19 results on '"Ophélie Nicolle"'
Search Results
2. Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2–Deficient Intestinal OrganoidsSummary
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Mohammed H. Mosa, Ophélie Nicolle, Sophia Maschalidi, Fernando E. Sepulveda, Aurelien Bidaud-Meynard, Constantin Menche, Birgitta E. Michels, Grégoire Michaux, Geneviève de Saint Basile, and Henner F. Farin
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Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border. Keywords: Microvillus Atrophy, Disease Modeling, Brush Border Formation, Apical Vesicular Transport
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- 2018
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- View/download PDF
3. The clathrin adaptor AP-1B independently controls proliferation and differentiation in the mammalian intestine
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Maela Duclos, Anne Bourdais, Ophélie Nicolle, Grégoire Michaux, and Aurélien Bidaud-Meynard
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Maintenance of the polarity of the epithelial cells facing the lumen of the small intestine is crucial to ensure the vectorial absorption of nutrients as well as the integrity of the apical brush border and the intestinal barrier. Polarized vesicular trafficking plays a key role in this process, and defective transport due to mutations in apical trafficking-related genes has been shown to affect nutrient absorption. Interestingly, it has been demonstrated that downregulation of the polarized sorting clathrin adaptor AP-1B led to both epithelial polarity and proliferation defects in the mouse intestine. This enlightened a new function of polarized trafficking in the gut epithelium and a novel link between trafficking, polarity, and proliferation. Here, using CRISPR-Cas9-mediated mutation of the AP-1B coding geneAp1m2in mouse intestinal organoids, we uncovered a novel proliferation pathway controlled by AP-1B. We showed that the polarity defects induced byAp1m2mutations led to a defective apical targeting of both Rab11+apical recycling endosomes and of the polarity determinant Cdc42. Moreover, we showed that these polarity defects were accompanied by an induction of YAP and EGFR/mTOR-dependent proliferation pathways. Finally, we showed that AP-1B additionally controlled a proliferation-independent differentiation pathway towards the secretory lineage. Overall, our results highlighted the pleiotropic roles played by AP-1B in the homeostasis of the gut epithelium.
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- 2023
4. PAR-4/LKB1 regulates intestinal cell number by restricting endoderm specification to the E lineage
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Flora Demouchy, Ophélie Nicolle, Grégoire Michaux, Anne Pacquelet, Institut de Génétique et Développement de Rennes (IGDR), and Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
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[SDV]Life Sciences [q-bio] - Abstract
The master kinase PAR-4/LKB1 appears as a major regulator of intestinal physiology. It is in particular mutated in the Peutz-Jeghers syndrome, an inherited disorder in which patients develop benign intestine polyps. Moreover, ectopic activation of PAR-4/LKB1 is sufficient to induce the polarized accumulation of apical and basolateral surface proteins and the formation of apical microvilli-like structures in intestinal epithelial cancer cell lines. InC. elegans, PAR-4 was shown to be required for the differentiation of intestinal cells. Here, we further examine the role of PAR-4 during intestinal development. We find that it is not required for the establishment of enterocyte polarity and plays only a minor role in brush border formation. By contrast,par-4mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a novel function of PAR-4 in preventing intestinal hyperplasia. Importantly, we find that the ability of PAR-4 to control intestinal cell number does not involve the regulation of cell proliferation but is rather due to its ability to restrict the expression of intestinal cell fate factors to the E blastomere lineage. We therefore propose that PAR-4 is required to regulateC. elegansintestine specification.
