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1. Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin‐9 dependent mechanism

Author

Anthony Lefebvre, Camille Trioën, Sarah Renaud, William Laine, Benjamin Hennart, Clément Bouchez, Bertrand Leroux, Delphine Allorge, Jérôme Kluza, Elisabeth Werkmeister, Guillaume Paul Grolez, Nadira Delhem, and Olivier Moralès

Subjects
extracellular vesicles, galectin‐9, IDO, IL‐4, IL4I1, mature regulatory dendritic cells, Cytology, QH573-671
Abstract

Abstract Nasopharyngeal carcinoma‐derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin‐9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV‐DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real‐time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV‐DCs using a proprietary anti‐Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV‐DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL‐4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV‐DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV‐DCs using an anti‐Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin‐9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.

Published
2023
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2. A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice

Author

Alexandre Quilbe, Rami Mustapha, Belinda Duchêne, Abhishek Kumar, Elisabeth Werkmeister, Emmanuelle Leteurtre, Olivier Moralès, Nicolas Jonckheere, Isabelle Van Seuningen, and Nadira Delhem

Subjects
pancreatic adenocarcinoma, galectin-9, regulatory T cells, immunotherapy, transgenic mouse model, pancreatic intraepithelial neoplasm (PanIN), Immunologic diseases. Allergy, RC581-607
Abstract

BackgroundPancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9).Materials and methodsCD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4–13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression.ResultsIHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN.ConclusionWe demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Published
2023
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4. Tirap controls Mycobacterium tuberculosis phagosomal acidification.

Author

Imène Belhaouane, Amine Pochet, Jonathan Chatagnon, Eik Hoffmann, Christophe J Queval, Nathalie Deboosère, Céline Boidin-Wichlacz, Laleh Majlessi, Valentin Sencio, Séverine Heumel, Alexandre Vandeputte, Elisabeth Werkmeister, Laurence Fievez, Fabrice Bureau, Yves Rouillé, François Trottein, Mathias Chamaillard, Priscille Brodin, and Arnaud Machelart

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.

Published
2023
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5. Rheological comparison between control and Dupuytren fibroblasts when plated in circular micropatterns using atomic force microscopy

Author

Sandra Pérez-Domínguez, Elisabeth Werkmeister, Maria Luisa Marini, Vincent Dupres, Sébastien Janel, Frank Lafont, and Manfred Radmacher

Subjects
AFM, Dupuytren’s disease, micropatterning, viscoelasticity, fibroblast, Physics, QC1-999
Abstract

In tissue, cells are obliged to confine and adapt to a specific geometric shape due to the surrounding environmental constraints. Under healthy conditions, fibroblasts present an elongated shape; however, changes in biochemical and physical properties of the extracellular matrix could distort the cell shape, inducing a pathological state. We have studied fibroblasts’ mechanical behavior under circular geometrical constraints. Circular micropatterns force fibroblasts to acquire a different shape from that of a healthy tissue, inducing a possible pathological condition. In total, three different fibroblast types from Dupuytren’s disorder, all obtained from the same patient, were confined in circular-shaped micropatterns of three different diameters (25, 35, and 45 µm), and mechanical properties were evaluated using an atomic force microscope (AFM). We found that control fibroblast mechanics (apparent Young’s modulus) increases with the increasing pattern diameter and comes together with a decrease in cell height and in loss tangent, translated into a more solid-like behavior. We hypothesize that these results resemble the transition toward the myofibroblast phenotype, ameliorating cytoskeleton formation and organization and enhancing cell contraction. Scar and Dupuytren fibroblasts did not display major changes in cell mechanics and cell height when changing the pattern diameter, suggesting that they are less affected by physical changes in the environment as they can adapt their shape to the geometrical dimensions. Therefore, our findings demonstrate that combining micropatterning and AFM measurements provides a powerful tool to study cell mechanics inducing constraints onto the cell, thus mimicking certain aspects of the tissue environment in both healthy and pathological states.

Published
2022
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6. Characterization of Two Parabacteroides distasonis Candidate Strains as New Live Biotherapeutics against Obesity

Author

Bernardo Cuffaro, Denise Boutillier, Jérémy Desramaut, Amin Jablaoui, Elisabeth Werkmeister, François Trottein, Anne-Judith Waligora-Dupriet, Moez Rhimi, Emmanuelle Maguin, and Corinne Grangette

Subjects
Parabacteroides, microbiota, live biotherapeutic products, probiotics, obesity, holobiont, Cytology, QH573-671
Abstract

The gut microbiota is now considered as a key player in the development of metabolic dysfunction. Therefore, targeting gut microbiota dysbiosis has emerged as a new therapeutic strategy, notably through the use of live gut microbiota-derived biotherapeutics. We previously highlighted the anti-inflammatory abilities of two Parabacteroides distasonis strains. We herein evaluate their potential anti-obesity abilities and show that the two strains induced the secretion of the incretin glucagon-like peptide 1 in vitro and limited weight gain and adiposity in obese mice. These beneficial effects are associated with reduced inflammation in adipose tissue and the improvement of lipid and bile acid metabolism markers. P. distasonis supplementation also modified the Actinomycetota, Bacillota and Bacteroidota taxa of the mice gut microbiota. These results provide better insight into the capacity of P. distasonis to positively influence host metabolism and to be used as novel source of live biotherapeutics in the treatment and prevention of metabolic-related diseases.

Published
2023
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7. 2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations

Author

Carole Trzaska, Séverine Amand, Christine Bailly, Catherine Leroy, Virginie Marchand, Evelyne Duvernois-Berthet, Jean-Michel Saliou, Hana Benhabiles, Elisabeth Werkmeister, Thierry Chassat, Romain Guilbert, David Hannebique, Anthony Mouray, Marie-Christine Copin, Pierre-Arthur Moreau, Eric Adriaenssens, Andreas Kulozik, Eric Westhof, David Tulasne, Yuri Motorin, Sylvie Rebuffat, and Fabrice Lejeune

Subjects
Science
Abstract

Nonsense mutations can be corrected by several molecules that activate readthrough of premature termination codon. Here, the authors report that 2,6-diaminopurine efficiently corrects UGA nonsense mutations with no significant toxicity.

Published
2020
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8. Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication.

Author

Kannan Venugopal, Sylia Chehade, Elisabeth Werkmeister, Nicolas Barois, Javier Periz, Frank Lafont, Isabelle Tardieux, Jamal Khalife, Gordon Langsley, Markus Meissner, and Sabrina Marion

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.

Published
2020
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9. ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease

Author

Florent Letronne, Geoffroy Laumet, Anne-Marie Ayral, Julien Chapuis, Florie Demiautte, Mathias Laga, Michel E. Vandenberghe, Nicolas Malmanche, Florence Leroux, Fanny Eysert, Yoann Sottejeau, Linda Chami, Amandine Flaig, Charlotte Bauer, Pierre Dourlen, Marie Lesaffre, Charlotte Delay, Ludovic Huot, Julie Dumont, Elisabeth Werkmeister, Franck Lafont, Tiago Mendes, Franck Hansmannel, Bart Dermaut, Benoit Deprez, Anne-Sophie Hérard, Marc Dhenain, Nicolas Souedet, Florence Pasquier, David Tulasne, Claudine Berr, Jean-Jacques Hauw, Yves Lemoine, Philippe Amouyel, David Mann, Rebecca Déprez, Frédéric Checler, David Hot, Thierry Delzescaux, Kris Gevaert, and Jean-Charles Lambert

Subjects
Alzheimer, APP, ADAM30, Amyloid, Metabolism, LTP, Medicine, Medicine (General), R5-920
Abstract

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development.

Published
2016
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10. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection

Author

Lamba Omar Sangaré, Tchilabalo Dilezitoko Alayi, Benoit Westermann, Agnes Hovasse, Fabien Sindikubwabo, Isabelle Callebaut, Elisabeth Werkmeister, Frank Lafont, Christian Slomianny, Mohamed-Ali Hakimi, Alain Van Dorsselaer, Christine Schaeffer-Reiss, and Stanislas Tomavo

Subjects
Science
Abstract

The retromer complex is a multi-protein component of the endosomal protein sorting machinery. Here, Sangaré et al. identify unique features in the retromer complex of the parasite Toxoplasma gondii, and show that it is crucial for the biogenesis of secretory organelles in this pathogen.

Published
2016
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11. Heparin-Binding Hemagglutinin Adhesin (HBHA) Is Involved in Intracytosolic Lipid Inclusions Formation in Mycobacteria

Author

Dominique Raze, Claudie Verwaerde, Gaspard Deloison, Elisabeth Werkmeister, Baptiste Coupin, Marc Loyens, Priscille Brodin, Carine Rouanet, and Camille Locht

Subjects
tuberculosis, Mycobacterium, lipids, dormancy, carbon storage, Microbiology, QR1-502
Abstract

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Published
2018
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12. Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division.

Author

Kannan Venugopal, Elisabeth Werkmeister, Nicolas Barois, Jean-Michel Saliou, Anais Poncet, Ludovic Huot, Fabien Sindikubwabo, Mohamed Ali Hakimi, Gordon Langsley, Frank Lafont, and Sabrina Marion

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis.

Published
2017
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13. Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator.

Author

Thomas Mouveaux, Gabrielle Oria, Elisabeth Werkmeister, Christian Slomianny, Barbara A Fox, David J Bzik, and Stanislas Tomavo

Subjects
Medicine, Science
Abstract

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

Published
2014
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14. A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatin-associated component, modulator of nucleolar architecture and parasite virulence.

Author

Alejandro Olguin-Lamas, Edwige Madec, Agnes Hovasse, Elisabeth Werkmeister, Isabelle Callebaut, Christian Slomianny, Stephane Delhaye, Thomas Mouveaux, Christine Schaeffer-Reiss, Alain Van Dorsselaer, and Stanislas Tomavo

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii.

