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Many endocytic uptake events depend on the ‘pinchase’ activity of dynamin. By measuring the orientation of single gold nanorods, a new study reveals that invaginated clathrin-coated endocytic pits undergo a strong rotational twist prior to or concomitant with their detachment.
Ludger Johannes; Cesar Augusto Valades-Cruz. The final twist in endocytic membrane scission. Nature Cell Biology 2021, 23, 812 -813.
AMA StyleLudger Johannes, Cesar Augusto Valades-Cruz. The final twist in endocytic membrane scission. Nature Cell Biology. 2021; 23 (8):812-813.
Chicago/Turabian StyleLudger Johannes; Cesar Augusto Valades-Cruz. 2021. "The final twist in endocytic membrane scission." Nature Cell Biology 23, no. 8: 812-813.
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.
Senthil Arumugam; Stefanie Schmieder; Weria Pezeshkian; Ulrike Becken; Christian Wunder; Dan Chinnapen; John Hjort Ipsen; Anne K. Kenworthy; Wayne Lencer; Satyajit Mayor; Ludger Johannes. Ceramide structure dictates glycosphingolipid nanodomain assembly and function. Nature Communications 2021, 12, 1 -12.
AMA StyleSenthil Arumugam, Stefanie Schmieder, Weria Pezeshkian, Ulrike Becken, Christian Wunder, Dan Chinnapen, John Hjort Ipsen, Anne K. Kenworthy, Wayne Lencer, Satyajit Mayor, Ludger Johannes. Ceramide structure dictates glycosphingolipid nanodomain assembly and function. Nature Communications. 2021; 12 (1):1-12.
Chicago/Turabian StyleSenthil Arumugam; Stefanie Schmieder; Weria Pezeshkian; Ulrike Becken; Christian Wunder; Dan Chinnapen; John Hjort Ipsen; Anne K. Kenworthy; Wayne Lencer; Satyajit Mayor; Ludger Johannes. 2021. "Ceramide structure dictates glycosphingolipid nanodomain assembly and function." Nature Communications 12, no. 1: 1-12.
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.
Ludger Johannes. The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery—The GL–Lect Hypothesis. Molecules 2021, 26, 3299 .
AMA StyleLudger Johannes. The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery—The GL–Lect Hypothesis. Molecules. 2021; 26 (11):3299.
Chicago/Turabian StyleLudger Johannes. 2021. "The Cellular and Chemical Biology of Endocytic Trafficking and Intracellular Delivery—The GL–Lect Hypothesis." Molecules 26, no. 11: 3299.
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 favour delivery vector optimization and the understanding of the enigmatic translocation process.
Marco Lucchino; Anne Billet; Siau‐Kun Bai; Estelle Dransart; Justine Hadjerci; Frédéric Schmidt; Christian Wunder; Ludger Johannes. Absolute Quantification of Drug Vector Delivery to the Cytosol. Angewandte Chemie 2021, 133, 14950 -14956.
AMA StyleMarco Lucchino, Anne Billet, Siau‐Kun Bai, Estelle Dransart, Justine Hadjerci, Frédéric Schmidt, Christian Wunder, Ludger Johannes. Absolute Quantification of Drug Vector Delivery to the Cytosol. Angewandte Chemie. 2021; 133 (27):14950-14956.
Chicago/Turabian StyleMarco Lucchino; Anne Billet; Siau‐Kun Bai; Estelle Dransart; Justine Hadjerci; Frédéric Schmidt; Christian Wunder; Ludger Johannes. 2021. "Absolute Quantification of Drug Vector Delivery to the Cytosol." Angewandte Chemie 133, no. 27: 14950-14956.
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 favour delivery vector optimization and the understanding of the enigmatic translocation process.
Marco Lucchino; Anne Billet; Siau‐Kun Bai; Estelle Dransart; Justine Hadjerci; Frédéric Schmidt; Christian Wunder; Ludger Johannes. Absolute Quantification of Drug Vector Delivery to the Cytosol. Angewandte Chemie International Edition 2021, 60, 14824 -14830.
