Mass Spectrometry and Proteomics Facility

Publications

Year of publication 2016

Lisa Prendergast, Sebastian Müller, Yiwei Liu, Hongda Huang, Florent Dingli, Damarys Loew, Isabelle Vassias, Dinshaw J Patel, Kevin F Sullivan, Geneviève Almouzni (2016 Jun 11)

The CENP-T/-W complex is a binding partner of the histone chaperone FACT.

Genes & development : 1313-26 : DOI : 10.1101/gad.275073.115 Learn more
Summary

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.

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Dorian Obino, Francesca Farina, Odile Malbec, Pablo J Sáez, Mathieu Maurin, Jérémie Gaillard, Florent Dingli, Damarys Loew, Alexis Gautreau, Maria-Isabel Yuseff, Laurent Blanchoin, Manuel Théry, Ana-Maria Lennon-Duménil (2016 Mar 19)

Actin nucleation at the centrosome controls lymphocyte polarity

Nature communications : 10969 : DOI : 10.1038/ncomms10969 Learn more
Summary

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome-regulated by the availability of the Arp2/3 complex-determines its capacity to polarize in response to external stimuli.

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Joanna Kowal, Guillaume Arras, Marina Colombo, Mabel Jouve, Jakob Paul Morath, Bjarke Primdal-Bengtson, Florent Dingli, Damarys Loew, Mercedes Tkach, Clotilde Théry (2016 Feb 8)

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.

PNAS : 113: E968-977 : DOI : 10.1073/pnas.1521230113 Learn more
Summary

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.

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Year of publication 2015

Anne Lafon, Surayya Taranum, Federico Pietrocola, Florent Dingli, Damarys Loew, Sandipan Brahma, Blaine Bartholomew, Manolis Papamichos-Chronakis (2015 Dec 15)

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

Molecular cell : 784-96 : DOI : 10.1016/j.molcel.2015.10.028 Learn more
Summary

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

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Aurelia Kuster, Sebastien Nola, Florent Dingli, Barbara Vacca, Christian Gauchy, Jean-Claude Beaujouan, Marcela Nunez, Thomas Moncion, Damarys Loew, Etienne Formstecher, Thierry Galli, Veronique Proux-Gillardeaux (2015 Sep 12)

The Q-soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Q-SNARE) SNAP-47 Regulates Trafficking of Selected Vesicle-associated Membrane Proteins (VAMPs).

The Journal of biological chemistry : 28056-69 : DOI : 10.1074/jbc.M115.666362 Learn more
Summary

SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.

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Sara Chiker, Vincent Pennaneach, Damarys Loew, Florent Dingli, Denis Biard, Fabrice P Cordelières, Simon Gemble, Sophie Vacher, Ivan Bieche, Janet Hall, Marie Fernet (2015 Aug 3)

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

Cell cycle (Georgetown, Tex.) : 3066-78 : DOI : 10.1080/15384101.2015.1078020 Learn more
Summary

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.

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Laura S Besnier, Philippe Cardot, Barbara Da Rocha, Anthony Simon, Damarys Loew, Christophe Klein, Béatrice Riveau, Michel Lacasa, Caroline Clair, Monique Rousset, Sophie Thenet (2015 Jul 31)

The cellular prion protein PrPc is a partner of the Wnt pathway in intestinal epithelial cells.

Molecular biology of the cell : 3313-28 : DOI : 10.1091/mbc.E14-11-1534 Learn more
Summary

We reported previously that the cellular prion protein (PrP(c)) is a component of desmosomes and contributes to the intestinal barrier function. We demonstrated also the presence of PrP(c) in the nucleus of proliferating intestinal epithelial cells. Here we sought to decipher the function of this nuclear pool. In human intestinal cancer cells Caco-2/TC7 and SW480 and normal crypt-like HIEC-6 cells, PrP(c) interacts, in cytoplasm and nucleus, with γ-catenin, one of its desmosomal partners, and with β-catenin and TCF7L2, effectors of the canonical Wnt pathway. PrP(c) up-regulates the transcriptional activity of the β-catenin/TCF7L2 complex, whereas γ-catenin down-regulates it. Silencing of PrP(c) results in the modulation of several Wnt target gene expressions in human cells, with different effects depending on their Wnt signaling status, and in mouse intestinal crypt cells in vivo. PrP(c) also interacts with the Hippo pathway effector YAP, suggesting that it may contribute to the regulation of gene transcription beyond the β-catenin/TCF7L2 complex. Finally, we demonstrate that PrP(c) is required for proper formation of intestinal organoids, indicating that it contributes to proliferation and survival of intestinal progenitors. In conclusion, PrP(c) must be considered as a new modulator of the Wnt signaling pathway in proliferating intestinal epithelial cells.

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Gentili M, Kowal 1, Tkach M, Satoh T, Lahaye X, Conrad C, Boyron M, Lombard B, Durand S, Kroemer G, Loew D, Dalod M, Théry C, Manel N. (2015 Jul 30)

Transmission of innate immune signaling by packaging of cGAMP in viral particles.

Science : DOI : 10.1126/science.aab3628 Learn more
Summary

Infected cells detect viruses through a variety of receptors that initiate cell-intrinsic innate defense responses. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a cytosolic sensor for many DNA viruses and HIV-1. In response to cytosolic viral DNA, cGAS synthesizes the second messenger 2’3′-cyclic GMP-AMP (cGAMP), which activates antiviral signaling pathways. We show that in cells producing virus, cGAS-synthesized cGAMP can be packaged in viral particles and extracellular vesicles. Viral particles efficiently delivered cGAMP to target cells. cGAMP transfer by viral particles to dendritic cells activated innate immunity and antiviral defenses. Finally, we show that cell-free murine cytomegalovirus and Modified Vaccinia Ankara virus contained cGAMP. Thus, transfer of cGAMP by viruses may represent a defense mechanism to propagate immune responses to uninfected target cells.

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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 Learn more
Summary

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 Learn more
Summary

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 Learn more
Summary

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 Learn more
Summary

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 Learn more
Summary

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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Guillaume Kellermann, Markus Kaiser, Florent Dingli, Olivier Lahuna, Delphine Naud-Martin, Florence Mahuteau-Betzer, Damarys Loew, Evelyne Ségal-Bendirdjian, Marie-Paule Teulade-Fichou, Sophie Bombard (2015 May 9)

Identification of human telomerase assembly inhibitors enabled by a novel method to produce hTERT.

Nucleic acids research : 43 : e99 : DOI : 10.1093/nar/gkv425 Learn more
Summary

Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors.

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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 Learn more
Summary

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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