Published
2011
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15. Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

Author

Anna Martinez, El Moukhtar Aliouat, Annie Standaert-Vitse, Elisabeth Werkmeister, Muriel Pottier, Claire Pinçon, Eduardo Dei-Cas, and Cécile-Marie Aliouat-Denis

Subjects
Medicine, Science
Abstract

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.

Published
2011
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16. Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni.

Author

Alvaro Baeza Garcia, Raymond J Pierce, Benjamin Gourbal, Elisabeth Werkmeister, Dominique Colinet, Jean-Marc Reichhart, Colette Dissous, and Christine Coustau

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions.

Published
2010
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17. A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis

Author

Amélie Dewitte, Elisabeth Werkmeister, François Pierre, Florent Sebbane, Sébastien Bontemps-Gallo, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), CHU Lille, Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), ANR-15-CE39-0017,LUCY,Illuminer les puces pour mieux comprendre les mécanismes complexes de transmission de Yersinia pestis(2015), ANR-21-CE15-0047,RESISTANT,Rôle d'un système de signalisation cellulaire dans l'adaptation de Yersinia pestis à son vecteur(2021), ANR-16-IDEX-0004,ULNE,ULNE(2016), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], and Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 [PLBS]

Subjects
Ecology, Yersinia pestis, flea, Applied Microbiology and Biotechnology, HmsHFRS, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, OmpR-envZ, bacteria, plague, vector-borne diseases, [SDV.BA.ZI]Life Sciences [q-bio]/Animal biology/Invertebrate Zoology, Food Science, Biotechnology
Abstract

International audience; Yersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague.

Published
2023

18. The endocytic recycling compartment serves as a viral factory for hepatitis E virus

Author

Cyrine Bentaleb, Kévin Hervouet, Claire Montpellier, Charline Camuzet, Martin Ferrié, Julien Burlaud-Gaillard, Stéphane Bressanelli, Karoline Metzger, Elisabeth Werkmeister, Maliki Ankavay, Nancy Leon Janampa, Julien Marlet, Julien Roux, Clarence Deffaud, Anne Goffard, Yves Rouillé, Jean Dubuisson, Philippe Roingeard, Cécile-Marie Aliouat-Denis, Laurence Cocquerel, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], Morphogénèse et antigénicité du VIH et du virus des Hépatites [MAVIVH - U1259 Inserm - CHRU Tours ], Institut de Biologie Intégrative de la Cellule [I2BC], Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 [PLBS], BIOTEM [Apprieu], Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Institute of Microbiology [University of Lausanne] (IMUL), Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), and This work has been funded by Inserm-Transfert (MAT-PI-17006), Pasteur Institute of Lille (DiagHepE), Region Hauts-de-France (DiagHepE), ANRS and the University of Tours.

Subjects
medicine.drug_class, viruses, Viral factories, [SDV]Life Sciences [q-bio], Endocytic recycling, RNA-dependent RNA polymerase, Infectious particles, Biology, medicine.disease_cause, Monoclonal antibody, Virus, Antibodies, Cellular and Molecular Neuroscience, 03 medical and health sciences, Hepatitis E virus, Viral factory, viral factories, medicine, Electron microscopy, Humans, AlphaFold2, Molecular Biology, 030304 developmental biology, Pharmacology, 0303 health sciences, 030306 microbiology, ORF2 capsid protein, Antibodies, Monoclonal, virus diseases, Biological Transport, Cell Biology, medicine.disease, Virology, digestive system diseases, 3. Good health, Endocytic recycling compartment, Rab11, Capsid, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Molecular Medicine, Capsid Proteins, Viral hepatitis, Viral Replication Compartments
Abstract

Background & AimsAlthough Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms.MethodsWe generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC).ResultsOne of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC.ConclusionsOur study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.Lay summaryHepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide but many steps of its lifecycle are still elusive. Thanks to the development of new antibodies that recognize the different forms of the HEV capsid protein, we were able to visualize vesicular and tubular structures that were established by the virus in the host cell. In addition, extensive efforts to identify these structures led us to conclude that HEV hijacks the endocytic recycling compartment of the cell to form this network of vesicles and tubules, which might be the hallmark of HEV infection.

Published
2022

19. Blocking bacterial entry at the adhesion step reveals dynamic recruitment of membrane and cytosolic probes

Author

Frank Lafont, Elisabeth Werkmeister, Yann Ciczora, Sébastien Janel, Magali Soyer, Michka Popoff, Assemblage et réplication du virus de l'hépatite C (ARVHC), Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Physique - IEMN (PHYSIQUE - IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), We thank A. Bongiovanni from the BICeL Facility for access to systems and technical advice. We would like to thank J. Warein for expert technical assistance, P.-H. Puech for valuable discussions and T. Melin, P-E Milhiet and G Tran Van Nhieu for critical reading of the manuscript., FundingThis study was supported by funds from Univ. Lille1 to M.P. and ANR (09-MIEN-020-01, 10-EQPX-04-01, 16), FEDER (12,001,407) to F.L., and LAFONT, Frank

Subjects
Cell signaling, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Glycosylphosphatidylinositols, Green Fluorescent Proteins, Cell, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Adhesion force, Polymerization, 03 medical and health sciences, Cytosol, 0302 clinical medicine, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Cell Adhesion, medicine, Fluorescence microscope, ubiquitin, Humans, Yersinia pseudotuberculosis, Super-resolution microscopy, Cytoskeleton, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Calcium signaling, TNF Receptor-Associated Factor 6, Host cell surface, 0303 health sciences, Binding Sites, biology, Cell Membrane, Ubiquitination, Cell Biology, General Medicine, Adhesion, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Actins, Cell biology, Correlative microscopy, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, medicine.anatomical_structure, Adsorption, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, AFM, Protein Processing, Post-Translational, 030217 neurology & neurosurgery, HeLa Cells
Abstract

International audience; Background: Bacterial invasion covers two steps: adhesion and entry per se. The cell signalling response is triggered upon pathogen interaction at the cell surface. This response continues when the pathogen is internalised. It is likely that these two steps activate different molecular machineries. So far, it has not been possible to easily follow in physiological conditions these events separately. We thus developed an approach to uncouple adhesion from entry using atomic force microscopy (AFM)-driven force and fluorescence measurements.Results: We report nanometric-scale, high-resolution, functional dynamic measurements of bacterial interaction with the host cell surface using photonic and adhesion force analyses. We describe how to achieve a precise monitoring of iterative cell–bacterium interactions to analyse host cell signalling responses to infection. By applying this method to Yersinia pseudotuberculosis, we first unveil glycosylphosphatidylinositol-anchored protein domains recruitment to the bacterium cell surface binding site and concomitant cytoskeleton rearrangements using super-resolution fluorescence microscopy. Second, we demonstrate the feasibility of monitoring post-translationally modified proteins, for example, via ubiquitylation, during the first step of infection.Conclusion: We provide an approach to discriminate between cellular signalling response activated at the plasma membrane during host–pathogen interaction and that is triggered during the internalisation of the pathogen within the cell.Significance: This approach adds to the technological arsenal to better understand and fight against pathogens and beyond the scope of microbiology to address conceptual issues of cell surface signalling.

Published
2019

20. Direct activation of the aryl hydrocarbon receptor by dog allergen participates in airway neutrophilic inflammation

Author

Fan Ying, Daniel Alvarez-Simon, Philippe Marquillies, Mélodie Bouté, Julie Nanou, Anne Tsicopoulos, Saliha Ait Yahia, Joanne Balsamelli, Patricia de Nadai, Han Vorng, Elisabeth Werkmeister, Cécile Chenivesse, and Camille Audousset

Subjects
Inflammation, biology, business.industry, Extramural, Immunology, Neutrophilic inflammation, Allergens, Aryl hydrocarbon receptor, medicine.disease_cause, medicine.disease, Asthma, Allergen, Dogs, Receptors, Aryl Hydrocarbon, biology.protein, Immunology and Allergy, Medicine, Animals, medicine.symptom, business, Receptor, Airway
Published
2021

21. A role for AKT1 in nonsense-mediated mRNA decay

Author

Catherine Leroy, Martine Palma, Sophie Salomé-Desnoulez, Marc Mongy, Fabrice Lejeune, Elisabeth Werkmeister, Hervé Le Hir, Rebekah Kong, LE HIR, Hervé, Cancer Heterogeneity, Plasticity and Resistance to Therapies - UMR 9020 - U 1277 (CANTHER), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Réseau International des Instituts Pasteur (RIIP), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), F.L. is supported by funding from La Ligue contre le Cancer, Vaincre la mucoviscidose, Association Française contre les Myopathies, GIP Cancéropôle Nord Ouest, M.P. received a PhD fellowship from the University of Lille, la Région Hauts-de-France, Contrat de Plan Etat-Région CPER Cancer 2015-2020. Funding for open access charge: CNRS institution., Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
AcademicSubjects/SCI00010, [SDV]Life Sciences [q-bio], Nonsense-mediated decay, AKT1, Biology, Phosphatidylinositol 3-Kinases, 03 medical and health sciences, 0302 clinical medicine, Genes, Reporter, Genetics, Humans, RNA, Messenger, Phosphorylation, Luciferases, Protein kinase A, Protein kinase B, PI3K/AKT/mTOR pathway, Cell Proliferation, Gene Library, 030304 developmental biology, 0303 health sciences, Kinase, TOR Serine-Threonine Kinases, Gene regulation, Chromatin and Epigenetics, RNA-Binding Proteins, Nonsense Mediated mRNA Decay, Cell biology, [SDV] Life Sciences [q-bio], Eukaryotic Initiation Factor-4E, HEK293 Cells, Codon, Nonsense, 030220 oncology & carcinogenesis, Trans-Activators, Regulatory Pathway, Proto-Oncogene Proteins c-akt, RNA Helicases, HeLa Cells, Signal Transduction
Abstract

Nonsense-mediated mRNA decay (NMD) is a highly regulated quality control mechanism through which mRNAs harboring a premature termination codon are degraded. It is also a regulatory pathway for some genes. This mechanism is subject to various levels of regulation, including phosphorylation. To date only one kinase, SMG1, has been described to participate in NMD, by targeting the central NMD factor UPF1. Here, screening of a kinase inhibitor library revealed as putative NMD inhibitors several molecules targeting the protein kinase AKT1. We present evidence demonstrating that AKT1, a central player in the PI3K/AKT/mTOR signaling pathway, plays an essential role in NMD, being recruited by the UPF3X protein to phosphorylate UPF1. As AKT1 is often overactivated in cancer cells and as this should result in increased NMD efficiency, the possibility that this increase might affect cancer processes and be targeted in cancer therapy is discussed.