AMA StyleMarco Lucchino, Anne Billet, Siau‐Kun Bai, Estelle Dransart, Justine Hadjerci, Frédéric Schmidt, Christian Wunder, Ludger Johannes. Absolute Quantification of Drug Vector Delivery to the Cytosol. Angewandte Chemie International Edition. 2021; 60 (27):14824-14830.
Chicago/Turabian StyleMarco Lucchino; Anne Billet; Siau‐Kun Bai; Estelle Dransart; Justine Hadjerci; Frédéric Schmidt; Christian Wunder; Ludger Johannes. 2021. "Absolute Quantification of Drug Vector Delivery to the Cytosol." Angewandte Chemie International Edition 60, no. 27: 14824-14830.
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.
Alena Ivashenka; Christian Wunder; Valerie Chambon; Roger Sandhoff; Richard Jennemann; Estelle Dransart; Katrina Podsypanina; Bérangère Lombard; Damarys Loew; Christophe Lamaze; Francoise Poirier; Hermann-Josef Gröne; Ludger Johannes; Massiullah Shafaq-Zadah. Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes. Communications Biology 2021, 4, 1 -15.
AMA StyleAlena Ivashenka, Christian Wunder, Valerie Chambon, Roger Sandhoff, Richard Jennemann, Estelle Dransart, Katrina Podsypanina, Bérangère Lombard, Damarys Loew, Christophe Lamaze, Francoise Poirier, Hermann-Josef Gröne, Ludger Johannes, Massiullah Shafaq-Zadah. Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes. Communications Biology. 2021; 4 (1):1-15.
Chicago/Turabian StyleAlena Ivashenka; Christian Wunder; Valerie Chambon; Roger Sandhoff; Richard Jennemann; Estelle Dransart; Katrina Podsypanina; Bérangère Lombard; Damarys Loew; Christophe Lamaze; Francoise Poirier; Hermann-Josef Gröne; Ludger Johannes; Massiullah Shafaq-Zadah. 2021. "Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes." Communications Biology 4, no. 1: 1-15.
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.
Juan Saez; Stephanie Dogniaux; Massiullah Shafaq-Zadah; Ludger Johannes; Claire Hivroz; Andrés Zucchetti. Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism. Cells 2021, 10, 359 .
AMA StyleJuan Saez, Stephanie Dogniaux, Massiullah Shafaq-Zadah, Ludger Johannes, Claire Hivroz, Andrés Zucchetti. Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism. Cells. 2021; 10 (2):359.
Chicago/Turabian StyleJuan Saez; Stephanie Dogniaux; Massiullah Shafaq-Zadah; Ludger Johannes; Claire Hivroz; Andrés Zucchetti. 2021. "Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism." Cells 10, no. 2: 359.
BackgroundTumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase.MethodsWe 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.ResultsWe 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.ConclusionsTogether, 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.
Marion V Guerin; Fabienne Regnier; Maxime Thoreau; Lene Vimeux; Matthieu Benard; Estelle Dransart; Hweixian L Penny; Ludger Johannes; Alain Trautmann; Nadege Bercovici. Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse. Journal for ImmunoTherapy of Cancer 2020, 8, e000996 .
AMA StyleMarion V Guerin, Fabienne Regnier, Maxime Thoreau, Lene Vimeux, Matthieu Benard, Estelle Dransart, Hweixian L Penny, Ludger Johannes, Alain Trautmann, Nadege Bercovici. Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse. Journal for ImmunoTherapy of Cancer. 2020; 8 (2):e000996.
Chicago/Turabian StyleMarion V Guerin; Fabienne Regnier; Maxime Thoreau; Lene Vimeux; Matthieu Benard; Estelle Dransart; Hweixian L Penny; Ludger Johannes; Alain Trautmann; Nadege Bercovici. 2020. "Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse." Journal for ImmunoTherapy of Cancer 8, no. 2: e000996.
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.
Massiullah Shafaq-Zadah; Estelle Dransart; Ludger Johannes. Quantitative Methods to Study and of Cargo Proteins. Methods in Molecular Biology 2020, 2233, 53 -70.
