Glycosylation-Dependent IFN-γR Partitioning in Lipid and Actin Nanodomains Is Critical for JAK Activation.

Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.

PSL Chemical Biology Symposia Third Edition: A Branch of Science in its Explosive Phase.

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.

Caveolae: The FAQs.

Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round-table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.

Small Molecule Inhibitors of Interferon-Induced JAK-STAT Signalling.

Interferons (IFN) are cytokines which, upon binding to cell surface receptors, trigger a series of downstream biochemical events including Janus Kinase (JAK) activation, phosphorylation of Signal Transducer and Activator of Transcription protein (STAT), translocation of pSTAT to the nucleus and transcriptional activation. Dysregulated IFN signalling has been linked to cancer progression and auto-immune diseases. Here, we report the serendipitous discovery of a small molecule that blocks IFNγ activation of JAK-STAT signalling. Further lead optimisation gave rise to a potent and more selective analogue that exerts its activity by a mechanism consistent with direct IFNγ targeting in vitro, which reduces bleeding in model of haemorrhagic colitis in vivo. This first-in-class small molecule also inhibits type I and III IFN-induced STAT phosphorylation in vitro. Our work provides the basis for the development of pan-IFN inhibitory drugs.

Lifetime estimation of moving subcellular objects in frequency-domain fluorescence lifetime imaging microscopy.

Fluorescence lifetime is usually defined as the average nanosecond-scale delay between excitation and emission of fluorescence. It has been established that lifetime measurements yield numerous indications on cellular processes such as interprotein and intraprotein mechanisms through fluorescent tagging and Förster resonance energy transfer. In this area, frequency-domain fluorescence lifetime imaging microscopy is particularly appropriate to probe a sample noninvasively and quantify these interactions in living cells. The aim is then to measure the fluorescence lifetime in the sample at each location in space from fluorescence variations observed in a temporal sequence of images obtained by phase modulation of the detection signal. This leads to a sensitivity of lifetime determination to other sources of fluorescence variations such as intracellular motion. In this paper, we propose a robust statistical method for lifetime estimation for both background and small moving structures with a focus on intracellular vesicle trafficking.

Membrane trafficking and signaling: two sides of the same coin.

Recent findings on clathrin-dependent and non clathrin-dependent endocytic routes are currently changing our classical view of endocytosis. Originally seen as a way for the cell to internalize membrane, receptors or various soluble molecules, this process is in fact directly linked to complex signaling pathways. Here, we review new insights in endocytosis and present latest development in imaging techniques that allow us to visualize and follow the dynamics of membrane-associated signaling events at the plasma membrane and other intracellular compartments. The immune synapse is taken as an illustration of the importance of membrane reorganization and proteins clustering to initiate and maintain signaling. Future challenges include understanding the crosslink between traffic and signaling and how all compartmentalized signals are integrated inside the cell at a higher level.

Cross-cultural adaptation and reliability of the Foot Posture Index (FPI-6) - French version.

PURPOSE: The 6-item Foot Posture Index (FPI-6) is a reliable tool for the evaluation of foot deformities. Our aim was to translate and cross-culturally validate the FPI-6 for use in French-speaking countries and to determine the intra-rater and inter-rater reliability of the French version. METHODS: Cross-cultural adaptation was performed according to guidelines. Two clinicians assessed the FPI-6 in 52 asymptomatic individuals. We evaluated intra- and inter-rater reliability with the intraclass correlation coefficients (ICC), correlations (p-value < 0.05) and Bland-Altman plots. Standard error of measurement (SEM) and minimum detectable change (MDC95) were determined. RESULTS: For the cross-cultural adaptation, we modified several items of the FPI-6 user guide and added footnotes to ensure correct interpretation. ICC of the total FPI-6 scores were 0.94 to 0.96 for the intra- and inter-rater reliability for dominant and non-dominant lower limb. Correlations were significant (p < 0.001); r 0.88 to 0.92. Total score SEM was 0.68 to 0.78 and MDC95 was 1.58 to 1.82. CONCLUSIONS: Intra- and inter-rater reliability of this French version of the FPI-6 was excellent for the total score and good to excellent for each item. The French FPI-6 can be used in French-speaking countries. The identification of SEM and MDC scores is useful for clinical interpretation.

The French version of the 6-item Foot Posture Index (FPI-6) can be used in clinical practice in French-speaking countriesIntra- and inter-rater reliability of total FPI score are excellentWe provide minimal detectable change value for clinical interpretation (1.58 to 1.82).

Lipid nanodomains and receptor signaling: From actin-based organization to membrane mechanics.