Published
2021

22. Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication

Author

Sabrina Marion, Kannan Venugopal, Sylia Chehade, Frank Lafont, Elisabeth Werkmeister, Nicolas Barois, Jamal Khalife, Isabelle Tardieux, Markus Meissner, Javier Periz, Gordon Langsley, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Ludwig-Maximilians-Universität München (LMU), Membrane Dynamics of Parasite-Host Cell Interactions [Grenoble], Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire de Biologie Comparative des Apicomplexes [Paris], Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), KV, SM and GL have been supported by the Laboratoire d’Excellence (LabEx) ParaFrap from the National Agency for Research ANR-11-LABX-0024 grant and by the ANR-14-CE14-0002-01 grant. SM has been supported by a joint Chaire d’Excellence from Université of Lille and the Centre National pour la Recherche Scientifique (CNRS)., ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Bodescot, Myriam, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, and Biologie Comparative des Apicomplexes = Comparative Biology of Apicomplexa Parasites

Subjects
QH301-705.5, Immunology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microbiology, Exocytosis, Microneme, 03 medical and health sciences, Virology, Genetics, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Secretion, Biology (General), Cell adhesion, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Molecular Biology, 030304 developmental biology, 0303 health sciences, Rhoptry, 030302 biochemistry & molecular biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, RC581-607, Entry into host, QR, 3. Good health, Transport protein, Cell biology, Parasitology, Dense granule, Immunologic diseases. Allergy, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract

International audience; Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.

Published
2020

23. 2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations

Author

Catherine Leroy, Séverine Amand, Anthony Mouray, David Tulasne, Andreas E. Kulozik, Christine Bailly, Thierry Chassat, Carole Trzaska, David Hannebique, Eric Adriaenssens, Jean-Michel Saliou, Elisabeth Werkmeister, Pierre-Arthur Moreau, Eric Westhof, Evelyne Duvernois-Berthet, Yuri Motorin, Romain Guilbert, Marie-Christine Copin, Virginie Marchand, Sylvie Rebuffat, Fabrice Lejeune, Hana Benhabiles, Cancer Heterogeneity, Plasticity and Resistance to Therapies - UMR 9020 - U 1277 (CANTHER), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Molécules de Communication et Adaptation des Micro-organismes (MCAM), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Physiologie moléculaire et adaptation (PhyMA), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Réseau International des Instituts Pasteur (RIIP), Laboratoire de Génie civil et Géo-environnement (LGCgE), Ecole nationale supérieure Mines-Télécom Lille Douai (IMT Lille Douai), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Lille-Université d'Artois (UA)-Université catholique de Lille (UCL)-École polytechnique universitaire de Lille (Polytech Lille), Hopp Children's Cancer Center Heidelberg [Heidelber, Germany] (KITZ), German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ)-Heidelberg University Hospital [Heidelberg], Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), F.L. is an Inserm researcher supported by fundings from Vaincre la mucoviscidose, the Association française contre les myopathies, the GIP Cancéropôle Nord Ouest, the Fondation Maladies Rares and the SATT Lutech, Authors would like to thank Dr. Jens Lykke-Andersen, Pr. P.A. Jänne and Pr. Lynne Maquat for reagents, Dr. Clément Carré, Dr. Bruno Lapeyre, Dr. Gabriele Neu-Yilik, Dr. Matthias Hentze, and Dr. Jean-Paul Renaud for helpful discussions. Authors also thank the Bicel facility for technical help, the PLEHTA for technical help on mouse managing and in vivo experiments, Valérie IGEL-BOURGUIGNON and Dr. Lilia AYADI from the Next-Generation Sequencing Core Facility of UMS2008 IBSLor (Université de Lorraine-CNRS-INSERM) for their help in RiboMethSeq library preparation and sequencing., Hétérogénéité, Plasticité et Résistance aux Thérapies des Cancers [Lille] (CANTHER), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université Lille Nord (France)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 (CIIL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Department of Pediatric Oncology, Alder Hey Children's Hospital, Alder Hey Children's Hospital, Centre National de la Recherche Scientifique (CNRS)-Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Médecine cellulaire et molécualire, PRES Université Lille Nord de France-Institut de Biologie de Lille-IFR 142, Université d'Artois (UA)-École polytechnique universitaire de Lille (Polytech Lille)-Université de Lille-Ecole nationale supérieure Mines-Télécom Lille Douai (IMT Lille Douai), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Yncréa Hauts-de-France, and Université catholique de Lille (UCL)-Université catholique de Lille (UCL)

Subjects
0301 basic medicine, Molecular biology, General Physics and Astronomy, chemistry.chemical_compound, Mice, RNA, Transfer, lcsh:Science, 2-Aminopurine, ComputingMilieux_MISCELLANEOUS, Genetics, Regulation of gene expression, tRNA Methyltransferases, Multidisciplinary, Molecular medicine, Drug discovery, Lepisma, 3. Good health, Gene Expression Regulation, Neoplastic, Drug screening, Codon, Nonsense, Cytosine, Science, Nonsense mutation, Mice, Nude, Biology, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Humans, Gene, RNA metabolism, 030102 biochemistry & molecular biology, 2,6-Diaminopurine, HEK 293 cells, fungi, RNA, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Genes, p53, Disease Models, Animal, 030104 developmental biology, HEK293 Cells, chemistry, Cancer cell, Mutation, lcsh:Q, Drug Screening Assays, Antitumor, HeLa Cells
Abstract

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2′-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations., Nonsense mutations can be corrected by several molecules that activate readthrough of premature termination codon. Here, the authors report that 2,6-diaminopurine efficiently corrects UGA nonsense mutations with no significant toxicity.

Published
2020

24. Phenotypic assays for Mycobacterium tuberculosis infection

Author

Vincent Delorme, Alain R. Baulard, Frank Lafont, Elisabeth Werkmeister, Nathalie Deboosere, Gaspard Deloison, Raffaella Iantomasi, Ok-Ryul Song, Christophe J. Queval, and Priscille Brodin

Subjects
0301 basic medicine, Histology, Tuberculosis, biology, Drug discovery, Intracellular parasite, 030106 microbiology, Cell Biology, biology.organism_classification, medicine.disease, Virology, Phenotype, 3. Good health, Pathology and Forensic Medicine, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, 030104 developmental biology, Drug development, medicine, Axenic, Intracellular
Abstract

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.

Published
2017

25. MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering

Author

Audrey Vinchent, Marie Lesaffre, Leroy Catherine, Ghaffar Muharram, David Tulasne, Zoulika Kherrouche, Anne Baranzelli, Rémi Montagne, Elisabeth Werkmeister, and Alexis B. Cortot

Subjects
0301 basic medicine, Lung Neoplasms, Blotting, Western, Fluorescent Antibody Technique, medicine.disease_cause, Receptor tyrosine kinase, 03 medical and health sciences, 0302 clinical medicine, Protein Domains, proteolytic cleavages, Cell Line, Tumor, medicine, Humans, Kinase activity, Mutation, biology, Calpain, Epithelial Cells, Proto-Oncogene Proteins c-met, Molecular biology, lung cancer, 030104 developmental biology, Oncology, Protein kinase domain, Gene Knockdown Techniques, 030220 oncology & carcinogenesis, Immunology, MET, receptor tyrosine kinase, biology.protein, Hepatocyte growth factor, hepatocyte growth factor/scatter factor, Carcinogenesis, Signal Transduction, Research Paper, medicine.drug
Abstract

// Remi Montagne 1 , Anne Baranzelli 1, 2 , Ghaffar Muharram 3 , Leroy Catherine 1 , Marie Lesaffre 1 , Audrey Vinchent 1 , Zoulika Kherrouche 1 , Elisabeth Werkmeister 1, 4 , Alexis B. Cortot 1, 2 , David Tulasne 1 1 University Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, F-59000 Lille, France 2 University Lille, CHU Lille, Thoracic Oncology Department, F-59000 Lille, France 3 University Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France 4 BioImaging Center Lille, Lille 59021, France Correspondence to: David Tulasne, email: david.tulasne@ibl.cnrs.fr Keywords: lung cancer, MET, receptor tyrosine kinase, hepatocyte growth factor/scatter factor, proteolytic cleavages Received: September 02, 2016 Accepted: December 26, 2016 Published: January 04, 2017 ABSTRACT The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

Published
2017

26. Rab11A regulates the constitutive secretory pathway during Toxoplasma gondii invasion of host cells and parasite replication

Author

Frank Lafont, Elisabeth Werkmeister, Javier Periz, Sabrina Marion, Gordon Langsley, Jamal Khalife, Nicolas Barois, S. Chehade, Isabelle Tardieux, Kannan Venugopal, and Markus Meissner

Subjects
0303 health sciences, Rhoptry, Constitutive secretory pathway, Motility, Toxoplasma gondii, Biology, biology.organism_classification, Transmembrane protein, Exocytosis, Cell biology, Microneme, 03 medical and health sciences, 0302 clinical medicine, Secretion, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

SummaryToxoplasma gondiipossesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect ofT. gondiivesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of DG and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile parasites suggesting that a Rab11A-dependent apically polarized transport of cargo regulates parasite motility.

