Chimie et Biologie de la Cellule

Galectins are carbohydrate-binding proteins that are involved in many physiological functions, such as inflammation, immune responses, cell migration, autophagy and signalling. They are also linked to diseases such as fibrosis, cancer and heart disease. How such a small family of only 15 members can have such widespread effects remains a conundrum. In this Cell Science at a Glance article, we summarise recent literature on the many cellular activities that have been ascribed to galectins. As shown on the accompanying poster, these include carbohydrate-independent interactions with cytosolic or nuclear targets and carbohydrate-dependent interactions with extracellular glycoconjugates. We discuss how these intra- and extracellular activities might be linked and point out the importance of unravelling molecular mechanisms of galectin function to gain a true understanding of their contributions to the physiology of the cell. We close with a short outlook on the organismal functions of galectins and a perspective on the major challenges in the field.

RNA interference (RNAi) is a fundamental cellular process for the posttranscriptional regulation of gene expression. RNAi can exogenously be modulated by small RNA oligonucleotides, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), or by antisense oligonucleotides. These small oligonucleotides provided the scientific community with powerful and versatile tools to turn off the expression of genes of interest, and hold out the promise of new therapeutic solutions against a wide range of gene-associated pathologies. However, unmodified nucleic acids are highly instable in biological systems, and their weak interaction with plasma proteins confers an unfavorable pharmacokinetics. In this review, we first provide an overview of the most efficient chemical strategies that, over the past 30 years, have been used to significantly improve the therapeutic potential of oligonucleotides. Oligonucleotides targeting and delivery technologies are then presented, including covalent conjugates between oligonucleotides and targeting ligand, and noncovalent association with lipid or polymer nanoparticles. Finally, we specifically focus on the endosomal escape step, which represents a major stumbling block for the effective use of oligonucleotides as therapeutic agents. The need for approaches to quantitatively measure endosomal escape and cytosolic arrival of biomolecules is discussed in the context of the development of efficient oligonucleotide targeting and delivery vectors.

Background Resident memory T lymphocytes (T RM ) are located in tissues and play an important role in immunosurveillance against tumors. The presence of T RM prior to treatment or their induction is associated to the response to anti-Programmed cell death protein 1 (PD-1)/Programmed death-ligand 1 (PD-L1) immunotherapy and the efficacy of cancer vaccines. Previous work by our group and others has shown that the intranasal route of vaccination allows more efficient induction of these cells in head and neck and lung mucosa, resulting in better tumor protection. The mechanisms of in vivo migration of these cells remains largely unknown, apart from the fact that they express the chemokine receptor CXCR6. Methods We used CXCR6 -deficient mice and an intranasal tumor vaccination model targeting the Human Papillomavirus (HPV) E7 protein expressed by the TC-1 lung cancer epithelial cell line. The role of CXCR6 and its ligand, CXCL16, was analyzed using multiparametric cytometric techniques and Luminex assays. Human biopsies obtained from patients with lung cancer were also included in this study. Results We showed that CXCR6 was preferentially expressed by CD8 + T RM after vaccination in mice and also on intratumoral CD8 + T RM derived from human lung cancer. We also demonstrate that vaccination of Cxcr6-deficient mice induces a defect in the lung recruitment of antigen-specific CD8 + T cells, preferentially in the T RM subsets. In addition, we found that intranasal vaccination with a cancer vaccine is less effective in these Cxcr6 -deficient mice compared with wild-type mice, and this loss of efficacy is associated with decreased recruitment of local antitumor CD8 + T RM . Interestingly, intranasal, but not intramuscular vaccination induced higher and more sustained concentrations of CXCL16, compared with other chemokines, in the bronchoalveolar lavage fluid and pulmonary parenchyma. Conclusions This work demonstrates the in vivo role of CXCR6-CXCL16 axis in the migration of CD8 + resident memory T cells in lung mucosa after vaccination, resulting in the control of tumor growth. This work reinforces and explains why the intranasal route of vaccination is the most appropriate strategy for inducing these cells in the head and neck and pulmonary mucosa, which remains a major objective to overcome resistance to anti-PD-1/PD-L1, especially in cold tumors.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