AMA StyleMassiullah Shafaq-Zadah, Estelle Dransart, Ludger Johannes. Quantitative Methods to Study and of Cargo Proteins. Methods in Molecular Biology. 2020; 2233 ():53-70.
Chicago/Turabian StyleMassiullah Shafaq-Zadah; Estelle Dransart; Ludger Johannes. 2020. "Quantitative Methods to Study and of Cargo Proteins." Methods in Molecular Biology 2233, no. : 53-70.
The broad field of Structural DNA Nanotechnology has diverged into various areas of applications ranging from computing, photonics, synthetic biology, biosensing to in‐vivo bioimaging and therapeutic delivery to name 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, which 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 evolution and biological applications of DNA nanotechnology, where integration of DNA structures with biomolecules can now uncover phenomena of interests to biologists and biomedical scientists. Finally, we conclude with the challenges, limitations, and perspectives of DNA nanodevices in fundamental and applied research.
Dhiraj Devidas Bhatia; Christian Wunder; Ludger Johannes. Self‐assembled, Programmable DNA Nanodevices for Biological and Biomedical Applications. ChemBioChem 2020, 22, 763 -778.
AMA StyleDhiraj Devidas Bhatia, Christian Wunder, Ludger Johannes. Self‐assembled, Programmable DNA Nanodevices for Biological and Biomedical Applications. ChemBioChem. 2020; 22 (5):763-778.
Chicago/Turabian StyleDhiraj Devidas Bhatia; Christian Wunder; Ludger Johannes. 2020. "Self‐assembled, Programmable DNA Nanodevices for Biological and Biomedical Applications." ChemBioChem 22, no. 5: 763-778.
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.
Massiullah Shafaq-Zadah; Estelle Dransart; Ludger Johannes. Clathrin-independent endocytosis, retrograde trafficking, and cell polarity. Current Opinion in Cell Biology 2020, 65, 112 -121.
AMA StyleMassiullah Shafaq-Zadah, Estelle Dransart, Ludger Johannes. Clathrin-independent endocytosis, retrograde trafficking, and cell polarity. Current Opinion in Cell Biology. 2020; 65 ():112-121.
Chicago/Turabian StyleMassiullah Shafaq-Zadah; Estelle Dransart; Ludger Johannes. 2020. "Clathrin-independent endocytosis, retrograde trafficking, and cell polarity." Current Opinion in Cell Biology 65, no. : 112-121.
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.
Annie Willysson; Anne-Lie Ståhl; Daniel Gillet; Julien Barbier; Jean-Christophe Cintrat; Valérie Chambon; Anne Billet; Ludger Johannes; Diana Karpman. Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit. Toxins 2020, 12, 449 .
AMA StyleAnnie Willysson, Anne-Lie Ståhl, Daniel Gillet, Julien Barbier, Jean-Christophe Cintrat, Valérie Chambon, Anne Billet, Ludger Johannes, Diana Karpman. Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit. Toxins. 2020; 12 (7):449.
Chicago/Turabian StyleAnnie Willysson; Anne-Lie Ståhl; Daniel Gillet; Julien Barbier; Jean-Christophe Cintrat; Valérie Chambon; Anne Billet; Ludger Johannes; Diana Karpman. 2020. "Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit." Toxins 12, no. 7: 449.
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.
Ludger Johannes; Anne Billet. Glycosylation and raft endocytosis in cancer. Cancer and Metastasis Reviews 2020, 39, 375 -396.
AMA StyleLudger Johannes, Anne Billet. Glycosylation and raft endocytosis in cancer. Cancer and Metastasis Reviews. 2020; 39 (2):375-396.
Chicago/Turabian StyleLudger Johannes; Anne Billet. 2020. "Glycosylation and raft endocytosis in cancer." Cancer and Metastasis Reviews 39, no. 2: 375-396.
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 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 non-diseased 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.
Tanja Bien; Markus Perl; Andrea C. Machmüller; Ulrich Nitsche; Anja Conrad; Ludger Johannes; Johannes Müthing; Jens Soltwisch; Klaus-Peter Janssen; Klaus Dreisewerd. MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue. Analytical Chemistry 2020, 92, 7096 -7105.
