Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins.

Membrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly.

Retromer Sets a Trap for Endosomal Cargo Sorting.

Membrane trafficking from endosomes to the trans-Golgi network or the plasma membrane is driven by the retromer complex. Through structural analysis of the cargo-bound complex, Lucas et al. describe a mechanism by which endosomal membrane recruitment and cargo recognition are integrated through cooperative interactions between retromer subunits.

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.

Building endocytic pits without clathrin.

How endocytic pits are built in clathrin- and caveolin-independent endocytosis still remains poorly understood. Recent insight suggests that different forms of clathrin-independent endocytosis might involve the actin-driven focusing of membrane constituents, the lectin-glycosphingolipid-dependent construction of endocytic nanoenvironments, and Bin-Amphiphysin-Rvs (BAR) domain proteins serving as scaffolding modules. We discuss the need for different types of internalization processes in the context of diverse cellular functions, the existence of clathrin-independent mechanisms of cargo recruitment and membrane bending from a biological and physical perspective, and finally propose a generic scheme for the formation of clathrin-independent endocytic pits.

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.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Enterococcus hirae and Barnesiella intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects.

The efficacy of the anti-cancer immunomodulatory agent cyclophosphamide (CTX) relies on intestinal bacteria. How and which relevant bacterial species are involved in tumor immunosurveillance, and their mechanism of action are unclear. Here, we identified two bacterial species, Enterococcus hirae and Barnesiella intestinihominis that are involved during CTX therapy. Whereas E. hirae translocated from the small intestine to secondary lymphoid organs and increased the intratumoral CD8/Treg ratio, B. intestinihominis accumulated in the colon and promoted the infiltration of IFN-γ-producing γδT cells in cancer lesions. The immune sensor, NOD2, limited CTX-induced cancer immunosurveillance and the bioactivity of these microbes. Finally, E. hirae and B. intestinihominis specific-memory Th1 cell immune responses selectively predicted longer progression-free survival in advanced lung and ovarian cancer patients treated with chemo-immunotherapy. Altogether, E. hirae and B. intestinihominis represent valuable "oncomicrobiotics" ameliorating the efficacy of the most common alkylating immunomodulatory compound.

Induced domain formation in endocytic invagination, lipid sorting, and scission.

Lipid segregation occurs in biological membranes, but how this plays into cellular processes like endocytosis has been unclear. Here, we discuss how the active or passive induction of lipid-protein domain formation in membranes can alter membrane mechanics and thus affect processes such as the generation of curvature, the scission of buds and tubules, and lipid sorting.

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.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

The roles of dynein and myosin VI motor proteins in endocytosis.

Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.

PSL Chemical Biology Symposia: Recent Progress in Ferroptosis.

This symposium is the 5th PSL (Paris Sciences & Lettres) Chemical Biology meeting (2015, 2016, 2019, 2023, 2024) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette, 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 hosted around 150 participants and was focused on the burgeoning field of ferroptosis, its mechanism and implications in health and disease. While not initially planned, it was felt that the next large Ferroptosis venue (CSHA, China) would not happen before late 2024. A discussion involving Conrad, Birsoy, Ubellacker, Brabletz and Rodriguez next to lake Como in Italy sponsored by the DKFZ, prompted us to fill in this gap and to organize a Ferroptosis meeting in Paris beforehand.

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.

Tracing the retrograde route in protein trafficking.

Retrograde transport, in which proteins and lipids are shuttled between endosomes and biosynthetic/secretory compartments such as the Golgi apparatus, is crucial for a diverse range of cellular functions. Mechanistic studies that explore the molecular machinery involved in this retrograde trafficking route are shedding light on the functions of transport proteins and are providing fresh insights into possible new therapeutic directions.

Site-specific N-glycan profiles of α5 ß1 integrin from rat liver.

BACKGROUND INFORMATION: Like most other cell surface proteins, α5 ß1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. RESULTS: Here, we have established the first comprehensive site-specific glycan map of α5 ß1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of ß1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 ß1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 ß1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. CONCLUSION: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 ß1 integrin activity and endocytic trafficking. SIGNIFICANCE: Glycosylation of α5 ß1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 ß1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins.

Glycosphingolipid metabolic reprogramming drives neural differentiation.

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.

High-Relaxivity Molecular MRI Contrast Agent to Target Gb3-Expressing Cancer Cells.

Targeted contrast agents (CAs) can improve magnetic resonance imaging (MRI) for accurate cancer diagnosis. In this work, we used the Shiga toxin B-subunit (STxB) as a targeting agent, which binds to Gb3, a glycosphingolipid highly overexpressed on the surface of tumor cells. We developed STxB-targeted MRI probes from cyclic peptide scaffolds functionalized with six to nine monoamide DO3A[Gd(III)] chelates. The influence of structural constraints on the longitudinal relaxivity (r1) of the CAs has been studied. The cyclic peptide carrying nine monoamide DO3A[Gd(III)] exhibited a r1 per compound of 32 and 93 mM-1s-1 at 9.4 and 1.5 T, respectively. Its conjugation to the pentameric STxB protein led to a 70 kDa compound with a higher r1 of 150 and 475 mM-1 s-1 at 9.4 and 1.5 T, respectively. Specific accumulation and cellular distribution of this conjugate in Gb3-expressing cancer cells were demonstrated using immunofluorescence microscopy and quantified by an inductively coupled plasma-mass spectrometry dosage of Gd(III). Such an agent should enable the in vivo detection by MRI of tumors expressing Gb3 receptors.

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.

Glycosylation and raft endocytosis in cancer.

Changes in glycosylation on proteins or lipids are one of the hallmarks of tumorigenesis. In many cases, it is still not understood how glycan information is translated into biological function. In this review, we discuss at the example of specific cancer-related glycoproteins how their endocytic uptake into eukaryotic cells is tuned by carbohydrate modifications. For this, we not only focus on overall uptake rates, but also illustrate how different uptake processes-dependent or not on the conventional clathrin machinery-are used under given glycosylation conditions. Furthermore, we discuss the role of certain sugar-binding proteins, termed galectins, to tune glycoprotein uptake by inducing their crosslinking into lattices, or by co-clustering them with glycolipids into raft-type membrane nanodomains from which the so-called clathrin-independent carriers (CLICs) are formed for glycoprotein internalization into cells. The latter process has been termed glycolipid-lectin (GL-Lect) hypothesis, which operates in a complementary manner to the clathrin pathway and galectin lattices.