CAVIN1-Mediated hERG Dynamics: A Novel Mechanism Underlying the Interindividual Variability in Drug-Induced Long QT.

BACKGROUND: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (IKr) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. METHODS: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. RESULTS: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and IKr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1, essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin. CONCLUSIONS: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Compressive stress triggers fibroblasts spreading over cancer cells to generate carcinoma in situ organization.

At the early stage of tumor progression, fibroblasts are located at the outer edges of the tumor, forming an encasing layer around it. In this work, we have developed a 3D in vitro model where fibroblasts' layout resembles the structure seen in carcinoma in situ. We use a microfluidic encapsulation technology to co-culture fibroblasts and cancer cells within hollow, permeable, and elastic alginate shells. We find that in the absence of spatial constraint, fibroblasts and cancer cells do not mix but segregate into distinct aggregates composed of individual cell types. However, upon confinement, fibroblasts enwrap cancer cell spheroid. Using a combination of biophysical methods and live imaging, we find that buildup of compressive stress is required to induce fibroblasts spreading over the aggregates of tumor cells. We propose that compressive stress generated by the tumor growth might be a mechanism that prompts fibroblasts to form a capsule around the tumor.

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.

A mechanosensitive caveolae-invadosome interplay drives matrix remodelling for cancer cell invasion.

Invadosomes and caveolae are mechanosensitive structures that are implicated in metastasis. Here, we describe a unique juxtaposition of caveola clusters and matrix degradative invadosomes at contact sites between the plasma membrane of cancer cells and constricting fibrils both in 2D and 3D type I collagen matrix environments. Preferential association between caveolae and straight segments of the fibrils, and between invadosomes and bent segments of the fibrils, was observed along with matrix remodelling. Caveola recruitment precedes and is required for invadosome formation and activity. Reciprocally, invadosome disruption results in the accumulation of fibril-associated caveolae. Moreover, caveolae and the collagen receptor ß1 integrin co-localize at contact sites with the fibrils, and integrins control caveola recruitment to fibrils. In turn, caveolae mediate the clearance of ß1 integrin and collagen uptake in an invadosome-dependent and collagen-cleavage-dependent mechanism. Our data reveal a reciprocal interplay between caveolae and invadosomes that coordinates adhesion to and proteolytic remodelling of confining fibrils to support tumour cell dissemination.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production.

OBJECTIVE: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. METHODS: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. RESULTS: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. CONCLUSIONS: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

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.

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.

Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.

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.

Caveolae promote successful abscission by controlling intercellular bridge tension during cytokinesis.

During cytokinesis, the intercellular bridge (ICB) connecting the daughter cells experiences pulling forces, which delay abscission by preventing the assembly of the ESCRT scission machinery. Abscission is thus triggered by tension release, but how ICB tension is controlled is unknown. Here, we report that caveolae, which are known to regulate membrane tension upon mechanical stress in interphase cells, are located at the midbody, at the abscission site, and at the ICB/cell interface in dividing cells. Functionally, the loss of caveolae delays ESCRT-III recruitment during cytokinesis and impairs abscission. This is the consequence of a twofold increase of ICB tension measured by laser ablation, associated with a local increase in myosin II activity at the ICB/cell interface. We thus propose that caveolae buffer membrane tension and limit contractibility at the ICB to promote ESCRT-III assembly and cytokinetic abscission. Together, this work reveals an unexpected connection between caveolae and the ESCRT machinery and the first role of caveolae in cell division.

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.

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

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.

Membrane tension buffering by caveolae: a role in cancer?

Caveolae are bulb-like invaginations made up of two essential structural proteins, caveolin-1 and cavins, which are abundantly present at the plasma membrane of vertebrate cells. Since their discovery more than 60 years ago, the function of caveolae has been mired in controversy. The last decade has seen the characterization of new caveolae components and regulators together with the discovery of additional cellular functions that have shed new light on these enigmatic structures. Early on, caveolae and/or caveolin-1 have been involved in the regulation of several parameters associated with cancer progression such as cell migration, metastasis, angiogenesis, or cell growth. These studies have revealed that caveolin-1 and more recently cavin-1 have a dual role with either a negative or a positive effect on most of these parameters. The recent discovery that caveolae can act as mechanosensors has sparked an array of new studies that have addressed the mechanobiology of caveolae in various cellular functions. This review summarizes the current knowledge on caveolae and their role in cancer development through their activity in membrane tension buffering. We propose that the role of caveolae in cancer has to be revisited through their response to the mechanical forces encountered by cancer cells during tumor mass development.

EHD2 is a Predictive Biomarker of Chemotherapy Efficacy in Triple Negative Breast Carcinoma.

EHD2 is a mechanotransducing ATPase localized in caveolae invaginations at the plasma membrane. EHD2 has recently been associated with several human cancers, however the significance of EHD2 transcript levels in cancer prognosis remains debated. Breast cancer is the most commonly occurring cancer in women and prognosis is variable depending on the subtypes. Triple negative breast cancer (TNBC) often has a poor therapeutic response. The aim of this study was to assess the prognostic significance of EHD2 transcripts and protein expression levels in breast carcinomas. We found that low EHD2 levels were associated with enhanced proliferation, migration and invasion of TNBC cells. EHD2 expression was significantly reduced in TNBC tissues and the loss of EHD2 led to higher expression of the pro-tumoral cytokine IL-8. In apparent contradiction with in vitro data, multivariate analysis of two independent cohorts of breast cancer patients revealed that low EHD2 was in fact associated with good prognosis in the highly proliferative TNBC subtype. Accordingly, TNBC low EHD2 expressers were found to benefit the most from chemotherapy when compared to all subtypes of breast cancers. Our study validates EHD2 expression level as an independent prognostic factor of metastasis-free survival and as a new predictive marker of chemotherapy efficacy in TNBC patients.

Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2.

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.

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.