Plateforme d’imagerie Cellulaire et Tissulaire

Publications

Année de publication : 2017

Julie Salomon, Cécile Gaston, Jérémy Magescas, Boris Duvauchelle, Danielle Canioni, Lucie Sengmanivong, Adeline Mayeux, Grégoire Michaux, Florence Campeotto, Julie Lemale, Jérôme Viala, Françoise Poirier, Nicolas Minc, Jacques Schmitz, Nicole Brousse, Benoit Ladoux, Olivier Goulet, Delphine Delacour (2017 Jan 14)

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Nature communications : 13998 : DOI : 10.1038/ncomms13998 En savoir plus
Résumé

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.

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Année de publication : 2016

Elodie Gazquez, Yuli Watanabe, Florence Broders-Bondon, Perrine Paul-Gilloteaux, Julie Heysch, Viviane Baral, Nadège Bondurand, Sylvie Dufour (2016 Dec 2)

Endothelin-3 stimulates cell adhesion and cooperates with β1-integrins during enteric nervous system ontogenesis.

Scientific reports : 37877 : DOI : 10.1038/srep37877 En savoir plus
Résumé

Endothelin-3 (EDN3) and β1-integrins are required for the colonization of the embryonic gut by enteric neural crest cells (ENCCs) to form the enteric nervous system (ENS). β1-integrin-null ENCCs exhibit migratory defects in a region of the gut enriched in EDN3 and in specific extracellular matrix (ECM) proteins. We investigated the putative role of EDN3 on ENCC adhesion properties and its functional interaction with β1-integrins during ENS development. We show that EDN3 stimulates ENCC adhesion to various ECM components in vitro. It induces rapid changes in ENCC shape and protrusion dynamics favouring sustained growth and stabilization of lamellipodia, a process coincident with the increase in the number of focal adhesions and activated β1-integrins. In vivo studies and ex-vivo live imaging revealed that double mutants for Itgb1 and Edn3 displayed a more severe enteric phenotype than either of the single mutants demonstrated by alteration of the ENS network due to severe migratory defects of mutant ENCCs taking place early during the ENS development. Altogether, our results highlight the interplay between the EDN3 and β1-integrin signalling pathways during ENS ontogenesis and the role of EDN3 in ENCC adhesion.

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Salomé Adam, Juliette Dabin, Odile Chevallier, Olivier Leroy, Céline Baldeyron, Armelle Corpet, Patrick Lomonte, Olivier Renaud, Geneviève Almouzni, Sophie E Polo (2016 Sep 20)

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Molecular cell : DOI : S1097-2765(16)30461-0 En savoir plus
Résumé

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

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Marco Biondini, Amel Sadou-Dubourgnoux, Perrine Paul-Gilloteaux, Giulia Zago, Melis D Arslanhan, François Waharte, Etienne Formstecher, Maud Hertzog, Jinchao Yu, Raphael Guerois, Alexis Gautreau, Giorgio Scita, Jacques Camonis, Maria Carla Parrini (2016 Sep 4)

Direct interaction between Exocyst and Wave complexes promotes cell protrusions and motility.

Journal of cell science : DOI : jcs.187336 En savoir plus
Résumé

Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave Regulatory Complex (WRC). The Exocyst complex, which functions in polarized exocytosis, has been involved in regulation of cell motility. Here we show a physical and functional connection between Exocyst and WRC. Purified components of Exocyst and WRC complexes directly associate in vitro and interactions interfaces are identified. The Exocyst/WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the Exocyst/WRC interaction leads to impaired migration. By time-lapse microscopy coupled to image correlation analysis, we visualize the traffic of WRC toward the front in nascent protrusions. Exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between Exocyst and WRC complexes provides a novel mechanistic insight into the spatio-temporal regulation of cell migration.

