Laboratoire de Spectrométrie de Masse Protéomique (LSMP)

Publications

Année de publication : 2015

Marie-Thérèse Prospéri, Priscilla Lépine, Florent Dingli, Perrine Paul-Gilloteaux, René Martin, Damarys Loew, Hans-Joachim Knölker, Evelyne Coudrier (2015 Jul 22)

Myosin 1b functions as an effector of EphB signaling to control cell repulsion.

The Journal of cell biology : 347-61 : DOI : 10.1083/jcb.201501018 En savoir plus
Résumé

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.

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Alessandra Lo Cicero, Cédric Delevoye, Floriane Gilles-Marsens, Damarys Loew, Florent Dingli, Christelle Guéré, Nathalie André, Katell Vié, Guillaume van Niel, Graça Raposo (2015 Jun 25)

Exosomes released by keratinocytes modulate melanocyte pigmentation.

Nature communications : 7506 : DOI : 10.1038/ncomms8506 En savoir plus
Résumé

Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

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Frédérique Rau, Jeanne Lainé, Laetitita Ramanoudjame, Arnaud Ferry, Ludovic Arandel, Olivier Delalande, Arnaud Jollet, Florent Dingli, Kuang-Yung Lee, Cécile Peccate, Stéphanie Lorain, Edor Kabashi, Takis Athanasopoulos, Taeyoung Koo, Damarys Loew, Maurice S Swanson, Elisabeth Le Rumeur, George Dickson, Valérie Allamand, Joëlle Marie, Denis Furling (2015 May 29)

Abnormal splicing switch of DMD’s penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

Nature communications : 7205 : DOI : 10.1038/ncomms8205 En savoir plus
Résumé

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1.

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Ewa Kotula, Nathalie Berthault, Celine Agrario, Marie-Christine Lienafa, Anthony Simon, Florent Dingli, Damarys Loew, Vonick Sibut, Simon Saule, Marie Dutreix (2015 May 29)

DNA-PKcs plays role in cancer metastasis through regulation of secreted proteins involved in migration and invasion.

Cell cycle (Georgetown, Tex.) : 1961-72 : DOI : 10.1080/15384101.2015.1026522 En savoir plus
Résumé

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. We used isogenic cell lines expressing different levels of DNA-PKcs to investigate the role of DNA-PKcs in metastatic development. We found that DNA-PKcs participates in melanoma primary tumor and metastasis development by stimulating angiogenesis, migration and invasion. Comparison of conditioned medium content from DNA-PKcs-proficient and deficient cells reveals that DNA-PKcs controls secretion of at least 103 proteins (including 44 metastasis-associated with FBLN1, SERPINA3, MMP-8, HSPG2 and the inhibitors of matrix metalloproteinases, such as α-2M and TIMP-2). High throughput analysis of secretomes, proteomes and transcriptomes, indicate that DNA-PKcs regulates the secretion of 85 proteins without affecting their gene expression. Our data demonstrate that DNA-PKcs has a pro-metastatic activity via the modification of the tumor microenvironment. This study shows for the first time a direct link between DNA damage repair and cancer metastasis and highlights the importance of DNA-PKcs as a potential target for anti-metastatic treatment.

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Alaguraj Veluchamy, Achal Rastogi, Xin Lin, Bérangère Lombard, Omer Murik, Yann Thomas, Florent Dingli, Maximo Rivarola, Sandra Ott, Xinyue Liu, Yezhou Sun, Pablo D Rabinowicz, James McCarthy, Andrew E Allen, Damarys Loew, Chris Bowler, Leïla Tirichine (2015 May 21)

An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.

Genome biology : 102 : DOI : 10.1186/s13059-015-0671-8 En savoir plus
Résumé

Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans.

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Mercè Guzmán-Vendrell, Sergio A Rincon, Florent Dingli, Damarys Loew, Anne Paoletti (2015 Apr 17)

Molecular control of the Wee1 regulatory pathway by the SAD kinase Cdr2.

Journal of cell science : 2842-53 : DOI : 10.1242/jcs.173146 En savoir plus
Résumé

Cell growth and division are tightly coordinated to maintain cell size constant during successive cell cycles. In Schizosaccharomyces pombe, the SAD kinase Cdr2 regulates the cell size at division and the positioning of the division plane. Cdr2 forms nodes on the medial cortex containing factors that constitute an inhibitory pathway for Wee1. This pathway is regulated by polar gradients of the DYRK kinase Pom1, and involves a direct inhibitor of Wee1, the SAD kinase Cdr1. Cdr2 also interacts with the anillin Mid1, which defines the division plane, and with additional components of the medial cortical nodes, including Blt1, which participate in the mitotic-promoting and cytokinetic functions of nodes. Here, we show that the interaction of Cdr2 with Wee1 and Mid1 requires the UBA domain of Cdr2, which is necessary for its kinase activity. In contrast, Cdr1 associates with the C-terminus of Cdr2, which is composed of basic and KA-1 lipid-binding domains. Mid1 also interacts with the C-terminus of Cdr2 and might bridge the N- and C-terminal domains, whereas Blt1 associates with the central spacer region. We propose that the association of Cdr2 effectors with different domains might constrain Cdr1 and Wee1 spatially to promote Wee1 inhibition upon Cdr2 kinase activation.

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Anne-Cécile Boulay, Aurélien Mazeraud, Salvatore Cisternino, Bruno Saubaméa, Phillipe Mailly, Laurent Jourdren, Corinne Blugeon, Virginie Mignon, Maria Smirnova, Alessia Cavallo, Pascal Ezan, Patrick Avé, Florent Dingli, Damarys Loew, Paulo Vieira, Fabrice Chrétien, Martine Cohen-Salmon (2015 Mar 13)

Immune quiescence of the brain is set by astroglial connexin 43.

