Trafic endocytique et ciblage intracellulaire

Publications de l’équipe

Année de publication : 2020

Forrester A, Rathjen SJ, Garcia Castillo MD, Bachert C, Couhert A, Tepshi L, Pichard S, Martinez J, Renard H-F, Valades Cruz CA, Dingli F, Loew D, Lamaze C, Cintrat JC, Linstedt AD, Gillet D, Barbier J, Johannes L (2020 May 29)

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

Nature Chemical BiologyNature : 16 : 327,336 : DOI : 10.1038/s41589-020-0474-4 En savoir plus
Résumé

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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Année de publication : 2019

Erik B. Watkins, Jaroslaw Majewski, Eva Y. Chi, Haifei Gao, Jean-Claude Florent, Ludger Johannes (2019 Sep 20)

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

Nano letters : 19 : DOI : 10.1021/acs.nanolett.9b03001 En savoir plus
Résumé

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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François Legoux, Déborah Bellet, Celine Daviaud, Yara El Morr, Aurelie Darbois, Kristina Niort, Emanuele Procopio, Marion Salou, Jules Gilet, Bernhard Ryffel, Aurélie Balvay, Anne Foussier, Manal Sarkis, Ahmed El Marjou, Frederic Schmidt, Sylvie Rabot, Olivier Lantz (2019 Aug 31)

Microbial metabolites control the thymic development of mucosal-associated invariant T cells.

Science (New York, N.Y.) : DOI : eaaw2719 En savoir plus
Résumé

How the microbiota modulate immune functions remains poorly understood. Mucosal-associated invariant T (MAIT) cells are implicated in mucosal homeostasis and absent in germ-free mice. Here, we show that commensal bacteria govern murine MAIT intrathymic development, as MAIT cells did not recirculate to the thymus. MAIT development required expression in bacteria, indicating that production of the MAIT antigen 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) was necessary. 5-OP-RU rapidly traveled from mucosal surfaces to the thymus, where it was captured by the major histocompatibility complex class Ib molecule MR1. This led to increased numbers of the earliest MAIT precursors and the expansion of more mature receptor-related orphan receptor γt-positive MAIT cells. Thus, a microbiota-derived metabolite controls development of mucosally targeted T cells, in a process blurring the distinction between exogenous and self-antigens.

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Année de publication : 2018

Patricia Bassereau, Rui Jin, Tobias Baumgart, Markus Deserno, Rumiana Dimova, Vadim A. Frolov, Pavel V. Bashkirov, Helmut Grubmüller, Reinhard Jahn, H. Jelger Risselada, Ludger Johannes, Michael M. Kozlov, Reinhard Lipowsky, Thomas J. Pucadyil, Wade F. Zeno, Jeanne C. Stachowiak, Dimitrios Stamou, Artù Breuer, Line Lauritsen, Camille Simon, Cécile Sykes, Gregory A. Voth, Thomas R Weikl (2018 Jul 20)

The 2018 biomembrane curvature and remodeling roadmap.

Journal of Physics D: Applied Physics : 51 : 343001 : DOI : 10.1088/1361-6463/aacb98 En savoir plus
Résumé

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 Journal of Physics D: Applied Physics 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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Silvia Serra, Ahmed Alouane, Thomas Le Saux, Steve Huvelle, Raphaël Plasson, Frédéric Schmidt, Ludovic Jullien, Raphaël Labruère (2018 Jun 7)

A chemically encoded timer for dual molecular delivery at tailored ranges and concentrations.

Chemical communications (Cambridge, England) : 6396-6399 : DOI : 10.1039/c8cc03253j En savoir plus
Résumé

Spatiotemporal control of molecular distribution is much in demand in many fields of chemistry. To address this goal, we exploit a low molecular weight branched self-immolative architecture, which acts as a triggerable chemically encoded timer for autonomous sequential release of two chemicals. Using a light-activated model liberating two distinct fluorophores, we generated a tunable spatially contrasted molecular distribution.

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Johannes Ludger, Pezeshkian W, Ipsen JH, Shillcock J (2018 May 7)

Clustering on Membranes: Fluctuations and More

Trends Cell BiolTrends Cell Biol : 28 : 405 415 : DOI : 10.1016/j.tcb.2018.01.009 En savoir plus
Résumé

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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Année de publication : 2017

Steve Huvelle, Ahmed Alouane, Thomas Le Saux, Ludovic Jullien, Frédéric Schmidt (2017 Mar 31)

Syntheses and kinetic studies of cyclisation-based self-immolative spacers.

Organic & biomolecular chemistry : 3435-3443 : DOI : 10.1039/c7ob00121e En savoir plus
Résumé

Kinetic analysis of the disassembly of self-immolative spacers based on cyclisation processes was performed. Five compounds were synthesized belonging to two different series, and their kinetic constants were determined. Electron-donating substituents gave a slight acceleration but the main effect was steric, and the Thorpe-Ingold effect was indeed particularly effective. Comparison with the self-immolative spacers based on elimination processes showed that cyclisations gave comparable or lower rate, but the corresponding spacers are more difficult to modulate.

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Weria Pezeshkian, Haifei Gao, Senthil Arumugam, Ulrike Becken, Patricia Bassereau, Jean-Claude Florent, John Hjort Ipsen, Ludger Johannes, Julian C Shillcock (2017 Jan 24)

Mechanism of Shiga Toxin Clustering on Membranes.

ACS Nano : 11 : 314-324 : DOI : 10.1021/acsnano.6b05706 En savoir plus
Résumé

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.