AMA StyleTanja Bien, Markus Perl, Andrea C. Machmüller, Ulrich Nitsche, Anja Conrad, Ludger Johannes, Johannes Müthing, Jens Soltwisch, Klaus-Peter Janssen, Klaus Dreisewerd. MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue. Analytical Chemistry. 2020; 92 (10):7096-7105.
Chicago/Turabian StyleTanja Bien; Markus Perl; Andrea C. Machmüller; Ulrich Nitsche; Anja Conrad; Ludger Johannes; Johannes Müthing; Jens Soltwisch; Klaus-Peter Janssen; Klaus Dreisewerd. 2020. "MALDI-2 Mass Spectrometry and Immunohistochemistry Imaging of Gb3Cer, Gb4Cer, and Further Glycosphingolipids in Human Colorectal Cancer Tissue." Analytical Chemistry 92, no. 10: 7096-7105.
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.
Henri-François Renard; François Tyckaert; Cristina Lo Giudice; Thibault Hirsch; Cesar Augusto Valades-Cruz; Camille Lemaigre; Massiullah Shafaq-Zadah; Christian Wunder; Ruddy Wattiez; Ludger Johannes; Pierre Van Der Bruggen; David Alsteens; Pierre Morsomme. Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166. Nature Communications 2020, 11, 1 -13.
AMA StyleHenri-François Renard, François Tyckaert, Cristina Lo Giudice, Thibault Hirsch, Cesar Augusto Valades-Cruz, Camille Lemaigre, Massiullah Shafaq-Zadah, Christian Wunder, Ruddy Wattiez, Ludger Johannes, Pierre Van Der Bruggen, David Alsteens, Pierre Morsomme. Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166. Nature Communications. 2020; 11 (1):1-13.
Chicago/Turabian StyleHenri-François Renard; François Tyckaert; Cristina Lo Giudice; Thibault Hirsch; Cesar Augusto Valades-Cruz; Camille Lemaigre; Massiullah Shafaq-Zadah; Christian Wunder; Ruddy Wattiez; Ludger Johannes; Pierre Van Der Bruggen; David Alsteens; Pierre Morsomme. 2020. "Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166." Nature Communications 11, no. 1: 1-13.
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. The ER exit site component Sec16A was identified as the target of Retro-2, a small-molecule inhibitor of protein toxins and pathogens. Retro-2 treatment alters retrograde early/maturing endosomes-to-Golgi trafficking of Shiga toxin.
Alison Forrester; Stefan J. Rathjen; Maria Daniela Garcia-Castillo; Collin Bachert; Audrey Couhert; Livia Tepshi; Sylvain Pichard; Jennifer Martinez; Mathilde Munier; Raphael Sierocki; Henri-François Renard; César Augusto Valades-Cruz; Florent Dingli; Damarys Loew; Christophe Lamaze; Jean-Christophe Cintrat; Adam D. Linstedt; Daniel Gillet; Julien Barbier; Ludger Johannes. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nature Chemical Biology 2020, 16, 327 -336.
AMA StyleAlison Forrester, Stefan J. Rathjen, Maria Daniela Garcia-Castillo, Collin Bachert, Audrey Couhert, Livia Tepshi, Sylvain Pichard, Jennifer Martinez, Mathilde Munier, Raphael Sierocki, Henri-François Renard, César Augusto Valades-Cruz, Florent Dingli, Damarys Loew, Christophe Lamaze, Jean-Christophe Cintrat, Adam D. Linstedt, Daniel Gillet, Julien Barbier, Ludger Johannes. Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2. Nature Chemical Biology. 2020; 16 (3):327-336.
Chicago/Turabian StyleAlison Forrester; Stefan J. Rathjen; Maria Daniela Garcia-Castillo; Collin Bachert; Audrey Couhert; Livia Tepshi; Sylvain Pichard; Jennifer Martinez; Mathilde Munier; Raphael Sierocki; Henri-François Renard; César Augusto Valades-Cruz; Florent Dingli; Damarys Loew; Christophe Lamaze; Jean-Christophe Cintrat; Adam D. Linstedt; Daniel Gillet; Julien Barbier; Ludger Johannes. 2020. "Functional dissection of the retrograde Shiga toxin trafficking inhibitor Retro-2." Nature Chemical Biology 16, no. 3: 327-336.