Published
2019
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27. Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication

Author

Kannan, Venugopal, Sylia, Chehade, Elisabeth, Werkmeister, Nicolas, Barois, Javier, Periz, Frank, Lafont, Isabelle, Tardieux, Jamal, Khalife, Gordon, Langsley, Markus, Meissner, and Sabrina, Marion

Subjects
Physiology, Cell Membranes, Protozoan Proteins, Microtubules, Microbiology, Parasite Replication, Cell Line, Host-Parasite Interactions, Toxoplasma Gondii, Virology, Cell Adhesion, Medicine and Health Sciences, Animals, Humans, Parasites, Vesicles, Transport Vesicles, Cytoskeleton, Secretion, Protozoans, Cell Membrane, Host Cells, Organisms, Membrane Proteins, Biology and Life Sciences, Eukaryota, Cell Biology, Parasitic Protozoans, Protein Transport, Cell Motility, rab GTP-Binding Proteins, Vacuoles, Parasitology, Cellular Structures and Organelles, Physiological Processes, Toxoplasma, Toxoplasmosis, Viral Transmission and Infection, Research Article
Abstract

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells., Author summary Toxoplasma gondii (T. gondii) is a highly prevalent parasite infecting a wide range of animals as well as humans. T. gondii secretes numerous virulent factors contained in specific organelles, termed the rhoptries, micronemes and dense granules. These factors are released upon host cell recognition and enable parasite invasion and subsequent development into an intracellular vacuole. In particular, dense granules contain critical effectors that modulate intrinsic defenses of infected host cells ensuring parasite survival and dissemination. The mechanisms regulating dense granule secretion have not been elucidated. In this study, we unraveled a novel role for the T. gondii GTPase Rab11A in promoting dense granule transport along the parasite cytoskeleton and their content release into the vacuolar space during parasite replication. We also found that T. gondii Rab11A regulates extracellular parasite motility and adhesion to host cells suggesting a broader role in distinct secretory pathways essential for parasite virulence.

Published
2019

28. Stiffness tomography of eukaryotic intracellular compartments by atomic force microscopy

Author

Michka Popoff, Séverine Divoux, Frank Lafont, Elisabeth Werkmeister, Sébastien Janel, Franck Perez, Nicolas Barois, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Plateforme BioImaging Center Lille (BICeL), Plateformes Lilloises en Biologie et Santé - UAR 2014 - US 41 (PLBS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Compartimentation et dynamique cellulaires (CDC), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), We thank A. Bongiovanni and J. Warein for expert technical assistance. Thanks to Y. Ciczora and S. Bovio for valuable discussions and L. Redondo-Morata for reading the manuscript. The work was funded by grants from the University Lille 1 (MP), ANR-10-EQPX-04-01, and FEDER 12001407 (FL)., ANR-10-EQPX-0004,Imaginex BioMed,Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution(2010), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Plateformes Lilloises en Biologie et Santé - UMS 2014 - US 41 (PLBS), LAFONT, Frank, and Equipements d'excellence - Plateau de microscopie de criblage à haut débit et d'analyse à très haute résolution - - Imaginex BioMed2010 - ANR-10-EQPX-0004 - EQPX - VALID

Subjects
Cytoplasm, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, [SPI.NANO] Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Cell, 02 engineering and technology, Microscopy, Atomic Force, 010402 general chemistry, Microtubules, 01 natural sciences, law.invention, law, Microscopy, Organelle, medicine, Humans, General Materials Science, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Cytoskeleton, ComputingMilieux_MISCELLANEOUS, Cell Nucleus, Viscosity, Chemistry, Cell Membrane, 021001 nanoscience & nanotechnology, Elasticity, 0104 chemical sciences, Characterization (materials science), medicine.anatomical_structure, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, Biophysics, Electron microscope, 0210 nano-technology, Intracellular, HeLa Cells
Abstract

International audience; Precise localization and biophysical characterization of cellular structures is a key to the understanding of biological processes happening both inside the cell and at the cell surface. Atomic force microscopy is a powerful tool to study the cell surface – topography, elasticity, viscosity, interactions – and also the viscoelastic behavior of the underlying cytoplasm, cytoskeleton or the nucleus. Here, we demonstrate the ability of atomic force microscopy to also map and characterize organelles and microorganisms inside cells, at the nanoscale, by combining stiffness tomography with super-resolution fluorescence and electron microscopy. By using this correlative approach, we could both identify and characterize intracellular compartments. The validation of this approach was performed by monitoring the stiffening effect according to the metabolic status of the mitochondria in living cells in real-time.

Published
2019

29. MAGP-1 and fibronectin control EGFL7 functions by driving its deposition into distinct endothelial extracellular matrix locations

Author

Elisabeth Werkmeister, Robert P. Mecham, Nelson Dusetti, Thomas J. Broekelmann, Odile Gayet, Gaëlle Villain, Fabrice Soncin, Chantal Havet, Etienne Lelièvre, Virginie Mattot, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP), Mécanismes de tumorigenèse et thérapies ciblées, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Washington University School of Medicine [Saint Louis, MO], Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), CHU Lille, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), and SONCIN, Fabrice

Subjects
0301 basic medicine, EGF Family of Proteins, RHOA, Leukocyte adhesion molecule, [SDV]Life Sciences [q-bio], extracellular matrix, Notch signaling pathway, Lysyl oxidase, Endothelial Growth Factors, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biochemistry, Protein-Lysine 6-Oxidase, Extracellular matrix, 03 medical and health sciences, Contractile Proteins, fibronectin, Basic Helix-Loop-Helix Transcription Factors, Cell Adhesion, Human Umbilical Vein Endothelial Cells, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Humans, MAGP-1, Molecular Biology, Extracellular Matrix Proteins, Receptors, Notch, biology, Chemistry, Calcium-Binding Proteins, Cell Biology, Fibronectins, Cell biology, Repressor Proteins, Endothelial stem cell, Fibronectin, 030104 developmental biology, biology.protein, EGFL7, RNA Splicing Factors, Signal Transduction
Abstract

International audience; The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.

Published
2018

30. MET amplification increases the metastatic spread of EGFR-mutated NSCLC

Author

Marie Christine Copin, Luc Stoven, Nathalie Marchand, David Tulasne, Alexis B. Cortot, Maeva Kyheng, Simon Baldacci, Vincent Cockenpot, Zoulika Kherrouche, and Elisabeth Werkmeister

Subjects
0301 basic medicine, Pulmonary and Respiratory Medicine, Cancer Research, Lung Neoplasms, Cell, Context (language use), Mice, SCID, medicine.disease_cause, 03 medical and health sciences, Mice, 0302 clinical medicine, In vivo, Cell Movement, Carcinoma, Non-Small-Cell Lung, Cell Line, Tumor, medicine, Animals, Humans, Epithelial–mesenchymal transition, Neoplasm Metastasis, Protein Kinase Inhibitors, Cell Proliferation, Mutation, business.industry, Gene Amplification, Proto-Oncogene Proteins c-met, Phenotype, In vitro, respiratory tract diseases, ErbB Receptors, 030104 developmental biology, medicine.anatomical_structure, Oncology, Cell culture, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis, Cancer research, business
Abstract

Background Five to 20% of metastatic EGFR-mutated non-small cell lung cancers (NSCLC) develop acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) through MET amplification. The effects of MET amplification on tumor and patient phenotype remain unknown. Methods We investigated,in vitro and in vivo, the impact of MET amplification on the biological properties of the HCC827 cell line, derived from an EGFR-mutated NSCLC. We further evaluated the time to new metastases after EGFR-TKI progression in EGFR-mutated NSCLC, exhibiting MET amplification or high MET overexpression. Results MET amplification significantly enhanced proliferation, anchorage independent growth, anoikis resistance, migration, and induced an epithelial to mesenchymal transition. In vivo, MET amplification significantly increased the tumor growth and metastatic spread. Treatment with a MET-TKI reversed this aggressive phenotype. We found that EGFR-mutated NSCLC patients exhibiting MET amplification on a re-biopsy, performed after EGFR-TKI progression, displayed a shorter time to new metastases after EGFR-TKI progression than patients with high MET overexpression but no MET amplification. Conclusion MET amplification increases metastatic spread even in the context of an already pre-existing strong driver mutation such as EGFR mutation. These results prompt development of therapeutic strategies aiming at preventing emergence of MET amplification.

