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.

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.

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.

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.

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.

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.

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.

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.

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription.

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress.

[Endosomal control of intracellular signaling]. / Contrôle endosomal de la signalisation intracellulaire.

Membrane receptors control essential processes such as cell growth, adhesion, differentiation and metabolism through the activation of specific signaling pathways. Nowadays, these receptors are not only known to signal from the plasma membrane but also from intracellular compartments. Indeed, after being internalized with their ligands via different endocytic pathways, some membrane receptors can initiate signal only after reaching the sorting endosome where they associate with specific protein partners. This review illustrates how this spatio-temporal regulation of signal transduction can occur, with several examples, including interferon receptors which activate JAK/STAT signaling pathways. The literature presented here explains why this control of signaling pathways occuring at the endosomal level creates a higher degree of tuning for the affected cellular processes.

The caveolae dress code: structure and signaling.

Over the past decade, interest in caveolae biology has peaked. These small bulb-shaped plasma membrane invaginations of 50-80nm diameter present in most cell types have been upgraded from simple membrane structures to a more complex bona fide organelle. However, although caveolae are involved in several essential cellular functions and pathologies, the underlying molecular mechanisms remain poorly defined. Following the identification of caveolins and cavins as the main caveolae constituents, recent studies have brought new insight into their structural organization as a coat. In this review, we discuss how these new data on caveolae can be integrated in the context of their role in signaling and pathophysiology.

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.

ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling.

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

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.

[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.