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.
Karl E. Johansson; Anne-Lie Ståhl; Ida Arvidsson; Sebastian Loos; Ashmita Tontanahal; Johan Rebetz; Milan Chromek; Ann-Charlotte Kristoffersson; Ludger Johannes; Diana Karpman. Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism. Scientific Reports 2019, 9, 1 -11.
AMA StyleKarl E. Johansson, Anne-Lie Ståhl, Ida Arvidsson, Sebastian Loos, Ashmita Tontanahal, Johan Rebetz, Milan Chromek, Ann-Charlotte Kristoffersson, Ludger Johannes, Diana Karpman. Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism. Scientific Reports. 2019; 9 (1):1-11.
Chicago/Turabian StyleKarl E. Johansson; Anne-Lie Ståhl; Ida Arvidsson; Sebastian Loos; Ashmita Tontanahal; Johan Rebetz; Milan Chromek; Ann-Charlotte Kristoffersson; Ludger Johannes; Diana Karpman. 2019. "Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism." Scientific Reports 9, no. 1: 1-11.
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.
Rebecca C. Gilson; Sachith D. Gunasinghe; Ludger Johannes; Katharina Gaus. Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling. Progress in Lipid Research 2019, 76, 101010 .
AMA StyleRebecca C. Gilson, Sachith D. Gunasinghe, Ludger Johannes, Katharina Gaus. Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling. Progress in Lipid Research. 2019; 76 ():101010.
Chicago/Turabian StyleRebecca C. Gilson; Sachith D. Gunasinghe; Ludger Johannes; Katharina Gaus. 2019. "Galectin-3 modulation of T-cell activation: mechanisms of membrane remodelling." Progress in Lipid Research 76, no. : 101010.
Bio-membranes 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 carbohy-drate 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.
Erik B. Watkins; Jaroslaw Majewski; Eva Y. Chi; Haifei Gao; Jean-Claude Florent; Ludger Johannes. Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature. Nano Letters 2019, 19, 7365 -7369.
AMA StyleErik B. Watkins, Jaroslaw Majewski, Eva Y. Chi, Haifei Gao, Jean-Claude Florent, Ludger Johannes. Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature. Nano Letters. 2019; 19 (10):7365-7369.
Chicago/Turabian StyleErik B. Watkins; Jaroslaw Majewski; Eva Y. Chi; Haifei Gao; Jean-Claude Florent; Ludger Johannes. 2019. "Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature." Nano Letters 19, no. 10: 7365-7369.
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.
Melissa Dewulf; Darius Vasco Köster; Bidisha Sinha; Christine Viaris De Lesegno; Valérie Chambon; Anne Bigot; Mona Bensalah; Elisa Negroni; Nicolas Tardif; Joanna Podkalicka; Ludger Johannes; Pierre Nassoy; Gillian Butler-Browne; Christophe Lamaze; Cedric M. Blouin. Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells. Nature Communications 2019, 10, 1974 .
AMA StyleMelissa Dewulf, Darius Vasco Köster, Bidisha Sinha, Christine Viaris De Lesegno, Valérie Chambon, Anne Bigot, Mona Bensalah, Elisa Negroni, Nicolas Tardif, Joanna Podkalicka, Ludger Johannes, Pierre Nassoy, Gillian Butler-Browne, Christophe Lamaze, Cedric M. Blouin. Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells. Nature Communications. 2019; 10 (1):1974.
Chicago/Turabian StyleMelissa Dewulf; Darius Vasco Köster; Bidisha Sinha; Christine Viaris De Lesegno; Valérie Chambon; Anne Bigot; Mona Bensalah; Elisa Negroni; Nicolas Tardif; Joanna Podkalicka; Ludger Johannes; Pierre Nassoy; Gillian Butler-Browne; Christophe Lamaze; Cedric M. Blouin. 2019. "Dystrophy-associated caveolin-3 mutations reveal that caveolae couple IL6/STAT3 signaling with mechanosensing in human muscle cells." Nature Communications 10, no. 1: 1974.