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- 2022
5. V0-ATPase downregulation induces MVID-like brush border defects independently of apical trafficking in the mammalian intestine
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Aurélien Bidaud-Meynard, Ophélie Nicolle, Anne Bourdais, Maela Duclos, Jad Saleh, Frank Ruemmele, Henner F Farin, Delphine Delacour, Despina Moshous, Grégoire Michaux, Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), École des Hautes Études en Santé Publique [EHESP] (EHESP), Université de Rennes (UR), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Service d'immuno-hématologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], Institute for Tumor Biology and Experimental Therapy [Frankfurt, Germany], German Cancer Consortium [Heidelberg] (DKTK), and Goethe-University Frankfurt am Main
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MVI ,[SDV]Life Sciences [q-bio] ,MVID ,microvillus inclusion ,microvillus inclusion disease - Abstract
SummaryIntestinal microvillus atrophy is a major cause of enteropathies such as idiopathic or congenital diarrhea that are often associated with severe morbidity. It can be caused by genetic disorders, inflammatory diseases, toxins or pathogens. In particular, Microvillus inclusion disease (MVID) is characterized by a chronic intractable diarrhea and a severe microvillus atrophy. It is triggered by mutations inMYO5B, STX3, MUNC18.2orUNC45Awhich alter epithelial polarity by affecting apical trafficking in intestinal epithelial cells. Furthermore, we recently established that the depletion of the V0sector of the V-ATPase complex induces an MVID-like phenotype inC. elegans. In this study we investigated the function of the V0-ATPase complex in mouse intestinal organoids. We found that its depletion also triggers a very severe microvillus atrophy in this model. Furthermore, we established that the polarity of intestinal cells is affected in a patient carrying mutations inTCIRG1which encodes a V0-ATPase subunit. However, V0- ATPase depletion does not recapitulate other MVID-specific phenotypes such as subapical vesicle accumulation and Rab11+ endosomes mislocalization. Finally, we found that the apical localization of the V0-ATPase is disrupted in MVID patients. Altogether these results suggest a role for the V0-ATPase in microvillus atrophy which might be independent from apical trafficking.
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- 2022
6. High-resolution dynamic mapping of the C. elegans intestinal brush border
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Grégoire Michaux, Anne Pacquelet, Camille N. Plancke, François Robin, Aurélien Bidaud-Meynard, Flora Demouchy, Ophélie Nicolle, Shashi Kumar Suman, Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Laboratoire de Biologie du Développement [Paris] (LBD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-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), Ligue Contre le Cancer, H2020 Marie Sk̢odowska-Curie Actions 844070, Fondation Maladies Rares 169608, Universitè de Rennes 1 17CQ436-S0, Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Laboratoire de Biologie du Développement [IBPS] (LBD)
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0303 health sciences ,Dynamic mapping ,Polarity ,Brush border ,Microvilli ,[SDV]Life Sciences [q-bio] ,High resolution ,Context (language use) ,Host defence ,Apical membrane ,Biology ,Intestine ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Enterocytes ,Animals ,Dynamic localization ,Membrane surface ,Caenorhabditis elegans ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology ,Developmental Biology - Abstract
The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption, and host defence. Studies on mammalian cultured epithelial cells uncovered some of the molecular players and physical constrains required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not been investigated in detail yet. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode C. elegans to build a high-resolution and dynamic localization map of known and new markers of the brush border. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation but become highly stable once microvilli are built. This new mapping tool will be instrumental to understand the molecular processes of microvilli growth and maintenance in vivo as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.
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- 2021
7. C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during
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João J, Ramalho, Jorian J, Sepers, Ophélie, Nicolle, Ruben, Schmidt, Janine, Cravo, Grégoire, Michaux, and Mike, Boxem
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Binding Sites ,Actins ,Actin Cytoskeleton ,Cytoskeletal Proteins ,Protein Domains ,Larva ,Mutagenesis, Site-Directed ,Animals ,Humans ,Amino Acid Sequence ,Intestinal Mucosa ,Phosphorylation ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Sequence Alignment ,Protein Binding - Abstract
ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP
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- 2020
8. In vivo control of the ezrin/radixin/moesin protein ERM-1 in C. elegans
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Ophélie Nicolle, Mike Boxem, João J. Ramalho, and Grégoire Michaux
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genetic structures ,stomatognathic system ,Chemistry ,Cytoplasm ,Mutant ,Morphogenesis ,Phosphorylation ,Context (language use) ,Threonine ,biochemical phenomena, metabolism, and nutrition ,Actin ,Function (biology) ,Cell biology - Abstract
ERM proteins are conserved regulators of cortical membrane specialization, that function as membrane–actin linkers and molecular hubs. Activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2-binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the singleC. elegansERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formationin vivo. Using CRISPR/Cas9-generatederm-1mutant alleles we demonstrate that PIP2-binding is critically required for ERM-1 function. In contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and drive lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.