Several mechanisms allow for cargo internalization into cells within membrane-bound endocytic carriers. How these internalization processes couple to specific pathways of intracellular distribution remains poorly explored. Here, we review uptake reactions that are independent of the conventional clathrin machinery. We discuss how these link to retrograde trafficking from endosomes to the Golgi apparatus and exemplify biological situations in which the polarized secretion capacity of the Golgi apparatus allows for retrograde cargoes to be delivered to specialized areas of the plasma membrane, such as the leading edge of migratory cells or the immunological synapse of immune cells. We also address the evidence that allows to position apicobasal polarity of epithelial cells in this context. The underlying theme is thereby the functional coupling between specific types of endocytosis to intracellular retrograde trafficking for protein cargoes that need to be localized in a highly polarized and dynamic manner to plasmalemmal subdomains.

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

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.

Endocytosis is an essential process where proteins and lipids are internalised from the plasma membrane in membrane-bound carriers, such as clathrin-coated vesicles. Once internalised into the cell these vesicles fuse with the endocytic network where their contents are sorted towards degradation in the lysosome or recycling to their origin. Initially, it was thought that cargo recycling is a passive process, but in recent years the identification and characterisation of specialised recycling complexes has established a hitherto unthought-of level of complexity that actively opposes degradation. This review will summarise recent developments regarding the composition and regulation of the recycling machineries and their relationship with the degradative pathways of the endosome.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasing 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 have 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.

integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4+ and CD8+ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8+ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer.

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.

Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis. Types of glycans on glycoproteins and glycolipids are reviewed from the angle of their interaction with glycan-binding proteins that are at the heart of galectin lattices and GL-Lect-driven endocytosis. Examples are given to show how these mechanisms affect cellular functions ranging from cell migration and signaling to vascularization and immune modulation. Finally, outstanding challenges on the link between glycosylation and endocytosis are discussed.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

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.

Photoactivatable fluorescent proteins (PA-FPs) have been widely used to assess the dynamics of cell biological processes. In addition, PA-FPs enabled single-molecule based super-resolution imaging (photoactivated localization microscopy) and thereby provided unprecedented structural insight. For the lack of tools, however, the fraction of PA-FPs that is, actually being switched on to fluoresce, that is, the photoactivation efficiency, has been difficult to assess. Uncertainty about photoactivation efficiency has hampered an understanding of the absolute amount of PA-FPs, that is, being examined. Here, we present internal rulers to assess photoactivation efficiencies of photoactivatable proteins. These internal rulers comprise a PA-FP that is genetically directly coupled to a spectrally distinct always-on fluorescent protein. Thus, these fluorescent proteins will be expressed in the bacterial and mammalian cell in a one-to-one ratio. With these tools, we describe photoactivation efficiencies of PA-GFP and PA-Cherry in intensity-based ratiometric ensemble studies and on the single-molecule level. In ratiometric ensemble studies, we show that photoactivation efficiency depends on how the PA-FPs are exposed to 405 nm light. Using a laser-scanning microscope, hundreds of iterative low-level exposures are up to four times more efficient than a short high-power exposure. Using wide-field illumination, photoactivation was similarly efficient and instantaneous. These findings suggest that the repetitive or stochastic exposure to photons of 405 nm light results in more efficient photoactivation than a continuous flow of photons. Because of the differential photoactivation efficiency, it is crucial to assess photoactivation efficiency for any given experimental set-up. The tools we provide can be applied to any genetically encoded photoactivatable protein. Determination of photoactivation efficiency is essential for an understanding of absolute molecule numbers in ensemble studies and, most importantly, quantitative superresolution imaging. © 2017 International Society for Advancement of Cytometry.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

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.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.