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.

Cells respond to mechanical stress by rapid disassembly of caveolae.

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.

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.

Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis.

Nascent transport intermediates detach from donor membranes by scission. This process can take place in the absence of dynamin, notably in clathrin-independent endocytosis, by mechanisms that are yet poorly defined. We show here that in cells scission of Shiga toxin-induced tubular endocytic membrane invaginations is preceded by cholesterol-dependent membrane reorganization and correlates with the formation of membrane domains on model membranes, suggesting that domain boundary forces are driving tubule membrane constriction. Actin triggers scission by inducing such membrane reorganization process. Tubule occurrence is indeed increased upon cellular depletion of the actin nucleator component Arp2, and the formation of a cortical actin shell in liposomes is sufficient to trigger the scission of Shiga toxin-induced tubules in a cholesterol-dependent but dynamin-independent manner. Our study suggests that membranes in tubular Shiga toxin-induced invaginations are poised to undergo actin-triggered reorganization leading to scission by a physical mechanism that may function independently from or in synergy with pinchase activity.

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.

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.

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.

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.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis.

During endocytosis, energy is invested to narrow the necks of cargo-containing plasma membrane invaginations to radii at which the opposing segments spontaneously coalesce, thereby leading to the detachment by scission of endocytic uptake carriers. In the clathrin pathway, dynamin uses mechanical energy from GTP hydrolysis to this effect, assisted by the BIN/amphiphysin/Rvs (BAR) domain-containing protein endophilin. Clathrin-independent endocytic events are often less reliant on dynamin, and whether in these cases BAR domain proteins such as endophilin contribute to scission has remained unexplored. Here we show, in human and other mammalian cell lines, that endophilin-A2 (endoA2) specifically and functionally associates with very early uptake structures that are induced by the bacterial Shiga and cholera toxins, which are both clathrin-independent endocytic cargoes. In controlled in vitro systems, endoA2 reshapes membranes before scission. Furthermore, we demonstrate that endoA2, dynamin and actin contribute in parallel to the scission of Shiga-toxin-induced tubules. Our results establish a novel function of endoA2 in clathrin-independent endocytosis. They document that distinct scission factors operate in an additive manner, and predict that specificity within a given uptake process arises from defined combinations of universal modules. Our findings highlight a previously unnoticed link between membrane scaffolding by endoA2 and pulling-force-driven dynamic scission.

A promotive effect for halofuginone on membrane repair and synaptotagmin-7 levels in muscle cells of dysferlin-null mice.

In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.

Meeting after meeting: 20 years of discoveries by the members of the Exocytosis-Endocytosis Club.

Twenty years ago, a group of French cell biologists merged two scientific clubs with the aim of bringing together researchers in the fields of Endocytosis and Exocytosis. Founded in 1997, the first annual meeting of the Exocytosis Club was held in 1998. The Endocytosis Club held quarterly meetings from its founding in 1999. The first joint annual meeting of the Exocytosis-Endocytosis Club took place in Paris in April, 2001. What started as a modest gathering of enthusiastic scientists working in the field of cell trafficking has gone from strength to strength, rapidly becoming an unmissable yearly meeting, vividly demonstrating the high quality of science performed in our community and beyond. On the occasion of the 20th meeting of our club, we want to provide historic insight into the fields of exocytosis and endocytosis, and by extension, to subcellular trafficking, highlighting how French scientists have contributed to major advances in these fields. Today, the Exocytosis-Endocytosis Club represents a vibrant and friendly community that will hold its 20th meeting at the Presqu'Ile de Giens, near Toulon in the South of France, on May 11-13, 2017.

Shiga toxin stimulates clathrin-independent endocytosis of the VAMP2, VAMP3 and VAMP8 SNARE proteins.

Endocytosis is an essential cellular process that is often hijacked by pathogens and pathogenic products. Endocytic processes can be classified into two broad categories, those that are dependent on clathrin and those that are not. The SNARE proteins VAMP2, VAMP3 and VAMP8 are internalized in a clathrin-dependent manner. However, the full scope of their endocytic behavior has not yet been elucidated. Here, we found that VAMP2, VAMP3 and VAMP8 are localized on plasma membrane invaginations and very early uptake structures that are induced by the bacterial Shiga toxin, which enters cells by clathrin-independent endocytosis. We show that toxin trafficking into cells and cell intoxication rely on these SNARE proteins. Of note, the cellular uptake of VAMP3 is increased in the presence of Shiga toxin, even when clathrin-dependent endocytosis is blocked. We therefore conclude that VAMP2, VAMP3 and VAMP8 are removed from the plasma membrane by non-clathrin-mediated pathways, in addition to by clathrin-dependent uptake. Moreover, our study identifies these SNARE proteins as the first transmembrane trafficking factors that functionally associate at the plasma membrane with the toxin-driven clathrin-independent invaginations during the uptake process.

Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.

Antigen-presenting cells have the remarkable capacity to transfer exogenous antigens to the cytosol for processing by proteasomes and subsequent presentation on major histocompatibility complex class-I (MHC-I) molecules, a process termed cross-presentation. This is the target of biomedical approaches that aim to trigger a therapeutic immune response. The receptor-binding B-subunit of Shiga toxin (STxB) has been developed as an antigen delivery tool for such immunotherapy applications. In this study, we have analyzed pathways and trafficking factors that are involved in this process. A covalent conjugate between STxB and saporin was generated to quantitatively sample the membrane translocation step to the cytosol in differentiated monocyte-derived THP-1 cells. We have found that retrograde trafficking to the Golgi complex was not required for STxB-saporin translocation to the cytosol or for STxB-dependent antigen cross-presentation. Depletion of endosomal Rab7 inhibited, and lowering membrane cholesterol levels favored STxB-saporin translocation. Interestingly, experiments with reducible and non-reducible linker-arm-STxB conjugates led to the conclusion that after translocation, STxB remains associated with the cytosolic membrane leaflet. In summary, we report new facets of the endosomal escape process bearing relevance to antigen cross-presentation.

Rab7 is functionally required for selective cargo sorting at the early endosome.

The small GTPases of the Rab family act as a molecular switch regulating various aspects of membrane trafficking through the selective recruitment of effector proteins. Whereas Rab7 has been classically involved in the regulation of transport within the endolysosomal network, persistent controversy remains as to whether Rab7 also plays a role in earlier steps of endosomal trafficking. In this study, we show that Rab7 depletion or inactivation results in enlargement of both early and late endosomes. Rab7 depletion led to the retention of a significant fraction of internalized low-density lipoproteins (LDL) mainly in enlarged early endosomes (EE). As a result, LDL processing and the transcriptional regulation of sterol-sensitive genes were impaired. We found that Rab7 activity was also required for the sorting of the mannose-6-phosphate receptor, the interferon alpha-receptor and the Shiga toxin B-subunit. In contrast, epidermal growth factor (EGF) sorting at the EE or the recycling of transferrin and LDL-R were not affected by Rab7 depletion. Our findings demonstrate that in addition to regulating late endosomes (LE) to lysosomes transport, Rab7 plays a functional role in the selective sorting of distinct cargos at the EE and that the Rab5 to Rab7 exchange occurs early in the endosomal maturation process.

Rab12 localizes to Shiga toxin-induced plasma membrane invaginations and controls toxin transport.

Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin-independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin-induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP-Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor-binding B-subunit of Shiga toxin reaching the trans-Golgi/TGN membranes was decreased in Rab12-depleted cells, and that cells were partially protected against intoxication by Shiga-like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady-state localizations of TGN46 and cation-independent mannose-6-phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

SNAP-tag based proteomics approach for the study of the retrograde route.

Proteomics is a powerful technique for protein identification at large scales. A number of proteomics approaches have been developed to study the steady state composition of intracellular compartments. Here, we report a novel vectorial proteomics strategy to identify plasma membrane proteins that undergo retrograde transport to the trans-Golgi network (TGN). This strategy is based on the covalent modification of the plasma membrane proteome with a membrane impermeable benzylguanine derivative. Benzylguanine-tagged plasma membrane proteins that are subsequently targeted to the retrograde route are covalently captured by a TGN-localized SNAP-tagged fusion protein, which allows for their identification. The approach was validated step-by-step using a well explored retrograde cargo protein, the B-subunit of Shiga toxin. It was then extended to the proteomics format. Among other hits we found one of the historically first identified cargo proteins that undergo retrograde transport, which further validated our approach. Most of the other hits were kinases, receptors or transporters. In conclusion, we have pioneered a vectorial proteomics approach that complements traditional methods for the study of retrograde protein trafficking. This approach is of generic nature and could in principle be extended to other endocytic pathways.

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.