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- 2020
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9. A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane
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Grégoire Michaux, Yann Le Cunff, Markus Heck, Ophélie Nicolle, Aurélien Bidaud-Meynard, Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Ligue Contre le Cancer, Centre National de la Recherche Scientifique, Université de Rennes 1, 844070, European Commission, 169608, Fondation maladies rares, Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)
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Vacuolar Proton-Translocating ATPases ,Brush border ,Endosome ,Enterocyte ,Polarity (physics) ,ATPase ,Epithelial cells ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Clathrin ,Animals, Genetically Modified ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Intestinal Mucosa ,Microvillus inclusion disease ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,030304 developmental biology ,Epithelial polarity ,0303 health sciences ,Trafficking ,biology ,Polarity ,Cell Membrane ,Cell Polarity ,Microvillus ,Cell biology ,Intestine ,Protein Transport ,Proton-Translocating ATPases ,medicine.anatomical_structure ,Enterocytes ,Intestinal Absorption ,biology.protein ,C. elegans ,030217 neurology & neurosurgery ,Developmental Biology ,Research Article ,Signal Transduction - Abstract
Intestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining a genetic RNA interference (RNAi) screen with in vivo super-resolution imaging in the Caenorhabditis elegans intestine, we found that the V0 sector of the vacuolar ATPase (V0-ATPase) controls a late apical trafficking step, involving Ras-related protein 11 (RAB-11)(+) endosomes and the N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) synaptosome-associated protein 29 (SNAP-29), and is necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids and clathrin in enterocyte polarity maintenance. Finally, we demonstrate that silencing of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from individuals with microvillus inclusion disease (MVID) and use this new in vivo MVID model to follow the dynamics of microvillus inclusions. Thus, we describe a new function for V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of the C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies.
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- 2019
10. The localisation of the apical Par/Cdc42 polarity module is specifically affected in microvillus inclusion disease
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André Le Bivic, Ophélie Nicolle, Marion Rabant, Dominique Massey-Harroche, Nicole Brousse, Olivier Goulet, Grégoire Michaux, and Frank M. Ruemmele
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0301 basic medicine ,Enterocyte ,Polarity (physics) ,Cell Biology ,General Medicine ,Anatomy ,Apical membrane ,Biology ,Microvillus ,Intestinal absorption ,Syntaxin 3 ,3. Good health ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Cell polarity ,medicine ,Epithelial polarity - Abstract
BACKGROUND INFORMATION: . Microvillus inclusion disease (MVID) is a genetic disorder affecting intestinal absorption. It is caused by mutations in MYO5B or syntaxin 3 (STX3) affecting apical membrane trafficking. Morphologically MVID is characterised by a depletion of apical microvilli and the formation of microvillus inclusions inside the cells, suggesting a loss of polarity. To investigate this hypothesis we examined the location of essential apical polarity determinants in five MVID patients. RESULTS: We found that the polarity determinants Cdc42, Par6B, PKCζ/ι and the structural proteins ezrin and phospho-ezrin were lost from the apical membrane and accumulated either in the cytoplasm or on the basal side of enterocytes in patients which suggests an inversion of cell polarity. Moreover microvilli-like structures were observed at the basal side in electron microscopy. We next performed Myo5B depletion in 3D-grown human Caco2 cells forming cysts and we found a direct link between the loss of Myo5B and the mislocalisation of the same apical proteins; furthermore we observed that a majority of cyst displayed an inverted polarity phenotype as seen in some patients. Finally we found that this loss of polarity was specific for MVID: tissue samples of patients with Myo5B independent absorption disorders showed normal polarity but we identified Cdc42 as a potentially essential biomarker for tricho-hepato-enteric syndrome. CONCLUSION: Our findings indicate that the loss of Myo5B induces a strong loss of enterocyte polarity, potentially leading to polarity inversion. SIGNIFICANCE: Our results show that polarity determinants could be useful markers to help establishing a diagnosis in patients. Furthermore they could be used to characterise other rare intestinal absorption diseases.