The plasma membrane serves as the primary barrier between the cell's interior and its external surroundings, which places it at the forefront of intercellular communication, receptor signal transduction and the integration of mechanical forces from outside. Most of these signals are largely dependent on the plasma membrane heterogeneity which relies on lipid-lipid and lipid-protein interactions and the lateral nano-distribution of lipids organized by the dynamic network of cortical actin. In this review, we undertake an in-depth exploration of recent discoveries, which contribute significantly to the evolution from raft model to lipid nanodomains. Specifically, we will focus on their role in membrane receptor-mediated signaling in the context of cell membrane mechanics.

[Clathrin-independent endocytosis: free the way!]. / Endocytose sans clathrine: la voie est libre !

Along the years, the interest paid to the study of endocytosis has never wavered as this process plays such an essential role in many cellular functions. Cell growth, adhesion and differentiation, regulation of signaling induced by membrane receptors or infection by viral particles are all dependent on the entry of molecules into the cell. Once the clathrin-dependent endocytosis well characterized, it has become apparent that other entry pathways also existed in the cell. This review is intended to provide an update on recent advances that establish with certainty the existence of endocytic pathways independent of clathrin and highlight their specific regulators.

Relationship between care pathway features and use or non-use of orthotic devices by individuals with Charcot-Marie-Tooth disease: a cross-sectional, exploratory study.

PURPOSE: Orthotic devices may be prescribed for the management of foot and ankle deformities caused by Charcot-Marie-Tooth disease (CMT). However, the actual use of these devices is variable. No studies have evaluated the impact of prescription, delivery and follow-up of orthotic devices on their use.We aimed to describe the relationship between the pathways followed by individuals with CMT and orthotic device use. MATERIALS AND METHODS: Exploratory, cross-sectional, 35-item survey of orthotic device management. Individuals with CMT were recruited from CMT-France Association. RESULTS: Of the 940 respondents, 795 were included, mean age of 52.9 (SD 16.9) years. Rate of orthotic device use was 49.2% (391/795). The most frequent reason for non-use was a poor fit. Non-use was related to the orthotic device type, the health professionals consulted, and the severity of the CMT-related impairments. Follow-up visits (38.7%), re-evaluation of orthotic devices (25.3%) and consultations with the Physical and Rehabilitation Medicine physician were infrequent (28.3%). CONCLUSIONS: Orthotic devices are massively underused. Follow-up and re-evaluation are infrequent. Care pathways, prescription and delivery of orthotic devices must be optimized to meet the expectations of people with CMT. Device fitting, individual needs, and changes in the clinical state must be re-evaluated regularly by specialists to improve orthotic device use.Implications for rehabilitationFoot deformities and foot drop contribute to disability in people with Charcot Marie Tooth Disease but use of orthotic devices is poor.To improve orthotic device use, regular multidisciplinary consultations and regular re-evaluation of the device by specialists in neuro-orthopaedics are important.The device fit should be regularly re-evaluated by the prescribing doctor or the practitioner who made the device.Regular multimodal evaluation of foot deformity, including muscle strength and length, and the individual's needs and expectations is also important to improve orthotic device use.

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 -ß receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/ß-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-ß, 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-ß.

Development of a preoperative questionnaire to improve satisfaction with hallux valgus repair: A Delphi study.

BACKGROUND: Satisfaction with hallux valgus repair is often poor, despite good surgical outcomes. Many tools have been developed to assess the outcome of the procedure; however none evaluate the association between the initial motive for repair and the reasons for post-surgical dissatisfaction. The aim of this study was to develop a new tool to analyse the subjective and objective expectations of individuals during a pre-operative consultation for hallux valgus repair in order to improve post-surgical satisfaction. METHODS: We first collected the reasons for dissatisfaction with repair from the medical files of dissatisfied individuals. Then, a steering committee of 4 French experts in the management of hallux valgus designed a questionnaire based on the reasons for dissatisfaction. We then used the DELPHI method to validate the questionnaire: we submitted the questionnaire to a panel of 34 francophone experts in hallux valgus repair for rating in 4 rounds. RESULTS: The medical files of 853 individuals were reviewed and a 52-item questionnaire relating to expectations from hallux valgus surgery was drafted. After the 4 rounds, a final 44 item questionnaire reached consensus. Thirteen items related to clinical and psychological profile, 5 to pain, 9 to physical activity, 4 to aesthetics and 13 to footwear. CONCLUSION: This tool should facilitate gathering of individuals' expectations from hallux valgus repair to ensure realistic goals and reduce post-surgical dissatisfaction.

Identification of a New Cholesterol-Binding Site within the IFN-γ Receptor that is Required for Signal Transduction.