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Année de publication : 2016

Ludger Johannes, Christian Wunder, Massiullah Shafaq-Zadah (2016 Dec 17)

Glycolipids and Lectins in Endocytic Uptake Processes.

Journal of molecular biology : DOI : S0022-2836(16)30453-3 En savoir plus
Résumé

A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns intra-membrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution of extracellular determinants to the construction of endocytic pits has received much less attention, depite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of membrane domains, termed rafts, whose contributions to predominantly clathrin-independent internalization processes is well recognized. Furthermore, sugar modifications on extracellular domains of proteins, and sugar-binding proteins, termed lectins, have also been linked to the uptake of endocytic cargoes at the plasma membrane. In this review, we first summarize these contributions by extracellular determinants to the endocytic process. We thus propose a molecular hypothesis – termed the GL-Lect hypothesis – on how GlycoLipids and Lectins drive the formation of compositional nanoenvrionments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, we position this hypothesis within the global context of endocytic pathway proposals that have emerged in recent years.

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Daniela Chmiest, Nanaocha Sharma, Natacha Zanin, Christine Viaris de Lesegno, Massiullah Shafaq-Zadah, Vonick Sibut, Florent Dingli, Philippe Hupé, Stephan Wilmes, Jacob Piehler, Damarys Loew, Ludger Johannes, Gideon Schreiber, Christophe Lamaze (2016 Dec 6)

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex.

Nature communications : 13476 : DOI : 10.1038/ncomms13476 En savoir plus
Résumé

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.

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Ludger Johannes, Christian Wunder (2016 Dec 3)

Retromer Sets a Trap for Endosomal Cargo Sorting.

Cell : 1452-1454 : DOI : S0092-8674(16)31598-7 En savoir plus
Résumé

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.

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Romain Daillère, Marie Vétizou, Nadine Waldschmitt, Takahiro Yamazaki, Christophe Isnard, Vichnou Poirier-Colame, Connie P M Duong, Caroline Flament, Patricia Lepage, Maria Paula Roberti, Bertrand Routy, Nicolas Jacquelot, Lionel Apetoh, Sonia Becharef, Sylvie Rusakiewicz, Philippe Langella, Harry Sokol, Guido Kroemer, David Enot, Antoine Roux, Alexander Eggermont, Eric Tartour, Ludger Johannes, Paul-Louis Woerther, Elisabeth Chachaty, Jean-Charles Soria, Encouse Golden, Silvia Formenti, Magdalena Plebanski, Mutsa Madondo, Philip Rosenstiel, Didier Raoult, Vincent Cattoir, Ivo Gomperts Boneca, Mathias Chamaillard, Laurence Zitvogel (2016 Oct 9)

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

Immunity : DOI : S1074-7613(16)30378-8 En savoir plus
Résumé

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.

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N Gupta, R Noël, A Goudet, K Hinsinger, A Michau, V Pons, H Abdelkafi, T Secher, A Shima, O Shtanko, Y Sakurai, S Cojean, S Pomel, V Liévin-Le Moal, V Leignel, J-A Herweg, A Fischer, L Johannes, Kate Harrison, Philippa M Beard, P Clayette, R Le Grand, J O Rayner, T Rudel, J Vacus, P M Loiseau, R A Davey, E Oswald, J-C Cintrat, J Barbier, D Gillet (2016 Oct 8)

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Chlamydiales and Leishmania.

Chemico-biological interactions : DOI : S0009-2797(16)30427-6 En savoir plus
Résumé

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

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Mijo Simunovic, Emma Evergren, Ivan Golushko, Coline Prévost, Henri-François Renard, Ludger Johannes, Harvey T McMahon, Vladimir Lorman, Gregory A Voth, Patricia Bassereau (2016 Oct 4)

How curvature-generating proteins build scaffolds on membrane nanotubes.

Proceedings of the National Academy of Sciences of the United States of America : 113 : DOI : 10.1073/pnas.1606943113 En savoir plus
Résumé

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein-membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30-40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.

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Christine Sedlik, Adèle Heitzmann, Sophie Viel, Rafik Ait Sarkouh, Cornélie Batisse, Frédéric Schmidt, Philippe De La Rochere, Nathalie Amzallag, Eduardo Osinaga, Pablo Oppezzo, Otto Pritsch, Xavier Sastre-Garau, Pascale Hubert, Sebastian Amigorena, Eliane Piaggio (2016 Sep 14)

Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen.

Oncoimmunology : e1171434 : DOI : 10.1080/2162402X.2016.1171434 En savoir plus
Résumé

Antibody-drug conjugates (ADC), combining the specificity of tumor recognition by monoclonal antibodies (mAb) and the powerful cytotoxicity of anticancer drugs, are currently under growing interest and development. Here, we studied the potential of Chi-Tn, a mAb directed to a glyco-peptidic tumor-associated antigen, to be used as an ADC for cancer treatment. First, we demonstrated that Chi-Tn specifically targeted tumor cells in vivo. Also, using flow cytometry and deconvolution microscopy, we showed that the Chi-Tn mAb is rapidly internalized – condition necessary to ensure the delivery of conjugated cytotoxic drugs in an active form, and targeted to early and recycling endosomes. When conjugated to saporin (SAP) or to auristatin F, the Chi-Tn ADC exhibited effective cytotoxicity to Tn-positive tumor cells in vitro, which correlated with the level of tumoral Tn expression. Furthermore, the Chi-Tn mAb conjugated to auristatin F also exhibited efficient antitumor activity in vivo, validating for the first time the use of an anti-Tn antibody as an effective ADC.

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