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- 2015
11. Force Transmission between Three Tissues Controls Bipolar Planar Polarity Establishment and Morphogenesis
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Sylvain Prigent, Ophélie Nicolle, Ghislain Gillard, Thibault Brugière, Grégoire Michaux, Mathieu Pinot, Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Biosit : biologie, santé, innovation technologique (SFR UMS CNRS 3480 - INSERM 018), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Plateforme Génomique Santé Biogenouest®, Chard-Hutchinson, Xavier, Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
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0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Morphogenesis ,morphogenesis ,Biology ,Mechanotransduction, Cellular ,General Biochemistry, Genetics and Molecular Biology ,Extracellular matrix ,Adherens junction ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Mechanotransduction ,Caenorhabditis elegans ,Actin ,planar polarity ,mechanotransduction ,Body Patterning ,Epidermis (botany) ,Hemidesmosome ,Adherens Junctions ,Cell biology ,Biomechanical Phenomena ,[SDV] Life Sciences [q-bio] ,030104 developmental biology ,Epidermal Cells ,C. elegans ,General Agricultural and Biological Sciences ,signaling ,Developmental biology ,030217 neurology & neurosurgery ,Muscle Contraction - Abstract
Summary How tissues from different developmental origins interact to achieve coordinated morphogenesis at the level of a whole organism is a fundamental question in developmental biology. While biochemical signaling pathways controlling morphogenesis have been extensively studied [ 1 , 2 , 3 ], morphogenesis of epithelial tissues can also be directed by mechanotransduction pathways physically linking two tissues [ 4 , 5 , 6 , 7 , 8 ]. C. elegans embryonic elongation requires the coordination of three tissues: muscles, the dorsal and ventral epidermis, and the lateral epidermis. Elongation starts by cell-shape changes driven by actomyosin contractions in the lateral epidermis [ 9 , 10 ]. At mid-elongation, muscles become connected to the apical surface of the dorsal and ventral epidermis by molecular tendons formed by muscle integrins, extracellular matrix, and C. elegans hemidesmosomes (CeHDs). The mechanical signal generated by the onset of muscle contractions in the antero-posterior axis from mid-elongation is translated into a biochemical pathway controlling the maturation of CeHDs in the dorsal and ventral epidermis [ 11 ]. Consistently, mutations affecting muscle contractions or molecular tendons lead to a mid-elongation arrest [ 12 ]. Here, we found that the mechanical force generated by muscle contractions and relayed by molecular tendons is transmitted by adherens junctions to lateral epidermal cells, where it establishes a newly identified bipolar planar polarity of the apical PAR module. The planar polarized PAR module is then required for actin planar organization, thus contributing to the determination of the orientation of cell-shape changes and the elongation axis of the whole embryo. This mechanotransduction pathway is therefore essential to coordinate the morphogenesis of three embryonic tissues.