The cytokine interferon-gamma (IFN-γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-γ exerts it signaling action by binding to a specific cell surface receptor, the IFN-γ receptor (IFN-γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN-γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFNγR2 interaction may represent a potential therapeutic strategy for various IFN-γ-dependent diseases.

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects.

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.

Filling up adipocytes with lipids. Lessons from caveolin-1 deficiency.

Caveolins are primarily known as the main constituents of the protein coat of caveolae invaginations at the plasma membrane. They have also been found at the surface of intracellular lipid droplets but their function in this lipid storage organelle remains poorly understood. This paper reviews recent studies in adipocytes, the specialized cell type for fatty acid storage, which suggest a role for caveolins in the formation, maintenance or mobilization of lipid droplet stores. These new functions emerged from studies of fat cells in which caveolin expression was invalidated, highlighting the metabolic phenotype of caveolin-deficient mice or human patients who develop progressive lipoatrophy.

GPMVs as a Tool to Study Caveolin-Interacting Partners.

Caveolins, major components of small plasma membrane invaginations called caveolae, play a role in signaling, particularly in mechanosignaling. These proteins are known to interact with a variety of effector molecules, including G-protein-coupled receptors, Src family kinases, ion channels, endothelial nitric oxide synthase (eNOS), adenylyl cyclases, protein kinase A (PKA), and mitogen-activated PKs (MAPKs). There is, however, speculation on the relevance of these interactions and the mechanisms by which caveolins may control intracellular signaling. This chapter introduces a method of isolation of giant plasma membrane-derived vesicles (GPMVs), which possess full complexity of membrane they originate from, thus comprising an excellent platform to revisit some of the previously described interactions in a cleaner environment and possibly identifying new binding partners. It is also a powerful technique for studying membrane mechanics, as it was previously used to demonstrate the role of caveolae in mechanoprotection.

Study of Caveolae-Dependent Mechanoprotection in Human Muscle Cells Using Micropatterning and Live-Cell Microscopy.

Caveolae are plasma membrane organelles that are, among many other features, involved in mechanosensing and mechanoprotection. Different tools have been developed to study caveolae-dependent mechanoprotection and had to be adapted to the tissue or cells studied, as these structures are found in almost every type of cells. This chapter focuses on a protocol combining the use of live-cell imaging, micropatterning, hypo-osmotic shock as a mechanical stress, and dyes such as calcein-AM and propidium iodide. We used this protocol for the in vitro study of the effect of mechanical stress on membrane integrity in human muscle cells from patients bearing caveolin-3 mutations.

Interferon Receptor Trafficking and Signaling: Journey to the Cross Roads.

Like most plasma membrane proteins, type I interferon (IFN) receptor (IFNAR) traffics from the outer surface to the inner compartments of the cell. Long considered as a passive means to simply control subunits availability at the plasma membrane, an array of new evidence establishes IFNAR endocytosis as an active contributor to the regulation of signal transduction triggered by IFN binding to IFNAR. During its complex journey initiated at the plasma membrane, the internalized IFNAR complex, i.e. IFNAR1 and IFNAR2 subunits, will experience post-translational modifications and recruit specific effectors. These finely tuned interactions will determine not only IFNAR subunits destiny (lysosomal degradation vs. plasma membrane recycling) but also the control of IFN-induced signal transduction. Finally, the IFNAR system perfectly illustrates the paradigm of the crosstalk between membrane trafficking and intracellular signaling. Investigating the complexity of IFN receptor intracellular routes is therefore necessary to reveal new insight into the role of IFNAR membrane dynamics in type I IFNs signaling selectivity and biological activity.

Coupling of melanocyte signaling and mechanics by caveolae is required for human skin pigmentation.

Tissue homeostasis requires regulation of cell-cell communication, which relies on signaling molecules and cell contacts. In skin epidermis, keratinocytes secrete factors transduced by melanocytes into signaling cues promoting their pigmentation and dendrite outgrowth, while melanocytes transfer melanin pigments to keratinocytes to convey skin photoprotection. How epidermal cells integrate these functions remains poorly characterized. Here, we show that caveolae are asymmetrically distributed in melanocytes and particularly abundant at the melanocyte-keratinocyte interface in epidermis. Caveolae in melanocytes are modulated by ultraviolet radiations and keratinocytes-released factors, like miRNAs. Preventing caveolae formation in melanocytes increases melanin pigment synthesis through upregulation of cAMP signaling and decreases cell protrusions, cell-cell contacts, pigment transfer and epidermis pigmentation. Altogether, we identify that caveolae serve as molecular hubs that couple signaling outputs from keratinocytes to mechanical plasticity of pigment cells. The coordination of intercellular communication and contacts by caveolae is thus crucial to skin pigmentation and tissue homeostasis.

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells.

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.