The Journal of neuroscience : the official journal of the Society for Neuroscience : 4427-39 : DOI : 10.1523/JNEUROSCI.2575-14.2015 En savoir plus
Résumé

In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood-brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.

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Serena Sanulli, Neil Justin, Aurélie Teissandier, Katia Ancelin, Manuela Portoso, Matthieu Caron, Audrey Michaud, Berangère Lombard, Simao T da Rocha, John Offer, Damarys Loew, Nicolas Servant, Michel Wassef, Fabienne Burlina, Steve J Gamblin, Edith Heard, Raphaël Margueron (2015 Mar 5)

Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Molecular cell : 769-83 : DOI : 10.1016/j.molcel.2014.12.020 En savoir plus
Résumé

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2’s enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

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Imène B Bouhlel, Midori Ohta, Adeline Mayeux, Nicole Bordes, Florent Dingli, Jérôme Boulanger, Guilhem Velve Casquillas, Damarys Loew, Phong T Tran, Masamitsu Sato, Anne Paoletti (2015 Mar 3)

Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast.

Journal of cell science : 1481-93 : DOI : 10.1242/jcs.159657 En savoir plus
Résumé

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.

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Michael Schertzer, Karina Jouravleva, Mylene Perderiset, Florent Dingli, Damarys Loew, Tangui Le Guen, Barbara Bardoni, Jean-Pierre de Villartay, Patrick Revy, Arturo Londoño-Vallejo (2015 Jan 27)

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

Nucleic acids research : 1834-47 : DOI : 10.1093/nar/gku1402 En savoir plus
Résumé

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.

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Année de publication : 2014

Guillaume van Niel, Ptissam Bergam, Aurelie Di Cicco, Ilse Hurbain, Alessandra Lo Cicero, Florent Dingli, Roberta Palmulli, Cecile Fort, Marie Claude Potier, Leon J Schurgers, Damarys Loew, Daniel Levy, Graça Raposo (2014 Nov 13)

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells.

Cell reports : 43-51 : DOI : 10.1016/j.celrep.2015.08.057 En savoir plus
Résumé

Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

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Dora Sabino, Delphine Gogendeau, Davide Gambarotto, Maddalena Nano, Carole Pennetier, Florent Dingli, Guillaume Arras, Damarys Loew, Renata Basto (2014 Sep 23)

Moesin is a major regulator of centrosome behavior in epithelial cells with extra centrosomes.

Current biology : CB : 879-89 : DOI : 10.1016/j.cub.2015.01.066 En savoir plus
Résumé

Centrosome amplification has severe consequences during development and is thought to contribute to a variety of diseases such as cancer and microcephaly. However, the adverse effects of centrosome amplification in epithelia are still not known. Here, we investigate the consequences of centrosome amplification in the Drosophila wing disc epithelium. We found that epithelial cells exhibit mechanisms of clustering but also inactivation of extra centrosomes. Importantly, these mechanisms are not fully efficient, and both aneuploidy and cell death can be detected. Epithelial cells with extra centrosomes generate tumors when transplanted into WT hosts and inhibition of cell death results in tissue over-growth and disorganization. Using SILAC-fly, we found that Moesin, a FERM domain protein, is specifically upregulated in wing discs with extra centrosomes. Moesin localizes to the centrosomes and mitotic spindle during mitosis, and we show that Moesin upregulation influences extra-centrosome behavior and robust bipolar spindle formation. This study provides a mechanistic explanation for the increased aneuploidy and transformation potential primed by centrosome amplification in epithelial tissues.

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Année de publication : 2012

Dorothée Buet, Isabelle Gallais, Evelyne Lauret, Nicole Denis, Bérangère Lombard, François Guillonneau, Olivier Kosmider, Damarys Loew, Isabelle Dusanter-Fourt, Christel Guillouf, Patrick Mayeux, Françoise Moreau-Gachelin (2012 Mar 23)

Cotargeting signaling pathways driving survival and cell cycle circumvents resistance to Kit inhibitors in leukemia.

Blood : 4228-41 : DOI : 10.1182/blood-2011-07-368316 En savoir plus
Résumé

Oncogenic mutations leading to persistent kinase activities are associated with malignancies. Therefore, deciphering the signaling networks downstream of these oncogenic stimuli remains a challenge to gather insights into targeted therapy. To elucidate the biochemical networks connecting the Kit mutant to leukemogenesis, in the present study, we performed a global profiling of tyrosine-phosphorylated proteins from mutant Kit-driven murine leukemia proerythroblasts and identified Shp2 and Stat5 as proximal effectors of Kit. Shp2 or Stat5 gene depletion by sh-RNA, combined with pharmacologic inhibition of PI3kinase or Mek/Erk activities, revealed 2 distinct and independent signaling pathways contributing to malignancy. We demonstrate that cell survival is driven by the Kit/Shp2/Ras/Mek/Erk1/2 pathway, whereas the G(1)/S transition during the cell cycle is accelerated by both the Kit/Stat5 and Kit/PI3K/Akt pathways. The combined use of the clinically relevant drugs NVP-BEZ235, which targets the cell cycle, and Obatoclax, which targets survival, demonstrated synergistic effects to inhibit leukemia cell growth. This synergy was confirmed with a human mast leukemia cell line (HMC-1.2) that expresses mutant Kit. The results of the present study using liquid chromatography/tandem mass spectrometry analysis have elucidated signaling networks downstream of an oncogenic kinase, providing a molecular rationale for pathway-targeted therapy to treat cancer cells refractory to tyrosine kinase inhibitors.

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