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- 2018
12. A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane
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Ophélie Nicolle, Markus Heck, Grégoire Michaux, and Aurélien Bidaud-Meynard
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0303 health sciences ,Brush border ,biology ,Chemistry ,Endosome ,Enterocyte ,Polarity (physics) ,ATPase ,Microvillus ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,In vivo ,medicine ,biology.protein ,030217 neurology & neurosurgery ,030304 developmental biology ,Epithelial polarity - Abstract
Intestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining genetic RNAi screen and in vivo super-resolution imaging in the C. elegans intestine, we uncovered that the V0 sector of the V-ATPase (V0-ATPase) controls a late apical trafficking step, involving RAB-11 endosomes and the SNARE SNAP-29, necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids in enterocyte polarity maintenance. Finally, we demonstrate that depletion of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from patients with Microvillus inclusion disease (MVID) and used this new in vivo MVID model to follow the dynamics of microvillus inclusions. Hence, we describe a new function for the V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies.Summary statementV0-ATPase controls a late apical trafficking step involved in the maintenance of the apical absorptive intestinal membrane and its depletion phenocopies the trafficking and structural defects of MVID in C. elegans.
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- 2018
13. Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study UsingCaenorhabditis elegans
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Grégoire Michaux, Ophélie Nicolle, Agnès Burel, Gareth Griffiths, and Irina Kolotuev
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biology ,ved/biology ,ved/biology.organism_classification_rank.species ,Cell Biology ,Anatomy ,Subcellular localization ,biology.organism_classification ,Biochemistry ,Protein subcellular localization prediction ,Green fluorescent protein ,Standard procedure ,Cell biology ,Immuno electron microscopy ,Structural Biology ,Microscopy ,Genetics ,Model organism ,Biological system ,Molecular Biology ,Caenorhabditis elegans - Abstract
Cryo-sectioning procedures, initially developed by Tokuyasu, have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample. Here, we describe our method that enables reproducible cryo-sectioning of Caenorhabditis elegans larvae/adults and embryos. We have established a chemical-fixation procedure in which flat embedding considerably simplifies manipulation and lateral orientation of larvae or adults. To bypass the limitations of chemical fixation, we have improved the hybrid cryo-immobilization-rehydration technique and reduced the overall time required to complete this procedure. Using our procedures, precise cryo-sectioning orientation can be combined with good ultrastructural preservation and efficient immuno-electron microscopy protein localization. Also, GFP fluorescence can be efficiently preserved, permitting a direct correlation of the fluorescent signal and its subcellular localization. Although developed for C. elegans samples, our method addresses the challenge of working with small asymmetric samples in general, and thus could be used to improve the efficiency of immuno-electron localization in other model organisms.
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- 2015
14. Coordinated morphogenesis through tension-induced planar polarity
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Grégoire Michaux, Ophélie Nicolle, Sylvain Prigent, Mathieu Pinot, Ghislain Gillard, and Thibault Brugière
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0303 health sciences ,Polarity (international relations) ,Epidermis (botany) ,Hemidesmosome ,030302 biochemistry & molecular biology ,Morphogenesis ,Biology ,Embryonic stem cell ,Cell biology ,03 medical and health sciences ,Mechanotransduction ,Developmental biology ,Actin ,030304 developmental biology - Abstract
Tissues from different developmental origins must interact to achieve coordinated morphogenesis at the level of a whole organism. C. elegans embryonic elongation is controlled by actomyosin dynamics which trigger cell shape changes in the epidermis and by muscle contractions, but how the two processes are coordinated is not known. We found that a tissue-wide tension generated by muscle contractions and relayed by tendon-like hemidesmosomes in the dorso-ventral epidermis is required to establish a planar polarity of the apical PAR module in the lateral epidermis. This planar polarized PAR module then controls actin planar organization, thus determining the orientation of cell shape changes and the elongation axis of the whole embryo. This trans-tissular mechanotransduction pathway thus contributes to coordinate the morphogenesis of three embryonic tissues.
