Publications by authors named "Ludger Johannes"

156 Publications

Ceramide structure dictates glycosphingolipid nanodomain assembly and function.

Nat Commun 2021 06 16;12(1):3675. Epub 2021 Jun 16.

Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology unit, Paris, Cedex, France.

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.
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http://dx.doi.org/10.1038/s41467-021-23961-9DOI Listing
June 2021

The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery-The GL-Lect Hypothesis.

Authors:
Ludger Johannes

Molecules 2021 May 31;26(11). Epub 2021 May 31.

Cellular and Chemical Biology Department, Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm, CEDEX 05, 75248 Paris, France.

Lipid membranes are common to all forms of life. While being stable barriers that delimitate the cell as the fundamental organismal unit, biological membranes are highly dynamic by allowing for lateral diffusion, transbilayer passage via selective channels, and in eukaryotic cells for endocytic uptake through the formation of membrane bound vesicular or tubular carriers. Two of the most abundant fundamental fabrics of membranes-lipids and complex sugars-are produced through elaborate chains of biosynthetic enzymes, which makes it difficult to study them by conventional reverse genetics. This review illustrates how organic synthesis provides access to uncharted areas of membrane glycobiology research and its application to biomedicine. For this Special Issue on Chemical Biology Research in France, focus will be placed on synthetic approaches (i) to study endocytic functions of glycosylated proteins and lipids according to the GlycoLipid-Lectin (GL-Lect) hypothesis, notably that of Shiga toxin; (ii) to mechanistically dissect its endocytosis and intracellular trafficking with small molecule; and (iii) to devise intracellular delivery strategies for immunotherapy and tumor targeting. It will be pointed out how the chemical biologist's view on lipids, sugars, and proteins synergizes with biophysics and modeling to "look" into the membrane for atomistic scale insights on molecular rearrangements that drive the biogenesis of endocytic carriers in processes of clathrin-independent endocytosis.
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http://dx.doi.org/10.3390/molecules26113299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198588PMC
May 2021

Absolute Quantification of Drug Vector Delivery to the Cytosol.

Angew Chem Int Ed Engl 2021 Jun 28;60(27):14824-14830. Epub 2021 May 28.

Cellular and Chemical Biology Unit, U1143 INSERM, UMR3666 CNRS, Institut Curie-, Université PSL, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP-tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B-subunit of Shiga toxin and the cell-penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.
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http://dx.doi.org/10.1002/anie.202102332DOI Listing
June 2021

CXCR6 deficiency impairs cancer vaccine efficacy and CD8 resident memory T-cell recruitment in head and neck and lung tumors.

J Immunother Cancer 2021 Mar;9(3)

Université de Paris, PARCC, INSERM U970, 75006 Paris, France

Background: Resident memory T lymphocytes (T) are located in tissues and play an important role in immunosurveillance against tumors. The presence of T 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 -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 after vaccination in mice and also on intratumoral CD8 T 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 subsets. In addition, we found that intranasal vaccination with a cancer vaccine is less effective in these -deficient mice compared with wild-type mice, and this loss of efficacy is associated with decreased recruitment of local antitumor CD8 T. 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.
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http://dx.doi.org/10.1136/jitc-2020-001948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949477PMC
March 2021

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism.

Cells 2021 Feb 9;10(2). Epub 2021 Feb 9.

Institut Curie, Université PSL, U932 INSERM, Integrative Analysis of T Cell Activation Team, 26 Rue d'Ulm, 75248 Paris CEDEX 05, France.

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.
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http://dx.doi.org/10.3390/cells10020359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916135PMC
February 2021

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

Commun Biol 2021 Feb 9;4(1):173. Epub 2021 Feb 9.

Institut Curie, Université PSL, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, Endocytic Trafficking and Intracellular Delivery Team, Paris, France.

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.
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http://dx.doi.org/10.1038/s42003-021-01693-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873212PMC
February 2021

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse.

J Immunother Cancer 2020 11;8(2)

Université de Paris, Institut Cochin, INSERM, CNRS, F-75014, Paris, France

Background: Tumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase.

Methods: We designed a mouse model of tumor relapse in which mice transplanted with E7 TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases.

Results: We show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival.

Conclusions: Together, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.
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http://dx.doi.org/10.1136/jitc-2020-000996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689071PMC
November 2020

Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins.

Methods Mol Biol 2021 ;2233:53-70

Institut Curie, PSL Research University, Cellular and Chemical Biology unit, Endocytic Trafficking and Intracellular Delivery team, U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm, Paris Cedex 05, France.

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5β1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.
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http://dx.doi.org/10.1007/978-1-0716-1044-2_4DOI Listing
March 2021

Self-assembled, Programmable DNA Nanodevices for Biological and Biomedical Applications.

