation of endocytic clusters and receptor uptake, highlighting the importance of clustering for CIE internalization.Moreover, we found that cholesterol depletion increases the proportion of large, non-endocytic clusters as well as IL-2R signaling. Disruption of the actin meshwork also promotes the formation of large clusters, yet it decreases IL-2R signaling. Thus, both factors regulate IL-2R endocytosis and signaling in a distinct manner. Our results provide new insights into the mechanisms regulating receptor signaling and CIE.La signalisation par le récepteur de l'interleukine-2 (IL-2R), est régulée par son endocytose indépendante de la clathrine (EIC) et sa dégradation ultérieure, tout en jouant un rôle essentiel dans l'immunité. L'endocytose indépendante de la clathrine étant dépourvue de manteau protéique induisant la formation des puits, la question qui se pose est de savoir comment la vésicule d’IL-2R est-elle initiée. Le regroupement local des protéines peut induire des changements de conformation de la membrane et créer une invagination. Sachant que l'IL-2R s'accumule à la base des protubérances membranaires avant d’initier sa vésicule d’endocytose, nous avons analysé si le regroupement des récepteurs pourrait favoriser son internalisation. Pour étudier l’importance du regroupement de l'IL-2R, nous avons généré une lignée lymphocytaire humaine éditée (CRISPR) pour exprimer GFP-IL-2Rᵧ. Nous avons ensuite analysé la stoechiométrie du récepteur au niveau de la membrane plasmique, par microscopie TIRF couplée à une méthode de suivi de GFP-IL-2Rᵧ en molécule unique et identifié des populations distinctes d’oligomérisation. L'IL-2Rᵧ semble atteindre la surface cellulaire sous forme d'oligomères pré-assemblés auxquels d'autres molécules sont ajoutées, atteignant ainsi une taille d'oligomérisation optimale qui est essentielle à son internalisation. La liaison de l'IL-2 favorise la formation de ces oligomères, compétents pour l’endocytose, ce qui souligne l'importance du regroupement des récepteurs dans l'internalisation EIC.De plus, nous avons constaté que la déplétion du cholestérol augmentait la proportion de gros oligomères, non compétents pour l’endocytose mais induisant une forte signalisation. La perturbation du réseau d'actine favorise également la formation de gros oligomères, mais cette fois en diminuant la signalisation de l’IL-2R. Ainsi, les deux facteurs régulent l'endocytose et la signalisation de l'IL-2R de manière distincte. Nos résultats apportent de nouvelles connaissances sur les mécanismes régulant la signalisation des récepteurs et l’EIC

Par ici la sortie ! Le SARS-CoV-2 utilise les lysosomes pour sortir de la cellule infectée

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Cytokine receptor cluster size impacts its endocytosis and signaling

International audienceThe interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ tagged with green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2–induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2–dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction

Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport

International audienceIntracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and-independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events and enhances the pathogen capacity to penetrate into the colonic tissue in vivo. bacteria | pathogen | secretion | endocytosis | polarit