Published
2018

31. ArfGAP1 restricts Mycobacterium tuberculosis entry by controlling the actin cytoskeleton

Author

Michka Popoff, Edouard Yeramian, Priscille Brodin, Samuel Jouny, Nathalie Deboosere, Frank Lafont, Elisabeth Werkmeister, Eik Hoffmann, Alain R. Baulard, Sabrina Marion, Vincent Delorme, Laurent Marsollier, Romain Veyron-Churlet, Ok-Ryul Song, Raffaella Iantomasi, Christophe J. Queval, Isabelle Ricard, ATOMycA (CRCINA-ÉQUIPE 6), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Financial support for this work was provided by the Korean National Research Foundation (K20802001409-09B1300-03700), the European Community (ERC-STG INTRACELLTB Grant no. 260901, MM4TB Grant no. 260872, CycloNHit no. 608407), the Agence Nationale de la Recherche (ANR-10-EQPX-04-01, ANR-14-CE14-0024, ANR-14-CE08-0017, ANR-14-CE14-0027-01, ANR-16-CE35-0009), the Projet Transversal de Recherche de l’Institut Pasteur (PTR441, PTR22-16), the Feder (12001407 (D-AL) Equipex Imaginex BioMed), and the Région Nord Pas de Calais (convention no. 12000080). SM is supported by the Laboratoire d’Excellence (LabEx) ParaFrap ANR-11-LABX-0024 and a Chaire d’Excellence (Université Lille Nord de France/CNRS)., ANR-14-CE14-0024,TB-MET,Rôle des métaux dans la virulence mycobactériennes et les défenses contre la tuberculose(2014), ANR-14-CE08-0017,ANTI-TB-NANO,Une galénique ' verte ' à base de nanoparticules de cyclodextrines pour un traitement plus efficace de la tuberculose(2014), European Project: 260901,EC:FP7:ERC,ERC-2010-StG_20091118,INTRACELLTB(2010), European Project: 260872,EC:FP7:HEALTH,FP7-HEALTH-2010-single-stage,MM4TB(2011), European Project: 608407,EC:FP7:PEOPLE,FP7-PEOPLE-2013-ITN,CYCLON HIT(2014), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bernardo, Elizabeth, Appel à projets générique - Rôle des métaux dans la virulence mycobactériennes et les défenses contre la tuberculose - - TB-MET2014 - ANR-14-CE14-0024 - Appel à projets générique - VALID, Appel à projets générique - Une galénique ' verte ' à base de nanoparticules de cyclodextrines pour un traitement plus efficace de la tuberculose - - ANTI-TB-NANO2014 - ANR-14-CE08-0017 - Appel à projets générique - VALID, A Chemical Genomics Approach of Intracellular Mycobacterium tuberculosis Towards Defining Specific Host Pathogen Interactions - INTRACELLTB - - EC:FP7:ERC2010-12-01 - 2015-11-30 - 260901 - VALID, More Medicines for Tuberculosis - MM4TB - - EC:FP7:HEALTH2011-02-01 - 2016-01-31 - 260872 - VALID, and NANOCARRIERS FOR THE DELIVERY OF ANTIMICROBIAL AGENTS TO FIGHT RESISTANCE MECHANISMS - CYCLON HIT - - EC:FP7:PEOPLE2014-03-01 - 2018-02-28 - 608407 - VALID

Subjects
0301 basic medicine, Polarity & Cytoskeleton, ArfGAP1, [SDV.CAN]Life Sciences [q-bio]/Cancer, phospho- lipase D1 Subject Categories Cell Adhesion, Biochemistry, Microbiology, Mycobacterium tuberculosis, 03 medical and health sciences, Shigella flexneri, [SDV.CAN] Life Sciences [q-bio]/Cancer, Arf1, Genetics, Gene silencing, Yersinia pseudotuberculosis, Molecular Biology, Actin, biology, epithelial cell, Actin cytoskeleton reorganization, biology.organism_classification, Actin cytoskeleton, 3. Good health, Cell biology, Virology & Host Pathogen Interaction, 030104 developmental biology, Phospholipase D1
Abstract

International audience; The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F-actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP-ribosylation factor 1 (Arf1) and phos-pholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M 3 R). We show that this pathway is controlled by Arf GTPase-activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.

Published
2018

32. Rab11A regulates the constitutive secretory pathway duringToxoplasma gondiiinvasion of host cells and parasite replication

Author

Kannan, Venugopal, primary, Sylia, Chehade, additional, Elisabeth, Werkmeister, additional, Nicolas, Barois, additional, Javier, Periz, additional, Frank, Lafont, additional, Isabelle, Tardieux, additional, Jamal, Khalife, additional, Langsley, Gordon, additional, Markus, Meissner, additional, and Sabrina, Marion, additional

Published
2019
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33. Structured illumination microscopy and correlative microscopy to study autophagy

Author

Laure-Anne Ligeon, Frank Lafont, Elisabeth Werkmeister, Nicolas Barois, and Antonino Bongiovanni

Subjects
Autophagosome, Cytoplasm, Autophagy, STED microscopy, Vacuole, Biology, Yersinia, General Biochemistry, Genetics and Molecular Biology, law.invention, Cell biology, Microscopy, Electron, law, Phagosomes, Microscopy, Ultrastructure, Electron microscope, Microtubule-Associated Proteins, Molecular Biology
Abstract

Autophagy is a predominant eukaryotic mechanism for the engulfment of "portions" of cytoplasm allowing their degradation to recycle metabolites. The autophagy is ubiquitous among the life kingdom revealing the importance of this pathway that appears more complex than previously thought. Several reviews have already addressed how to monitor this pathway and have highlighted the existence of new routes such as the LC3-associated phagocytosis (LAP) and the non-canonical autophagy. The principal difference between autophagosomes and LAP vacuoles is that the former has two limiting membranes positives for LC3 whereas the latter has one. Herein, we propose to emphasize the use of correlative light electron microscopy (CLEM) to answer some autophagy's related questions. The structured illumination microscopy (SIM) relatively easy to implement allows to better observe the Atg proteins recruitment and localization during the autophagy process. While LC3 recruitment is performed using light microscopy the ultrastructural morphological analysis of LC3-vacuoles is ascertained by electron microscopy. Hence, these combined and correlated approaches allow to tackle the LAP vs. autophagosome issue.

Published
2015

34. CLAFEM: Correlative light atomic force electron microscopy

Author

Sébastien, Janel, Elisabeth, Werkmeister, Antonino, Bongiovanni, Frank, Lafont, and Nicolas, Barois

Subjects
Microscopy, Fluorescence, Yersinia pseudotuberculosis, Animals, Humans, Microscopy, Atomic Force, Cell Line
Abstract

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

Published
2017

35. ArfGAP1 restricts

Author

Ok-Ryul, Song, Christophe J, Queval, Raffaella, Iantomasi, Vincent, Delorme, Sabrina, Marion, Romain, Veyron-Churlet, Elisabeth, Werkmeister, Michka, Popoff, Isabelle, Ricard, Samuel, Jouny, Nathalie, Deboosere, Frank, Lafont, Alain, Baulard, Edouard, Yeramian, Laurent, Marsollier, Eik, Hoffmann, and Priscille, Brodin

Subjects
Receptor, Muscarinic M3, GTPase-Activating Proteins, Scientific Reports, Mycobacterium tuberculosis, Actins, Polymerization, Shigella flexneri, Pulmonary Alveoli, Actin Cytoskeleton, Gene Expression Regulation, Species Specificity, A549 Cells, Yersinia pseudotuberculosis, Host-Pathogen Interactions, Phospholipase D, Humans, ADP-Ribosylation Factor 1, RNA, Small Interfering, Signal Transduction
Abstract

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.

Published
2017

36. Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division

Author

Frank Lafont, Elisabeth Werkmeister, Jean-Michel Saliou, Kannan Venugopal, Sabrina Marion, Anaïs F. Poncet, Gordon Langsley, Mohamed-Ali Hakimi, Ludovic Huot, Nicolas Barois, and Fabien Sindikubwabo

Subjects
0301 basic medicine, Life Cycles, Cell division, Clathrin adaptor complex, Cell Membranes, Protozoan Proteins, Gene Expression, Golgi Apparatus, Parasitic Cell Cycles, Mass Spectrometry, Toxoplasma Gondii, Gene Knockout Techniques, 0302 clinical medicine, Cell Cycle and Cell Division, lcsh:QH301-705.5, Protozoans, 3. Good health, Transport protein, Cell biology, Protein Transport, Cell Processes, Cellular Structures and Organelles, Toxoplasma, Cell Division, trans-Golgi Network, Research Article, lcsh:Immunologic diseases. Allergy, Protein subunit, Parasitic Life Cycles, Immunology, Adaptor Protein Complex 1, Endosomes, Biology, Microbiology, Clathrin, Models, Biological, Parasite Replication, Microneme, 03 medical and health sciences, Virology, Genetics, Animals, Vesicles, Molecular Biology, Cytokinesis, Organelles, Rhoptry, Organisms, Biology and Life Sciences, Membrane Proteins, Cell Biology, Parasitic Protozoans, 030104 developmental biology, lcsh:Biology (General), Vacuoles, biology.protein, Parasitology, lcsh:RC581-607, 030217 neurology & neurosurgery, Developmental Biology
Abstract

Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis., Author summary The phylum Apicomplexa comprises a large group of obligate intracellular parasites of wide human and agricultural significance. Most notable are Plasmodium, the causative agent of malaria, and Toxoplasma gondii, one of the most common human parasites, responsible for disease of the developing fetus and immune-compromised individuals. Apicomplexa are characterized by the presence of an apical complex consisting of secretory organelles named micronemes (MIC) and rhoptries (ROP). MIC and ROP proteins, released upon host cell recognition, are essential for host cell invasion and parasite survival. After invasion, these organelles are neo-synthesized at each parasite replication cycle. In our study, we demonstrate a crucial role for the T. gondii clathrin adaptor complex AP1 in the vesicular transport of neo-synthesized MIC and ROP proteins, thereby regulating mature apical organelle formation. In addition, we unravel an original role for TgAP1 in the late stages of the parasite division process during daughter cell segregation. Therefore, our study provides new insights into key regulatory mechanisms of the vesicular trafficking system essential for host invasion and intracellular survival of Toxoplasma gondii.