Chembiochem 2021 Mar 15;22(5):763-778. Epub 2021 Jan 15.

Cellular and Chemical Biology Unit, Endocytic Trafficking and Intracellular Delivery Team U1143 INSERM UMR 3666 CNRS, Institut Curie, PSL Research University, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

The broad field of structural DNA nanotechnology has diverged into various areas of applications ranging from computing, photonics, synthetic biology, and biosensing to in-vivo bioimaging and therapeutic delivery, to name but a few. Though the field began to exploit DNA to build various nanoscale architectures, it has now taken a new path to diverge from structural DNA nanotechnology to functional or applied DNA nanotechnology. More recently a third sub-branch has emerged-biologically oriented DNA nanotechnology, which seeks to explore the functionalities of combinatorial DNA devices in various biological systems. In this review, we summarize the key developments in DNA nanotechnology revealing a current trend that merges the functionality of DNA devices with the specificity of biomolecules to access a range of functions in biological systems. This review seeks to provide a perspective on the evolution and biological applications of DNA nanotechnology, where the integration of DNA structures with biomolecules can now uncover phenomena of interest to biologists and biomedical scientists. Finally, we conclude with the challenges, limitations, and perspectives of DNA nanodevices in fundamental and applied research.
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http://dx.doi.org/10.1002/cbic.202000372DOI Listing
March 2021

Clathrin-independent endocytosis, retrograde trafficking, and cell polarity.

Curr Opin Cell Biol 2020 08 17;65:112-121. Epub 2020 Jul 17.

Institut Curie, PSL Research University, Cellular and Chemical Biology Unit, INSERM U1143, CNRS UMR3666, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ceb.2020.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588825PMC
August 2020

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Toxins (Basel) 2020 07 10;12(7). Epub 2020 Jul 10.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden.

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.
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http://dx.doi.org/10.3390/toxins12070449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404996PMC
July 2020

Glycosylation and raft endocytosis in cancer.

Cancer Metastasis Rev 2020 06;39(2):375-396

Cellular and Chemical Biology Unit, INSERM U1143, CNRS UMR3666, Institut Curie, PSL Research University, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

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.
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http://dx.doi.org/10.1007/s10555-020-09880-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311491PMC
June 2020

MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue.

Anal Chem 2020 05 4;92(10):7096-7105. Epub 2020 May 4.

Institute of Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany.

The main cellular receptors of Shiga toxins (Stxs), the neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer/CD77) and globotetraosylceramide (Gb4Cer), are significantly upregulated in about half of the human colorectal carcinomas (CRC) and in other cancers. Therefore, conjugates exploiting the Gb3Cer/Gb4Cer-binding B subunit of Stx (StxB) have attracted great interest for both diagnostic and adjuvant therapeutic interventions. Moreover, fucosylated GSLs were recognized as potential tumor-associated targets. One obstacle to a broader use of these receptor/ligand systems is that the contribution of specific GSLs to tumorigenesis, in particular, in the context of an altered lipid metabolism, is only poorly understood. A second is that also nondiseased organs (e.g., kidney) and blood vessels can express high levels of certain GSLs, not least Gb3Cer/Gb4Cer. Here, we used, in a proof-of-concept study, matrix-assisted laser desorption/ionization mass spectrometry imaging combined with laser-induced postionization (MALDI-2-MSI) to simultaneously visualize the distribution of several Gb3Cer/Gb4Cer lipoforms and those of related GSLs (e.g., Gb3Cer/Gb4Cer precursors and fucosylated GSLs) in tissue biopsies from three CRC patients. Using MALDI-2 and StxB-based immunofluorescence microscopy, Gb3Cer and Gb4Cer were mainly found in dedifferentiated tumor cell areas, tumor stroma, and tumor-infiltrating blood vessels. Notably, fucosylated GSL such as Fuc-(n)Lc4Cer generally showed a highly localized expression in dysplastic glands and indian file-like cells infiltrating adipose tissue. Our "molecular histology" approach could support stratifying patients for intratumoral GSL expression to identify an optimal therapeutic strategy. The improved chemical coverage by MALDI-2 can also help to improve our understanding of the molecular basis of tumor development and GSL metabolism.
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http://dx.doi.org/10.1021/acs.analchem.0c00480DOI Listing
May 2020

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

Nat Commun 2020 03 19;11(1):1457. Epub 2020 Mar 19.

UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Croix du Sud 4-5, B-1348, Louvain-la-Neuve, Belgium.

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.
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http://dx.doi.org/10.1038/s41467-020-15303-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081352PMC
March 2020

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

Nat Chem Biol 2020 03 17;16(3):327-336. Epub 2020 Feb 17.

