Role of STAM in the endosomal regulation of IFN-induced JAK/STAT signaling pathway

La première implication des récepteurs aux interférons de type 1 (IFNAR1) dans le contrôle de la voie Jak/STAT induite par les IFNs a été établie par mon laboratoire il y a une dizaine d'années (Marchetti et al., 2006). Une des questions fondamentales était alors de déterminer comment et pourquoi l'endocytose des IFNARs pouvait contrôler la voie Jak/STAT. Deux acteurs clés du tri endosomal ont attiré notre attention : Hrs (Hepatocyte growth factor-Regulated tyrosine kinase Substrate) et STAM (Signal Transducing Adaptor Molecule). Ces deux protéines constituent le complexe ESCRT-0 (Endosomal Sorting Complexes Required for Transport-0) les localisant, de manière idéale, à l'interface entre signalisation cellulaire et trafic membranaire.La combinaison de la biologie moléculaire et cellulaire, de la biochimie et de la microscopie à fluorescence, nous a permis d'établir que STAM s'associe au complexe IFNAR1 à la membrane plasmique afin d'y exercer son effet inhibiteur sur la signalisation Jak/STAT. Cette inhibition est levée lorsqu'IFNAR est libéré dans l'endosome et qu'il peut ainsi être recruté par Hrs sous la dépendance de l'IFN-α. En nous basant sur des expériences de déplétion par shRNA ou d'inhibition pharmacologique, nous avons identifié PTP1B (Protein Tyrosine Phosphatase 1B) en tant qu'activateur de la voie Jak/STAT induite par les IFNs. Le blocage sélectif de l'endocytose d'IFNAR par déplétion de clathrin, nous a permis de montrer que l'activation de PTP1B est inhibée à la membrane plasmique. Cela a été confirmé par des expériences d'interaction protéine-protéine (Proximity Ligation Assay) indiquant que STAM est constitutivement associé à IFNAR1, tandis que l'interaction entre IFNAR1 et Hrs a seulement lieu à l'endosome.Ainsi, nos résultats permettent d'établir un modèle dans lequel, à la membrane plasmique, STAM est un frein permanent à la signalisation Jak/STAT, qui est levé après l'endocytose d'IFNAR et sa libération dans l'endosome de tri. De plus, nous avons montré que l'interaction Hrs/STAM dans l'endosome précoce permet de différencier, de manière sélective, l'activation de la signalisation Jak/STAT médiée par l'IFN-α ou l'IFN-β.A decade ago, my laboratory established the first role of type I IFNs receptor (IFNAR) endocytosis in the control of Jak/STAT signaling induced by type 1 IFNs (Marchetti et al., 2006). A salient question is now to elucidate why and how IFNAR endocytosis could control the Jak/STAT pathway. Two key players of endosomal sorting retained our interest: Hrs (Hepatocyte growth factor-Regulated tyrosine kinase Substrate) and STAM (Signal Transducing Adaptor Molecule). These two classical components of the ESCRT-0 (Endosomal Sorting Complexes Required for Transport-0) complex were ideally placed at the interface between signaling and membrane trafficking. By using a combination of molecular and cellular biology, biochemistry, and fluorescent microscopy, we could establish that STAM binds to the IFNAR complex at the plasma membrane to exert an inhibitory effect on Jak/STAT signaling. This inhibition is removed when IFNAR is delivered to the sorting endosome by interacting with Hrs upon IFN-α stimulation. Based on shRNA down-expression and pharmacological inhibition, we further involve the PTP1B (Protein Tyrosine Phosphatase 1B) as it activates Jak/STAT signaling upon IFN stimulation. We could also show that PTP1B activation is inhibited by STAM at the plasma membrane from experiments where IFNAR endocytosis was blocked by siRNA-mediated clathrin down-expression. This was further confirmed by protein-protein interaction experiments (Proximity Ligation Assay) showing that STAM was constitutively associated with IFNAR1, whereas the interaction between IFNAR1 and Hrs occured only at the sorting endosome. Our results therefore allow to draw a model where STAM is a constitutive handbrake on Jak/STAT signaling at the plasma membrane that is released after IFNAR endocytosis and delivery to the sorting endosome. We further show that Hrs/STAM interaction at the early endosome allows to selectively distinguish the activation of Jak/STAT signaling mediated by IFN-α or IFN-β

Contrôle endosomal de la signalisation intracellulaire

Les récepteurs membranaires contrôlent les mécanismes essentiels tels que la croissance, l’adhésion, la différenciation et le métabolisme cellulaires via l’activation de voies de signalisation spécifiques. Il apparaît désormais que ces récepteurs ne signalent pas seulement depuis la surface des cellules, mais également, depuis des compartiments intracellulaires, en particulier les endosomes, seulement après avoir été internalisés avec leurs ligands via des voies d’endocytose différentes. Cette synthèse illustre comment une telle compartimentation spatio-temporelle de la transduction du signal permet un degré supplémentaire de régulation des processus cellulaires engagés

Rac1, actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3), which controls the cell surface homeostasis of the tumor marker CD166/ALCAM. Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here,we investigate the role in this mechanism of actin, Rho GTPases and microtubules, which are major actors of CIE processes. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers and that its perturbation strongly inhibits the uptake process of CD166. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms

Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

n their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius–dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P2. In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling

C-type lectin receptor CLEC4A2 promotes tissue adaptation of macrophages and protects against atherosclerosis

Macrophages are integral to the pathogenesis of atherosclerosis, but the contribution of distinct macrophage subsets to disease remains poorly defined. Using single cell technologies and conditional ablation via a LysMCre+ Clec4a2flox/DTR mouse strain, we demonstrate that the expression of the C-type lectin receptor CLEC4A2 is a distinguishing feature of vascular resident macrophages endowed with athero-protective properties. Through genetic deletion and competitive bone marrow chimera experiments, we identify CLEC4A2 as an intrinsic regulator of macrophage tissue adaptation by promoting a bias in monocyte-to-macrophage in situ differentiation towards colony stimulating factor 1 (CSF1) in vascular health and disease. During atherogenesis, CLEC4A2 deficiency results in loss of resident vascular macrophages and their homeostatic properties causing dysfunctional cholesterol metabolism and enhanced toll-like receptor triggering, exacerbating disease. Our study demonstrates that CLEC4A2 licenses monocytes to join the vascular resident macrophage pool, and that CLEC4A2-mediated macrophage homeostasis is critical to combat cardiovascular disease.publishedVersionPeer reviewe

Budget-Friendly Generation, Biochemical Analyses, and Lentiviral Transduction of Patient-Derived Colon Organoids.

For the past decade, three-dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two-dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ-restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so-called patient-derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient-derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget-friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D-culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost-effective techniques to achieve immunoblot and high-resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of PDCOs from biopsies Basic Protocol 2: Long-term maintenance and expansion of PDCOs in BME domes Basic Protocol 3: Cryopreservation and thawing of PDCOs Basic Protocol 4: Lentiviral transduction of PDCOs Basic Protocol 5: Immunoblot and evaluation of variability between donors Basic Protocol 6: Immunofluorescence labeling and high-resolution microscopy of PDCOs Basic Protocol 7: Transcriptomic analyses of PDCOs by RT-qPCR

STAM and Hrs interact sequentially with IFN-α Receptor to control spatiotemporal JAK–STAT endosomal activation

Activation of the JAK-STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -β receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/β-induced JAK-STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM-IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-β, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK-STAT signalling by IFN-α and IFN-β