Published
2017

37. Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models

Author

Nicolas Malmanche, Pierre Dourlen, Marc Gistelinck, Florie Demiautte, Nichole Link, Cloé Dupont, Lies Vanden Broeck, Elisabeth Werkmeister, Philippe Amouyel, Antonino Bongiovanni, Hélène Bauderlique, Dieder Moechars, Anne Royou, Hugo J. Bellen, Frank Lafont, Patrick Callaerts, Jean-Charles Lambert, and Bart Dermaut

Subjects
PROTEIN-TAU, Cell Survival, Mitosis, tau Proteins, COPY-NUMBER VARIATION, Article, Cell Line, CELL-CYCLE ACTIVATION, Neural Stem Cells, mental disorders, Medicine and Health Sciences, Animals, Humans, Protein Isoforms, Photoreceptor Cells, TAU PROMOTES NEURODEGENERATION, PHOSPHORYLATION, IN-VIVO, GENE-EXPRESSION, Neurons, Gene Expression Regulation, Developmental, Biology and Life Sciences, PROGRESSIVE SUPRANUCLEAR PALSY, Aneuploidy, ALZHEIMERS-DISEASE, Phenotype, Tauopathies, MITOSIS, Mutation
Abstract

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development. ispartof: Scientific Reports vol:7 pages:40764- ispartof: location:England status: published

Published
2017

38. CLAFEM

Author

Sébastien Janel, Nicolas Barois, Frank Lafont, Elisabeth Werkmeister, and Antonino Bongiovanni

Subjects
0301 basic medicine, Super-resolution microscopy, Scanning confocal electron microscopy, Nanotechnology, Conductive atomic force microscopy, Biology, Dark field microscopy, Cell biology, 03 medical and health sciences, 030104 developmental biology, Light sheet fluorescence microscopy, Microscopy, Scanning ion-conductance microscopy, Energy filtered transmission electron microscopy
Abstract

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

Published
2017

39. Non-Invasive Second Harmonic Generation (SHG) in Macroscopy (MacroSHG) as Bio-Diagnosis to Image Collagen Network Organization in Extracellular Matrix

Author

Dominique Dumas, Sébastien Hupont, Patrick Menu, Didier Mainard, Céline Huselstein, Marthe Rousseau, Natalia de Isla, and Elisabeth Werkmeister

Subjects
Coupling (electronics), Extracellular matrix, animal structures, Materials science, Two-photon excitation microscopy, Collagen network, Second-harmonic generation, Matrix (biology), Signal, Fluorescence, Biomedical engineering
Abstract

In this work, we have considered a new multimodality imaging for macroscopy based on Second Harmonic Generation (SHG) method to monitor invasivelessly the matrix collagen. As the triple helicoidally structure of collagen molecules appearing as not centrosymetric, very organized and spatially oriented, collagen fibrils give rise to a very strong SHG signal and can be imaged without any exogenous dye. To integrate a multidimensional scale with a large field of view (non-sliced samples), we have adapted and validated an instrumental coupling between a two photon excitation laser and a macroscope to collect cartography of SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for respectively Fluorescence (F) and Second Harmonic Generation (SHG) values. For various sample where protein collagen is the major component of extracellular matrix (vessel, skin, carotide vessel, rat femoral head cartilage, mouse tumor, human wharton’s jelly and rat tendon) or not (nacre), we compared the index distribution obtained with MacroSHG. In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to Multimodality approaches (SHG-TCSPC) could be an innovative and non-invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

Published
2014

40. Phenotypic assays for Mycobacterium tuberculosis infection

Author

Ok-Ryul, Song, Nathalie, Deboosere, Vincent, Delorme, Christophe J, Queval, Gaspard, Deloison, Elisabeth, Werkmeister, Frank, Lafont, Alain, Baulard, Raffaella, Iantomasi, and Priscille, Brodin

Subjects
Male, Mice, Inbred C57BL, Mice, RAW 264.7 Cells, Macrophages, Drug Discovery, Antitubercular Agents, Animals, Tuberculosis, Biological Assay, Mycobacterium tuberculosis, Cells, Cultured, Fluorescence
Abstract

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.

Published
2016

41. EWI-2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry

Author

Cyril Couturier, Laura Kaestner, Eric Rubinstein, Laurence Cocquerel, Thomas F. Baumert, Costin-Ioan Popescu, Pierre-Emmanuel Milhiet, Claire Montpellier, Elisabeth Werkmeister, Jean Dubuisson, Patrice Rassam, Birke Andrea Tews, and Julie Potel

Subjects
0303 health sciences, viruses, Hepatitis C virus, 030302 biochemistry & molecular biology, Immunology, Colocalization, chemical and pharmacologic phenomena, Context (language use), biochemical phenomena, metabolism, and nutrition, Biology, medicine.disease_cause, Microbiology, Molecular biology, biological factors, 3. Good health, Cell biology, 03 medical and health sciences, Antigen, Tetraspanin, Cell culture, Virology, medicine, Receptor, 030304 developmental biology, CD81
Abstract

CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin-enriched areas in the plasma membrane. In our study, we combined single-molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI-2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI-2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81 molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI-2wint promotes CD81 clustering and confinement in CD81-enriched areas. In addition, we showed that EWI-2wint influences the colocalization of CD81 with Claudin-1 - a co-receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI-2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.

Published
2013

42. Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

Author

Christian Slomianny, Elisabeth Werkmeister, Mathieu Gissot, Thomas Mouveaux, Kevin M. Lesage, Franck Lafont, Jean-Michel Saliou, Maurine Bonabaud, Marine Rohmer, Flavie Courjol, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 (PHYCELL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioCampus (BCM), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Protozoan Proteins, Toxoplasma gondii, Biology, RNA Transport, 03 medical and health sciences, Cellular and Molecular Neuroscience, Gene expression, medicine, RNA, Ribosomal, 18S, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Parasites, Nuclear protein, Nuclear pore, Molecular Biology, Pharmacology, Cell Nucleus, Microscopy, Nucleoporins, High-Throughput Nucleotide Sequencing, Cell Biology, Chromatin, Transport protein, Cell biology, Nuclear pore complex, Nuclear Pore Complex Proteins, Cell nucleus, Protein Transport, 030104 developmental biology, medicine.anatomical_structure, Phenotype, Gene Expression Regulation, Gene Knockdown Techniques, Nuclear Pore, Molecular Medicine, Nucleoporin, Nuclear transport, CRISPR-Cas Systems, Apicomplexa, Toxoplasma, Protein Binding
Abstract

International audience; The nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes.

Published
2016

43. ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease

Author

Pierre Dourlen, Yves Lemoine, Amandine Flaig, Frédéric Checler, Anne Marie Ayral, Jean-Charles Lambert, Kris Gevaert, Mathias Laga, Fanny Eysert, Tiago Mendes, David M. A. Mann, Linda Chami, Anne Sophie Hérard, Thierry Delzescaux, Franck Lafont, Florie Demiautte, Franck Hansmannel, Benoit Deprez, Charlotte Delay, David Hot, Florence Leroux, Bart Dermaut, Philippe Amouyel, Ludovic Huot, Yoann Sottejeau, Florence Pasquier, Nicolas Souedet, Florent Letronne, Charlotte Bauer, Marc Dhenain, Marie Lesaffre, Geoffroy Laumet, Nicolas Malmanche, David Tulasne, Claudine Berr, Rebecca Deprez, Julie Dumont, Elisabeth Werkmeister, Michel E. Vandenberghe, Jean Jacques Hauw, Julien Chapuis, Epidémiologie des maladies chroniques : impact des interactions gène environnement sur la santé des populations, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Droit et Santé, Facteurs de Risque et Déterminants Moléculaires des Maladies liées au Vieillissement - U 1167 (RID-AGE), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Vlaams Instituut voor Biotechnologie [Ghent, Belgique] (VIB), Universiteit Gent = Ghent University (UGENT), Laboratoire des Maladies Neurodégénératives - UMR 9199 (LMN), Service MIRCEN (MIRCEN), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Médicaments et molécules pour agir sur les Systèmes Vivants - U 1177 (M2SV), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Mécanismes de la Tumorigénèse et Thérapies Ciblées - UMR 8161 (M3T), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Réseau International des Instituts Pasteur (RIIP), Nutrition-Génétique et Exposition aux Risques Environnementaux (NGERE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Service d'Hépato-gastro-entérologie [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), CEA [Fontenay-aux-Roses] (UGRA / SETA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Troubles cognitifs dégénératifs et vasculaires - U 1171 - EA 1046 (TCDV), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Droit et Santé-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Neuropsychiatrie : recherche épidémiologique et clinique (PSNREC), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Laboratoire de Neuropathologie Raymond Escourolle [CHU Pitié-Salpétriêre], Université Pierre et Marie Curie - Paris 6 (UPMC)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Institute of Brain, Behaviour and Mental Health, University of Manchester [Manchester], Hérard, Anne-Sophie, Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Troubles cognitifs dégénératifs et vasculaires - U 1171 (TCDV), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), CDithem Platform, IGM, CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U1172 Inserm - U837 (JPArc), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Lille Nord de France (COMUE)-Université de Lille, Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Récepteurs nucléaires, maladies cardiovasculaires et diabète - U 1011 (RNMCD), Institut Fédératif de Recherche 142, Institut Fédératif de Recherche, Center for Medical Genetics [Ghent], Ghent University Hospital, Département de neurologie, Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Neuropathologie Raymond Escourolle, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Division of Neuroscience and Experimental Psychology [Salford, UK], University of Manchester [Manchester]-Salford Royal NHS Foundation Trust [Salford, UK], Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Dept Med Prot Res, Flanders Institute for Biotechnology, Département Imagerie et Simulation pour le Contrôle (DISC), Laboratoire d'Intégration des Systèmes et des Technologies (LIST), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Laboratoire des Maladies Neurodégénératives - UMR 9199 [LMN], Institut de pharmacologie moléculaire et cellulaire [IPMC], Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer - U837 [JPArc], Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 [CIIL], Récepteurs nucléaires, maladies cardiovasculaires et diabète - U 1011 [RNMCD], Facteurs de Risque et Déterminants Moléculaires des Maladies liées au Vieillissement - U 1167 [RID-AGE], Nutrition-Génétique et Exposition aux Risques Environnementaux [NGERE], CEA [Fontenay-aux-Roses] [UGRA / SETA], and Institut de biologie de Lille - IBL [IBLI]