Institut Curie, PSL Research University, Cellular and Chemical Biology unit, U1143 INSERM, UMR3666 CNRS, Endocytic Trafficking and Intracellular Delivery team, Paris, France.

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.
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http://dx.doi.org/10.1038/s41589-020-0474-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039708PMC
March 2020

Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling.

Prog Lipid Res 2019 10 1;76:101010. Epub 2019 Nov 1.

EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney 2052, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney 2052, Australia. Electronic address:

Galectin-3 (Gal3) is a multifaceted protein which belongs to a family of lectins and binds β-galactosides. Gal3 expression is altered in many types of cancer, with increased expression generally associated with poor prognosis. Although the mechanisms remain unknown, Gal3 has been implicated in several biological processes involved in cancer progression, including suppression of T cell-mediated immune responses. Extracellular Gal3 binding to the plasma membrane of T cells alters membrane organization and the formation of an immunological synapse. Its multivalent capacity allows Gal3 to interact specifically with different membrane proteins and lipids, influencing endocytosis, trafficking and T cell receptor signalling. The ability of Gal3 to inhibit T cell responses may provide a mechanism by which Gal3 aids in cancer progression. In this review, we seek to give an overview of the mechanisms by which Gal3 alters the spatial organization of cell membranes and how these processes impact on T cell activation.
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http://dx.doi.org/10.1016/j.plipres.2019.101010DOI Listing
October 2019

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Sci Rep 2019 10 7;9(1):14362. Epub 2019 Oct 7.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1038/s41598-019-50692-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779916PMC
October 2019

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Nano Lett 2019 10 25;19(10):7365-7369. Epub 2019 Sep 25.

Cellular and Chemical Biology Unit , Institut Curie, PSL Research University , U1143 INSERM, UMR3666 CNRS , 26 rue d'Ulm , 75248 Paris Cedex 05, France.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.
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http://dx.doi.org/10.1021/acs.nanolett.9b03001DOI Listing
October 2019

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

Kidney Int 2019 08 28;96(2):327-341. Epub 2019 Feb 28.

Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany; Institute of Pharmacology, University of Marburg, Marburg, Germany. Electronic address:

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.
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http://dx.doi.org/10.1016/j.kint.2019.02.010DOI Listing
August 2019

Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells.

Nat Commun 2019 04 29;10(1):1974. Epub 2019 Apr 29.

Membrane Dynamics and Mechanics of Intracellular Signaling Laboratory, Institut Curie - Centre de Recherche, PSL Research University, CNRS UMR3666, INSERM U1143, Paris, 75248, France.

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.
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http://dx.doi.org/10.1038/s41467-019-09405-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488599PMC
April 2019

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chembiochem 2019 04 25;20(7):968-973. Epub 2019 Feb 25.

PSL Université Paris, Institut Curie, CNRS UMR3666, INSERM U1143, 75005, Paris, France.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.
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http://dx.doi.org/10.1002/cbic.201900092DOI Listing
April 2019

The 2018 biomembrane curvature and remodeling roadmap.

J Phys D Appl Phys 2018 Aug 20;51(34). Epub 2018 Jul 20.

Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo- and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.
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http://dx.doi.org/10.1088/1361-6463/aacb98DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333427PMC
August 2018

Retro Styles for Vesicle Coats.

Biochemistry 2019 02 17;58(6):433-434. Epub 2019 Jan 17.

Cellular and Chemical Biology Unit , Institut Curie, PSL Research University , U1143 INSERM, UMR3666 CNRS, 26 rue d'Ulm , Paris 75248 Cedex 05 , France.

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http://dx.doi.org/10.1021/acs.biochem.8b01271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372322PMC
February 2019

EHD2 is a mechanotransducer connecting caveolae dynamics with gene transcription.

J Cell Biol 2018 12 22;217(12):4092-4105. Epub 2018 Oct 22.

Membrane Dynamics and Mechanics of Intracellular Signaling Laboratory, Centre de Recherche, Institut Curie, PSL Research University, Paris, France

Caveolae are small invaginated pits that function as dynamic mechanosensors to buffer tension variations at the plasma membrane. Here we show that under mechanical stress, the EHD2 ATPase is rapidly released from caveolae, SUMOylated, and translocated to the nucleus, where it regulates the transcription of several genes including those coding for caveolae constituents. We also found that EHD2 is required to maintain the caveolae reservoir at the plasma membrane during the variations of membrane tension induced by mechanical stress. Metal-replica electron microscopy of breast cancer cells lacking EHD2 revealed a complete absence of caveolae and a lack of gene regulation under mechanical stress. Expressing EHD2 was sufficient to restore both functions in these cells. Our findings therefore define EHD2 as a central player in mechanotransduction connecting the disassembly of the caveolae reservoir with the regulation of gene transcription under mechanical stress.
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http://dx.doi.org/10.1083/jcb.201801122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279385PMC
December 2018

Rapalog combined with CCR4 antagonist improves anticancer vaccines efficacy.