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Marina Casiraghi, Marjorie Damian, Ewen Lescop, Elodie Point, Karine Moncoq, Nelly Morellet, Daniel Levy, Jacky Marie, Eric Guittet, Jean-Louis Banères, Laurent J Catoire (2016 Aug 5)

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

Journal of the American Chemical Society : 11170-5 : DOI : 10.1021/jacs.6b04432 En savoir plus
Résumé

Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

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Megan K Dennis, Cédric Delevoye, Amanda Acosta-Ruiz, Ilse Hurbain, Maryse Romao, Geoffrey G Hesketh, Philip S Goff, Elena V Sviderskaya, Dorothy C Bennett, J Paul Luzio, Thierry Galli, David J Owen, Graça Raposo, Michael S Marks (2016 Aug 3)

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.

The Journal of cell biology : 293-308 : DOI : 10.1083/jcb.201605090 En savoir plus
Résumé

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1-dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3-dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky-Pudlak syndrome variants.

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Katarzyna S Kopanska, Yara Alcheikh, Ralitza Staneva, Danijela Vignjevic, Timo Betz (2016 Jun 9)

Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion.

PloS one : e0156442 : DOI : 10.1371/journal.pone.0156442 En savoir plus
Résumé

The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid’s surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor.

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Léa Ripoll, Xavier Heiligenstein, Graça Raposo, Cédric Delevoye (2016 May 18)

Illuminating the dark side of recycling endosomes.

Cell cycle (Georgetown, Tex.) : 1309-10 : DOI : 10.1080/15384101.2016.1160682 En savoir plus
Résumé

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Selma Maacha, Océane Anezo, Malika Foy, Géraldine Liot, Laurence Mery, Cécile Laurent, Xavier Sastre-Garau, Sophie Piperno-Neumann, Nathalie Cassoux, Nathalie Planque, Simon Saule (2016 Apr 21)

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Promotes Human Uveal Melanoma Aggressiveness Through Membrane Accumulation of Matrix Metalloproteinase 14 (MMP14).

Investigative ophthalmology & visual science : 1982-90 : DOI : 10.1167/iovs.15-18780 En savoir plus
Résumé

To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness.

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Florence Broders-Bondon, Perrine Paul-Gilloteaux, Elodie Gazquez, Julie Heysch, Matthieu Piel, Roberto Mayor, John D Lambris, Sylvie Dufour (2016 Apr 5)

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

Developmental biology : 85-99 : DOI : 10.1016/j.ydbio.2016.03.022 En savoir plus
Résumé

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

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Guido Tiana, Assaf Amitai, Tim Pollex, Tristan Piolot, David Holcman, Edith Heard, Luca Giorgetti (2016 Mar 31)

Structural Fluctuations of the Chromatin Fiber within Topologically Associating Domains.

Biophysical journal : 1234-45 : DOI : 10.1016/j.bpj.2016.02.003 En savoir plus
Résumé

Experiments based on chromosome conformation capture have shown that mammalian genomes are partitioned into topologically associating domains (TADs), within which the chromatin fiber preferentially interacts. TADs may provide three-dimensional scaffolds allowing genes to contact their appropriate distal regulatory DNA sequences (e.g., enhancers) and thus to be properly regulated. Understanding the cell-to-cell and temporal variability of the chromatin fiber within TADs, and what determines them, is thus of great importance to better understand transcriptional regulation. We recently described an equilibrium polymer model that can accurately predict cell-to-cell variation of chromosome conformation within single TADs, from chromosome conformation capture-based data. Here we further analyze the conformational and energetic properties of our model. We show that the chromatin fiber within TADs can easily fluctuate between several conformational states, which are hierarchically organized and are not separated by important free energy barriers, and that this is facilitated by the fact that the chromatin fiber within TADs is close to the onset of the coil-globule transition. We further show that in this dynamic state the properties of the chromatin fiber, and its contact probabilities in particular, are determined in a nontrivial manner not only by site-specific interactions between strongly interacting loci along the fiber, but also by nonlocal correlations between pairs of contacts. Finally, we use live-cell experiments to measure the dynamics of the chromatin fiber in mouse embryonic stem cells, in combination with dynamical simulations, and predict that conformational changes within one TAD are likely to occur on timescales that are much shorter than the duration of one cell cycle. This suggests that genes and their regulatory elements may come together and disassociate several times during a cell cycle. These results have important implications for transcriptional regulation as they support the concept of highly dynamic interactions driven by a complex interplay between site-specific interactions and the intrinsic biophysical properties of the chromatin fiber.