Subjects
0301 basic medicine, Patch-Clamp Techniques, ADAM, A Disintegrin and Metalloproteinase Domain, lcsh:Medicine, Cathepsin D, COFRADIC, combined fractional diagonal chromatography, ALPHA-SECRETASE, Mice, 0302 clinical medicine, Pepstatins, Amyloid precursor protein, BRAIN, RNA, Small Interfering, Tissues and Organs (q-bio.TO), GENE-EXPRESSION, lcsh:R5-920, ROLES, IRS4, insulin receptor substrate 4, P3 peptide, Brain, Long-term potentiation, General Medicine, AMYLOID PRECURSOR PROTEIN, Alpha secretase, BACE, Beta-site APP cleaving enzyme 1, Neurons and Cognition (q-bio.NC), [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], RNA Interference, Macrolides, LTP, lcsh:Medicine (General), GKAP1, G kinase-anchoring protein 1, Research Paper, Genetically modified mouse, Amyloid, CTSD, cathepsin D, Down-Regulation, Mice, Transgenic, Biology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, TD2, Type 2 diabetes, Alzheimer Disease, APP, amyloid precursor protein, Cell Line, Tumor, Animals, Humans, Secretion, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Amino Acid Sequence, TRANSGENIC MOUSE MODEL, Amyloid beta-Peptides, PLA, proximity ligation assay, lcsh:R, LTP, long term potentiation, Biology and Life Sciences, Quantitative Biology - Tissues and Organs, ADAM30, DEGRADATION, CamKIIα, Ca2 +/calmodulin-dependent protein kinase II alpha, Molecular biology, Mice, Inbred C57BL, MICE, ADAM Proteins, 030104 developmental biology, HEK293 Cells, Metabolism, Microscopy, Fluorescence, Quantitative Biology - Neurons and Cognition, FOS: Biological sciences, COGNITIVE DECLINE, biology.protein, Alzheimer, BSA, bovine serum albumin, MMP, metalloproteinase, INHIBITORS, Lysosomes, APP, MRI, magnetic resonance imaging, 030217 neurology & neurosurgery
Abstract

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development., Highlights • By transcriptomic analyses, low levels of ADAM30 expression was observed in the brain of AD cases (compared with controls). • Activation of cathepsin D by ADAM30 is required to modulate the APP metabolism in vitro, i.e. decrease in Ab secretion. • In AD-like mice, neuronal ADAM30 expression led to lower soluble Aβ42, amyloid plaques and to rescue long term potentiation The amyloid precursor protein (APP) processing is central in the etiology of Alzheimer's disease (AD). Characterizing the actors of this metabolism is thus an important challenge. We searched for proteins involved in the APP processing and we selected the A disintegrin-like and metalloprotease 30 (ADAM30). Our results revealed an axis in APP metabolism pathway through an ADAM30-dependent cathepsin D activation. When activated following ADAM30 expression, a decrease in Aβ secretion occurred in vitro and in vivo. ADAM30 under-expression observed in AD brains and thus the impairment of the ADAM30-dependent pathway may favor Aβ peptide production and, ultimately, AD development.

Published
2016

44. Unconventional endosome-like compartment and retromer complex in Toxoplasma gondii govern parasite integrity and host infection

Author

Mohamed-Ali Hakimi, Frank Lafont, Elisabeth Werkmeister, Fabien Sindikubwabo, Lamba Omar Sangaré, Alain Van Dorsselaer, Tchilabalo Dilezitoko Alayi, Christian Slomianny, Isabelle Callebaut, Agnès Hovasse, Christine Schaeffer-Reiss, Benoit Westermann, Stanislas Tomavo, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Protéomique et Peptides Modifiés (P3M), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de Lille - UMS 3702 (IBL), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 (PHYCELL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Bioimaging platform (IBL), Université de Lille, HAL UPMC, Gestionnaire, and Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Genetics, Multidisciplinary, Retromer, Endosome, Science, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Endocytic recycling, General Chemistry, Biology, General Biochemistry, Genetics and Molecular Biology, Transmembrane protein, Major facilitator superfamily, 3. Good health, Cell biology, Retromer complex, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 030104 developmental biology, parasitic diseases, Organelle biogenesis, Biogenesis
Abstract

Membrane trafficking pathways play critical roles in Apicomplexa, a phylum of protozoan parasites that cause life-threatening diseases worldwide. Here we report the first retromer-trafficking interactome in Toxoplasma gondii. This retromer complex includes a trimer Vps35–Vps26–Vps29 core complex that serves as a hub for the endosome-like compartment and parasite-specific proteins. Conditional ablation of TgVps35 reveals that the retromer complex is crucial for the biogenesis of secretory organelles and for maintaining parasite morphology. We identify TgHP12 as a parasite-specific and retromer-associated protein with functions unrelated to secretory organelle formation. Furthermore, the major facilitator superfamily homologue named TgHP03, which is a multiple spanning and ligand transmembrane transporter, is maintained at the parasite membrane by retromer-mediated endocytic recycling. Thus, our findings highlight that both evolutionarily conserved and unconventional proteins act in concert in T. gondii by controlling retrograde transport that is essential for parasite integrity and host infection.

Published
2016

45. Toxoplasma Sortilin-like Receptor Regulates Protein Transport and Is Essential for Apical Secretory Organelle Biogenesis and Host Infection

Author

Christine Schaeffer-Reiss, Tchilabalo Dilezitoko Alayi, Alain Van Dorsselear, Agnès Hovasse, Isabelle Callebaut, Rajshekhar Y. Gaji, Pierre-Julien Sloves, Thomas Mouveaux, Vern B. Carruthers, Stéphane Delhaye, Elisabeth Werkmeister, Christian Slomianny, Stanislas Tomavo, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 (PHYCELL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Department of Microbiology and Immunology, University of Michigan Medical School, University of Michigan [Ann Arbor], University of Michigan System-University of Michigan System, Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cancer Research, Biology, Models, Biological, Microbiology, Microneme, Mice, 03 medical and health sciences, 0302 clinical medicine, Virology, Immunology and Microbiology(all), Protein Interaction Mapping, Animals, Humans, Molecular Biology, Cells, Cultured, 030304 developmental biology, Organelles, Mice, Inbred BALB C, 0303 health sciences, Rhoptry, Membrane Proteins, Signal transducing adaptor protein, Survival Analysis, Transmembrane protein, 3. Good health, Cell biology, Transport protein, Adaptor Proteins, Vesicular Transport, Protein Transport, Toxoplasmosis, Animal, Cytoplasm, Female, Parasitology, Organelle biogenesis, Toxoplasma, 030217 neurology & neurosurgery, Biogenesis, Protein Binding
Abstract

International audience; Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles.TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.

Published
2012
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46. Collagenous Extracellular Matrix of Cartilage Submitted to Mechanical Forces Studied by Second Harmonic Generation Microscopy

Author

Dominique Dumas, Jean-François Stoltz, Patrick Netter, Elisabeth Werkmeister, Natalia de Isla, Plate-forme Imagerie et Biophysique Cellulaire et Tissulaire (PTIBC-IBISA Nancy), Université Henri Poincaré - Nancy 1 (UHP), Physiopathologie, Pharmacologie et Ingénierie articulaires (PPIA), Université Henri Poincaré - Nancy 1 (UHP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de physiologie et Pathologie articulaire, Faculte de Medecine, Vandoeuvre-les-Nancy, Laboratoire Énergies et Mécanique Théorique et Appliquée (LEMTA ), and Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0206 medical engineering, Nanotechnology, 02 engineering and technology, MESH: Extracellular Matrix, Biochemistry, Extracellular matrix, 03 medical and health sciences, MESH: Osteoarthritis, MESH: Collagen, Osteoarthritis, Microscopy, Collagen network, Cartilaginous Tissue, medicine, Physical and Theoretical Chemistry, MESH: Biomechanics, 030304 developmental biology, 0303 health sciences, Chemistry, Cartilage, General Medicine, Second Harmonic Generation Microscopy, 020601 biomedical engineering, Biomechanical Phenomena, Extracellular Matrix, Autofluorescence, Microscopy, Fluorescence, Multiphoton, medicine.anatomical_structure, MESH: Cartilage, MESH: Microscopy, Fluorescence, Multiphoton, Collagenase, [SDV.IB]Life Sciences [q-bio]/Bioengineering, Collagen, Biomedical engineering, medicine.drug
Abstract

International audience; Osteoarthritis is a degenerative pathology leading to degradation of the extracellular matrix (ECM). Similar effects can be visualized when applying mechanical or biochemical constraints on cartilaginous tissue. Here, we characterized modification of the ECM appearing under mechanical compression and/or biochemical action (hypoxia environment, nitric oxide and collagenase action). In recent decades, multiphoton microscopy has proved its interest for observing living, thick and opaque biological tissues. Thus, the main components of the cartilaginous ECM can be observed without fluorescent labeling. In particular, the collagen network emits strong second harmonic generation (SHG) signal which could be collected at half of the excitation wavelength. Combining autofluorescence and SHG signal detection enables to obtain complementary structural information. Here, we proved that multiphoton microscopy represents an appropriate tool for ex vitro cartilage imaging. First, we showed that SHG signal specifically comes from collagen (collagenase digestion). Further, we verified that the use of an appropriate band-pass filter enables to reject the autofluorescence from the ECM. Once this specificity was shown, we followed modification of the cartilage ECM submitted to mechanical or biochemical constraints (compression, enzymatic digestion). By performing textural analysis of SHG images (Haralick's method), we showed the restructuration of the collagen network according to constraints.