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- 2017
15. Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells
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Ghislain Gillard, Massiullah Shafaq-Zadah, Grégoire Michaux, Raghida Damaj, Jacques Pecreaux, Ophélie Nicolle, Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes 1 (UR1), and Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )
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[SDV]Life Sciences [q-bio] ,Morphogenesis ,Embryonic Development ,Clathrin ,Cell Adhesion ,Animals ,Caenorhabditis elegans Proteins ,Caenorhabditis elegans ,Molecular Biology ,Actin ,Epithelial polarity ,Armadillo Domain Proteins ,Microscopy, Confocal ,biology ,integumentary system ,Cadherin ,Embryogenesis ,Cell Polarity ,E-cadherin ,biology.organism_classification ,Cadherins ,AP-1 ,Embryonic stem cell ,Membrane traffic ,Cell biology ,Transcription Factor AP-1 ,Microscopy, Electron ,biology.protein ,RNA Interference ,Epidermis ,Developmental Biology - Abstract
International audience; E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.
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- 2015
16. Adaptation of Cryo-Sectioning for IEM Labeling of Asymmetric Samples: A Study Using Caenorhabditis elegans
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Ophélie, Nicolle, Agnès, Burel, Gareth, Griffiths, Grégoire, Michaux, and Irina, Kolotuev
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Animals ,Caenorhabditis elegans ,Cryoultramicrotomy - Abstract
Cryo-sectioning procedures, initially developed by Tokuyasu, have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample. Here, we describe our method that enables reproducible cryo-sectioning of Caenorhabditis elegans larvae/adults and embryos. We have established a chemical-fixation procedure in which flat embedding considerably simplifies manipulation and lateral orientation of larvae or adults. To bypass the limitations of chemical fixation, we have improved the hybrid cryo-immobilization-rehydration technique and reduced the overall time required to complete this procedure. Using our procedures, precise cryo-sectioning orientation can be combined with good ultrastructural preservation and efficient immuno-electron microscopy protein localization. Also, GFP fluorescence can be efficiently preserved, permitting a direct correlation of the fluorescent signal and its subcellular localization. Although developed for C. elegans samples, our method addresses the challenge of working with small asymmetric samples in general, and thus could be used to improve the efficiency of immuno-electron localization in other model organisms.
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- 2015
17. UNC45A deficiency causes microvillus inclusion disease–like phenotype by impairing myosin VB–dependent apical trafficking
- Author
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Rémi Duclaux-Loras, Corinne Lebreton, Jérémy Berthelet, Fabienne Charbit-Henrion, Ophelie Nicolle, Céline Revenu de Courtils, Stephanie Waich, Taras Valovka, Anis Khiat, Marion Rabant, Caroline Racine, Ida Chiara Guerrera, Júlia Baptista, Maxime M. Mahe, Michael W. Hess, Béatrice Durel, Nathalie Lefort, Céline Banal, Mélanie Parisot, Cecile Talbotec, Florence Lacaille, Emmanuelle Ecochard-Dugelay, Arzu Meltem Demir, Georg F. Vogel, Laurence Faivre, Astor Rodrigues, Darren Fowler, Andreas R. Janecke, Thomas Müller, Lukas A. Huber, Fernando Rodrigues-Lima, Frank M. Ruemmele, Holm H. Uhlig, Filippo Del Bene, Grégoire Michaux, Nadine Cerf-Bensussan, and Marianna Parlato
- Subjects
Gastroenterology ,Medicine - Abstract
Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.