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Tao Zou, Fatimata Dembele, Anne Beugnet, Lucie Sengmanivong, Ario de Marco, Min-Hui Li (2016 Mar 24)

Nanobody-functionalized polymersomes.

Journal of controlled release : official journal of the Controlled Release Society : e79-80 : DOI : 10.1016/j.jconrel.2015.05.132 En savoir plus
Résumé

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Floris Bosveld, Olga Markova, Boris Guirao, Charlotte Martin, Zhimin Wang, Anaëlle Pierre, Maria Balakireva, Isabelle Gaugue, Anna Ainslie, Nicolas Christophorou, David K Lubensky, Nicolas Minc, Yohanns Bellaïche (2016 Feb 18)

Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis.

Nature : 495-8 : DOI : 10.1038/nature16970 En savoir plus
Résumé

The orientation of cell division along the long axis of the interphase cell–the century-old Hertwig’s rule–has profound roles in tissue proliferation, morphogenesis, architecture and mechanics. In epithelial tissues, the shape of the interphase cell is influenced by cell adhesion, mechanical stress, neighbour topology, and planar polarity pathways. At mitosis, epithelial cells usually adopt a rounded shape to ensure faithful chromosome segregation and to promote morphogenesis. The mechanisms underlying interphase cell shape sensing in tissues are therefore unknown. Here we show that in Drosophila epithelia, tricellular junctions (TCJs) localize force generators, pulling on astral microtubules and orienting cell division via the Dynein-associated protein Mud independently of the classical Pins/Gαi pathway. Moreover, as cells round up during mitosis, TCJs serve as spatial landmarks, encoding information about interphase cell shape anisotropy to orient division in the rounded mitotic cell. Finally, experimental and simulation data show that shape and mechanical strain sensing by the TCJs emerge from a general geometric property of TCJ distributions in epithelial tissues. Thus, in addition to their function as epithelial barrier structures, TCJs serve as polarity cues promoting geometry and mechanical sensing in epithelial tissues.

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Carlos A Niño, David Guet, Alexandre Gay, Sergine Brutus, Frédéric Jourquin, Shweta Mendiratta, Jean Salamero, Vincent Géli, Catherine Dargemont (2016 Jan 20)

Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket.

The Journal of cell biology : 167-80 : DOI : 10.1083/jcb.201506130 En savoir plus
Résumé

The nuclear pore complex (NPC) serves as both the unique gate between the nucleus and the cytoplasm and a major platform that coordinates nucleocytoplasmic exchanges, gene expression, and genome integrity. To understand how the NPC integrates these functional constraints, we dissected here the posttranslational modifications of the nuclear basket protein Nup60 and analyzed how they intervene to control the plasticity of the NPC. Combined approaches highlight the role of monoubiquitylation in regulating the association dynamics of Nup60 and its partner, Nup2, with the NPC through an interaction with Nup84, a component of the Y complex. Although major nuclear transport routes are not regulated by Nup60 modifications, monoubiquitylation of Nup60 is stimulated upon genotoxic stress and regulates the DNA-damage response and telomere repair. Together, these data reveal an original mechanism contributing to the plasticity of the NPC at a molecular-organization and functional level.

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Delphine Benarroch-Popivker, Sabrina Pisano, Aaron Mendez-Bermudez, Liudmyla Lototska, Parminder Kaur, Serge Bauwens, Nadir Djerbi, Chrysa M Latrick, Vincent Fraisier, Bei Pei, Alexandre Gay, Emilie Jaune, Kevin Foucher, Julien Cherfils-Vicini, Eric Aeby, Simona Miron, Arturo Londoño-Vallejo, Jing Ye, Marie-Hélène Le Du, Hong Wang, Eric Gilson, Marie-Josèphe Giraud-Panis (2016 Jan 18)

TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection.

Molecular cell : 274-86 : DOI : 10.1016/j.molcel.2015.12.009 En savoir plus
Résumé

The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.

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