Published
2010

47. Metamplification induces an aggressive phenotype in EGFR tyrosine kinase inhibitors resistant non-small-cell lung cancer

Author

Luc Stoven, Elisabeth Werkmeister, David Tulasne, Alexis B. Cortot, Nathalie Marchand, Simon Baldacci, and Zoulika Kherrouche

Subjects
Cell division, biology, business.industry, Clone (cell biology), Context (language use), medicine.disease, Phenotype, Molecular biology, respiratory tract diseases, Tumor progression, Cell culture, Cancer research, biology.protein, Medicine, Epidermal growth factor receptor, business, Lung cancer
Abstract

Introduction: Treatment of Non-Small-Cell Lung Cancer (NSCLC) harboring an Epidermal Growth Factor Receptor (EGFR) mutation relies on EGFR tyrosine kinase inhibitors (TKI). However all patients treated with EGFR TKI eventually present tumor progression, because of mechanisms of resistance such as amplification of the met receptor gene. There is currently no data on cellular phenotypic changes of tumor cells induced by met amplification in this context. Objective: to determine whether met amplification triggers a more aggressive tumor phenotype in TKI resistant EGFR-mutated NSCLC. Methods: The proliferation and migration capacities were studied in HCC827 cell line, derived from a mutated EGFR NSCLC, and his daughter cell line HCC827 GR6 which is resistant to EGFR TKI through amplification of the met gene. Tumor growth of both strains was analyzed in a mouse model of ectopic xenograft. Results: in vitro, we found a similar proliferation in the two cell lines and a significant increase in migration and anchorage-independant growth in the met amplified cell line. Treatment with PHA-665752, a MET TKI, significantly reduced the migration advantage of met amplified cells. In vivo, we observed a significant increase in tumor growth with the met amplified cell line. In vivo results regarding the metastatic potential of the two cell lines and the impact of EGFR and MET TKI will be presented during the meeting. Conclusion: met amplification confers an aggressive phenotype to TKI resistant EGFR-mutated NSCLC. These results pave the way for an early use of MET inhibitors in association with EGFR TKI in order to prevent emergence of an aggressive tumor clone.

Published
2015

48. PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met-addicted tumor cells

Author

Luc Stoven, Yvan de Launoit, Elisabeth Werkmeister, David Tulasne, Alexis B. Cortot, Zoulika Kherrouche, Anne Chotteau-Lelievre, Didier Monté, Mécanismes de tumorigenèse et thérapies ciblées, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé-Université de Lille, Sciences et Technologies, Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé, Site de Recherche Intégrée en Cancérologie (SIRIC-ONCOLille), Université de Lille, Sciences et Technologies-Université de Lille, Sciences Humaines et Sociales-Centre Régional de Lutte contre le Cancer Oscar Lambret [Lille] (UNICANCER/Lille), Université Lille Nord de France (COMUE)-UNICANCER-Université Lille Nord de France (COMUE)-UNICANCER-Cancéropole Nord-Ouest-Université de Lille, Droit et Santé-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Service de pneumologie et oncologie thoracique, Hôpital Calmette-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), This work was supported by the CNRS, the Institut Pasteur de Lille, and INSERM, and by grants from the 'Ligue contre le Cancer, comité Nord et comité Aisne', the 'Association pour la Recherche sur le Cancer', the 'Institut National du Cancer (PLBIO 2012 – DA N°2012‐116)', the 'Cancéropôle Nord‐Ouest' and the SIRIC ONCOLille. We thank the Microscopy‐Imaging‐Cytometry Facility of the BioImaging Center Lille Nord‐de‐France for access to instruments and technical advice., We would like to thank M. Duterque‐Coquillaud and N. Malaquin for providing several primers and antibodies, Professor P.A. Jänne for providing HCC827 and HCC‐GR6 cell lines, J.L. Baert, A. Verger and E. Lelievre for helpful discussions., Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), and Université de Lille-UNICANCER-Université de Lille-UNICANCER-Cancéropole Nord-Ouest-Université de Lille, Droit et Santé-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)

Subjects
Cancer Research, [SDV]Life Sciences [q-bio], Receptor tyrosine kinase, MESH: Gene Amplification, ETV1, MESH: Neoplasms/metabolism, Cell Movement, Neoplasms, MESH: Cell Movement, Lung, Migration, MESH: Transcription Factors/metabolism, biology, MESH: Neoplasms/pathology, PEA3, General Medicine, MESH: Gene Expression Regulation, Neoplastic, Articles, Proto-Oncogene Proteins c-met, MESH: Neoplasm Invasiveness/genetics, Gene Expression Regulation, Neoplastic, Oncology, MESH: Proto-Oncogene Proteins c-met/genetics, Met, Molecular Medicine, Signal transduction, Tyrosine kinase, Signal Transduction, MESH: Cell Line, Tumor, MESH: Proto-Oncogene Proteins c-met/metabolism, EGFR, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Cell Line, Tumor, Genetics, Gene silencing, Humans, Neoplasm Invasiveness, MESH: Transcription Factors/genetics, Transcription factor, MESH: Humans, Gene Amplification, Lung tumorigenesis, MESH: Signal Transduction, body regions, tumorigenesis, Hepatocyte Growth Factor Receptor, Cancer cell, biology.protein, Cancer research, MESH: Neoplasm Invasiveness/pathology, MESH: Neoplasms/genetics, ETS, Transcription Factors
Abstract

International audience; Various solid tumors including lung or gastric carcinomas display aberrant activation of the Met receptor which correlates with aggressive phenotypes and poor prognosis. Although downstream signaling of Met is well described, its integration at the transcriptional level is poorly understood. We demonstrate here that in cancer cells harboring met gene amplification, inhibition of Met activity with tyrosine kinase inhibitors or specific siRNA drastically decreased expression of ETV1, ETV4 and ETV5, three transcription factors constituting the PEA3 subgroup of the ETS family, while expression of the other members of the family were less or not affected. Similar link between Met activity and PEA3 factors expression was found in lung cancer cells displaying resistance to EGFR targeted therapy involving met gene amplification. Using silencing experiments, we demonstrate that the PEA3 factors are required for efficient migration and invasion mediated by Met, while other biological responses such as proliferation or unanchored growth remain unaffected. PEA3 overexpression or silencing revealed that they participated in the regulation of the MMP2 target gene involved in extracellular matrix remodeling. Our results demonstrated that PEA3-subgroup transcription factors are key players of the Met signaling integration involved in regulation of migration and invasiveness.

Published
2015

49. Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to LC3-associated pathways involving single- or double-membrane vacuoles

Author

Thierry Galli, Laure-Anne Ligeon, Nicolas Barois, Delphine-Armelle Lacorre, Frank Lafont, Elisabeth Werkmeister, Véronique Proux-Gillardeaux, Antonino Bongiovanni, and Kevin Moreau

Subjects
Autophagosome, Vesicle-Associated Membrane Protein 3, media_common.quotation_subject, Vacuole, Yersinia, Cell Line, R-SNARE Proteins, Phagosomes, Autophagy, Yersinia pseudotuberculosis, Humans, Internalization, Molecular Biology, Phagosome, media_common, biology, Macrophages, Cell Biology, biology.organism_classification, Basic Research Paper, Cell biology, Microscopy, Electron, Membrane protein, Vacuoles, Microtubule-Associated Proteins, Signal Transduction
Abstract

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3′s association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis’ intravesicular life cycle.

Published
2014

50. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival

Author

Pascual Sanz, M. Adelaida García-Gimeno, Jérôme Vicogne, Benjamin Bertin, Jamal Khalife, Alexis Verger, Aline Fréville, Elisabeth Werkmeister, Christine Pierrot, Muriel Montbarbon, Hadidjatou Kalamou, Isabelle Landrieu, Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Instituto de biomedicina [Valencia] (IBV), Consejo Superior de Investigaciones Científicas [Spain] (CSIC), Institut de biologie de Lille - IBL (IBLI), Université de Lille, Sciences et Technologies-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé, Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé-Université de Lille, Sciences et Technologies, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Cell Nucleus, Protein family, Phosphatase, Plasmodium falciparum, Protozoan Proteins, PP1 regulator, Saccharomyces cerevisiae, Biochemistry, Microbiology, Protein–protein interaction, 03 medical and health sciences, Protein phosphatase 1, MESH: Intracellular Signaling Peptides and Proteins, Protein Phosphatase 1, parasitic diseases, MESH: Protein Binding, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Enzyme Inhibitors, Molecular Biology, MESH: Protozoan Proteins, MESH: Plasmodium falciparum, 030304 developmental biology, Cell Nucleus, 0303 health sciences, MESH: Genetic Complementation Test, MESH: Humans, biology, Cell growth, MESH: Protein Phosphatase 1, 030302 biochemistry & molecular biology, Genetic Complementation Test, Intracellular Signaling Peptides and Proteins, Cell Biology, biology.organism_classification, Inhibitor-3, MESH: Saccharomyces cerevisiae, Yeast, In vitro, 3. Good health, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Enzyme Inhibitors, Protein Binding
Abstract

16 páginas, 9 figuras. Contiene en material suplementario: Figs. S1–S3 and Tables S1 and S2."This research was originally published in Journal of Biological Chemistry. Fréville A, Landrieu I, García-Gimeno MA, Vicogne J, Montbarbon M, Bertin B, Verger A, Kalamou H, Sanz P, Werkmeister E, Pierrot C, Khalife J. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival. J Biol Chem. 2012 Jan 6;287(2):1306-21. © the American Society for Biochemistry and Molecular Biology", Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1., The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry, and the University of Sciences and Technologies of Lille I.This work was supported in part by Inserm, Institut Pasteur de Lille, Université Lille Nord de France, and TGE RMN THC Grant FR-3050, France

Published
2011

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