- Published
- 2022
- Full Text
- View/download PDF
18. Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis
- Author
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Ophélie Nicolle, Martine Bonnaure-Mallet, Astrid Rouillon, Vincent Meuric, Fatiha Chandad, Zohreh Tamanai-Shacoori, Hélène Guyodo, Brébion, Alice, Microbiologie : Risques Infectieux, Université de Rennes (UR)-CHU Pontchaillou [Rennes]-Faculté de Chirurgie Dentaire de Rennes-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Conseil régional de Bretagne, Laboratoires Expanscience, Fondation Langlois, Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-CHU Pontchaillou [Rennes]-Faculté de Chirurgie Dentaire de Rennes-Faculté d'Odontologie-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes (UR)-CHU Pontchaillou [Rennes]-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Université de Rennes - UFR d'Odontologie (UR Odontologie), and Université de Rennes (UR)-Université de Rennes (UR)
- Subjects
Gene Expression ,MESH: Flow Cytometry ,MESH: Streptococcus gordonii ,MESH: Bacteriological Techniques ,biofilm ,Green fluorescent protein ,MESH: Recombinant Proteins ,Genes, Reporter ,Immunology and Allergy ,MESH: Microscopy, Confocal ,Anaerobiosis ,Promoter Regions, Genetic ,0303 health sciences ,Microscopy, Confocal ,green fluorescent protein (GFP) ,Streptococcus gordonii ,General Medicine ,MESH: Genetics, Microbial ,Flow Cytometry ,Recombinant Proteins ,SNAP-tag ,MESH: Staining and Labeling ,Infectious Diseases ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,Biochemistry ,MESH: Porphyromonas gingivalis ,Anaerobic bacteria ,Porphyromonas gingivalis ,Plasmids ,Alkyltransferase ,Genetics, Microbial ,Microbiology (medical) ,MESH: Gene Expression ,Green Fluorescent Proteins ,Immunology ,MESH: Biofilms ,Biology ,Dental plaque ,Microbiology ,Fluorescence ,O(6)-Methylguanine-DNA Methyltransferase ,03 medical and health sciences ,MESH: Green Fluorescent Proteins ,MESH: Plasmids ,MESH: Anaerobiosis ,MESH: Promoter Regions, Genetic ,medicine ,Humans ,Molecular Biology ,[SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology ,030304 developmental biology ,Bacteriological Techniques ,MESH: Humans ,Staining and Labeling ,MESH: O(6)-Methylguanine-DNA Methyltransferase ,030306 microbiology ,MESH: Molecular Biology ,MESH: Fluorescence ,MESH: Genes, Reporter ,Biofilm ,biology.organism_classification ,medicine.disease ,Biofilms ,confocal laser scanning microscopy (CLSM) - Abstract
International audience; Porphyromonas gingivalis is an anaerobic periodontal pathogen that resides in the complex multispecies microbial biofilm known as dental plaque. Effective reporter tools are increasingly needed to facilitate physiological and pathogenetic studies of dental biofilm. Fluorescent proteins are ideal reporters for conveniently monitoring biofilm growth, but are restricted by several environmental factors, such as a requirement of oxygen to emit fluorescence. We developed a fluorescent reporter plasmid, known as the SNAP-tag, for labeling P. gingivalis cells, which encode an engineered version of the human DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase. Fluorescent substrates containing O(6)-benzylguanine covalently and specifically bind to the enzyme via stable thioether bonds. For the present study, we constructed a replicative plasmid carrying SNAP26b under the control of the P. gingivalis endogenous trxB promoter. The P. gingivalis-expressing SNAP26 protein was successfully labeled with specific fluorophores under anaerobic conditions. Porphyromonas gingivalis biofilm formation was investigated using flow cells and confocal laser scanning microscopy. A specific distribution of a strong fluorescence signal was demonstrated in P. gingivalis-SNAP26 monospecies and bispecies biofilms with Streptococcus gordonii-GFPmut3(*). These findings show that the SNAP-tag can be applied to studies of anaerobic bacteria in biofilm models and is a useful and advantageous alternative to existing labeling strategies.
- Published
- 2010
19. Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells
- Author
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Ophélie Nicolle, Raghida Damaj, Massiullah Shafaq-Zadah, Jacques Pecreaux, Ghislain Gillard, and Grégoire Michaux
- Subjects
integumentary system ,biology ,Cadherin ,Embryogenesis ,Morphogenesis ,Cell Biology ,Embryonic stem cell ,Clathrin ,3. Good health ,Cell biology ,In vivo ,biology.protein ,Gene ,Actin - Abstract
E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.
- Published
- 2015
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