Int J Cancer 2018 12 1;143(11):3008-3018. Epub 2018 Oct 1.

Université Bourgogne Franche-Comté, INSERM, EFS BFC, UMR1098, Interactions Hôte-Greffon-Tumeur/Ingénierie Cellulaire et Génique, Besançon, France.

mTOR pathway inhibitors such as rapalogs represent a promising tool to induce functional memory CD8 T cells. In our study, we investigated the combination of temsirolimus with anticancer vaccines. Using various designs of cancer vaccines (short and long peptides or the B subunit of Shiga toxin as an antigen delivery vector) and tumor models (melanoma, lung and colon cancer), we showed that the administration of temsirolimus efficiently decreased tumor growth and enhanced tumor-specific CD8 T-cell responses induced by vaccination. Furthermore, tumor-specific CD8 T cells induced by the bi-therapy (vaccine + temsirolimus) exhibit phenotypic characteristics of central memory (CD127 CD62L ) CD8 T cells compared to vaccination alone. We demonstrated that regulatory CD4 T cells (T ) expansion in vivo limits the efficacy of the bi-therapy by altering the antitumor CD8 T-cell responses. Finally, the use of a small molecule CCR4 antagonist to prevent T induction considerably improved the efficacy of the bi-therapy by enhancing CD8 T cells-mediated antitumor immunity. Taken together, our study highlights the potential interest of combining cancer vaccines with drugs that promote memory CD8 T cells and inhibit T .
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http://dx.doi.org/10.1002/ijc.31842DOI Listing
December 2018

Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs.

Nucleic Acid Ther 2018 06;28(3):178-193

Institut Curie, PSL Research University , Cellular and Chemical Biology, U1143 INSERM, UMR3666 CNRS, Paris, France .

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.
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http://dx.doi.org/10.1089/nat.2017.0716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6000866PMC
June 2018

Galectins at a glance.

J Cell Sci 2018 05 1;131(9). Epub 2018 May 1.

Sect. MIG (Microbiology, Immunology, Glycobiology), Dept Laboratory Medicine, Lund University, POB 117, 22100 Lund, Sweden

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.
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http://dx.doi.org/10.1242/jcs.208884DOI Listing
May 2018

MicroRNA 199a-5p Attenuates Retrograde Transport and Protects against Toxin-Induced Inhibition of Protein Biosynthesis.

Mol Cell Biol 2018 06 15;38(11). Epub 2018 May 15.

Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, Connecticut, USA

Retrograde transport (RT) allows cells to retrieve receptors and other cellular cargoes for delivery to the Golgi apparatus, contributing to the maintenance of cellular homeostasis. This transport route is also commonly used by several bacterial toxins to exert their deleterious actions on eukaryotic cells. While the retrograde transport process has been well characterized, the contribution of microRNAs (miRNAs) in regulating this cellular transport mechanism remains unknown. Here, we determined that and , members of the intronic miRNA family, coordinate genes regulating RT and endosome trafficking. We demonstrate that miR-199a-5p attenuates the expression of Vps26A, Rab9B, and M6PR, thereby controlling RT from endosomes to the -Golgi network (TGN). Importantly, we found that overexpression of a Vps26A construct resistant to the inhibitory action of miR-199a-5p abrogates the effect of miR-199a-5p on RT. Finally, we demonstrate that miR-199-5p overexpression attenuates Shiga toxin type 1 (Stx1)-mediated inhibition of protein biosynthesis. In summary, our work identifies the first noncoding RNA that influences RT and reduces the inhibition of protein biosynthesis caused by bacterial toxins.
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http://dx.doi.org/10.1128/MCB.00548-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5954181PMC
June 2018

Clustering on Membranes: Fluctuations and More.

Trends Cell Biol 2018 05 1;28(5):405-415. Epub 2018 Mar 1.

Blue Brain Project, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. Electronic address:

Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions.
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http://dx.doi.org/10.1016/j.tcb.2018.01.009DOI Listing
May 2018

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

J Exp Med 2018 04 12;215(4):1245-1265. Epub 2018 Feb 12.

Crosstalk between T Cells and Dendritic Cells Group, Institut Curie, Paris Sciences and Lettres Research University, INSERM U932, Paris, France

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble -ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4 T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.
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http://dx.doi.org/10.1084/jem.20162042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881459PMC
April 2018