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1. High-Content RNAi Phenotypic Screening Unveils the Involvement of Human Ubiquitin-Related Enzymes in Late Cytokinesis

Author

Mikaël Boullé, Laurianne Davignon, Keïs Nabhane Saïd Halidi, Salomé Guez, Emilie Giraud, Marcel Hollenstein, and Fabrice Agou

Subjects
cytokinesis, CEP55, ubiquitin signaling, RNAi, image analysis tool, high-content screening, Cytology, QH573-671
Abstract

CEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first structural and functional link between ubiquitin signaling and ESCRT-mediated severing of the intercellular bridge. So far, high-content screens focusing on cytokinesis have used multinucleation as the endpoint readout. Here, we report an automated image-based detection method of intercellular bridges, which we applied to further our understanding of late cytokinetic signaling by performing an RNAi screen of ubiquitin ligases and deubiquitinases. A secondary validation confirmed four candidate genes, i.e., LNX2, NEURL, UCHL1 and RNF157, whose downregulation variably affects interconnected phenotypes related to CEP55 and its UBDs, as follows: decreased recruitment of CEP55 to the midbody, increased number of midbody remnants per cell, and increased frequency of intercellular bridges or multinucleation events. This brings into question the Notch-dependent or independent contributions of LNX2 and NEURL proteins to late cytokinesis. Similarly, the role of UCHL1 in autophagy could link its function with the fate of midbody remnants. Beyond the biological interest, this high-content screening approach could also be used to isolate anticancer drugs that act by impairing cytokinesis and CEP55 functions.

Published
2022
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2. Two NEMO-like Ubiquitin-Binding Domains in CEP55 Differently Regulate Cytokinesis

Author

Keïs Nabhane Said Halidi, Elisabeth Fontan, Alix Boucharlat, Laurianne Davignon, Marine Charpentier, Mikaël Boullé, Robert Weil, Alain Israël, Emmanuel Laplantine, and Fabrice Agou

Subjects
Science
Abstract

Summary: CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOACEP55 or ZFCEP55 domains, display severe abscission defects. In addition, the ZFCEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZFCEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscission and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome. : Biochemistry; Cell Biology; Structural Biology; Protein Structure Aspects Subject Areas: Biochemistry, Cell Biology, Structural Biology, Protein Structure Aspects

Published
2019
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3. Pinpointing Cell Identity in Time and Space

Author

Anca F. Savulescu, Caron Jacobs, Yutaka Negishi, Laurianne Davignon, and Musa M. Mhlanga

Subjects
spatiotemporal localization, cell subtype classification, spatial transcriptomics, MRNA subcellular localization, cell subtype, Biology (General), QH301-705.5
Abstract

Mammalian cells display a broad spectrum of phenotypes, morphologies, and functional niches within biological systems. Our understanding of mechanisms at the individual cellular level, and how cells function in concert to form tissues, organs and systems, has been greatly facilitated by centuries of extensive work to classify and characterize cell types. Classic histological approaches are now complemented with advanced single-cell sequencing and spatial transcriptomics for cell identity studies. Emerging data suggests that additional levels of information should be considered, including the subcellular spatial distribution of molecules such as RNA and protein, when classifying cells. In this Perspective piece we describe the importance of integrating cell transcriptional state with tissue and subcellular spatial and temporal information for thorough characterization of cell type and state. We refer to recent studies making use of single cell RNA-seq and/or image-based cell characterization, which highlight a need for such in-depth characterization of cell populations. We also describe the advances required in experimental, imaging and analytical methods to address these questions. This Perspective concludes by framing this argument in the context of projects such as the Human Cell Atlas, and related fields of cancer research and developmental biology.

Published
2020
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4. The lncRNA Connection Between Cellular Metabolism and Epigenetics in Trained Immunity

Author

Ezio T. Fok, Laurianne Davignon, Stephanie Fanucchi, and Musa M. Mhlanga

Subjects
trained immunity, epigenetics, metabolism, long non-coding, inflammation, nuclear architecture, Immunologic diseases. Allergy, RC581-607
Abstract

Trained immunity describes the ability of innate immune cells to form immunological memories of prior encounters with pathogens. Recollection of these memories during a secondary encounter manifests a broadly enhanced inflammatory response characterized by the increased transcription of innate immune genes. Despite this phenomenon having been described over a decade ago, our understanding of the molecular mechanisms responsible for this phenotype is still incomplete. Here we present an overview of the molecular events that lead to training. For the first time, we highlight the mechanistic role of a novel class of long non-coding RNAs (lncRNAs) in the establishment and maintenance of discrete, long lasting epigenetic modifications that are causal to the trained immune response. This recent insight fills in significant gaps in our understanding of trained immunity and reveals novel ways to exploit trained immunity for therapeutic purposes.

Published
2019
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5. Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

Author

Christophe Vanbelle, Clémentine Le Nevé, Laura Broutier, Nicolas Gadot, Laurianne Davignon, Julie Valantin, Léa Luciana, Laura Francols, and Alejandro Lopez Gonzalez

Subjects
General Immunology and Microbiology, General Chemical Engineering, General Neuroscience, 3D reconstruction, Spheroid, Cell Culture Techniques, Regenerative medicine, General Biochemistry, Genetics and Molecular Biology, law.invention, Staining, Organoids, 3D cell culture, Imaging, Three-Dimensional, Confocal microscopy, law, Cell culture, Spheroids, Cellular, Organoid, Humans, Biomedical engineering
Abstract

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 µm to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 µm depth) by confocal microscopy.

Published
2021

6. Two NEMO-like Ubiquitin-Binding Domains in CEP55 Differently Regulate Cytokinesis

Author

Elisabeth Fontan, Marine Charpentier, Alain Israël, Alix Boucharlat, Mikaël Boulle, Fabrice Agou, Keïs Nabhane Said Halidi, Robert Weil, Laurianne Davignon, Emmanuel Laplantine, Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris], Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Signalisation et Pathogénèse, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Département de Biologie cellulaire et infection - Department of Cell Biology and infection (BCI), Funding was supplied by Ligue Nationale Contre le Cancer, Global Care Initiative, and Institut Carnot Pasteur MS (to F.A.), Ligue Nationale Contre le Cancer comité du Val-d'Oise and Fondation ARC pour la recherche sur le cancer (to E.L.)., We thank N. Tarantino for technical assistance, A. Haouz (Crystallography Core Facility), P. England (Molecular Biophysics Core Facility), S. Petres (Production and Purification of Recombinant Proteins Core Facility), as well as M. Matondo, T. Chaze, and J. Chamot-Rooke (UtechS MSBio). We acknowledge A. Echard and M. Mhlanga for their advice and support., Institut Pasteur [Paris] (IP), Collège Doctoral, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cell division, Ubiquitin binding, [SDV]Life Sciences [q-bio], Mutant, 02 engineering and technology, Biochemistry, Article, 03 medical and health sciences, Abscission, Ubiquitin, Structural Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], lcsh:Science, Multidisciplinary, biology, Chemistry, Cell Biology, 021001 nanoscience & nanotechnology, Protein Structure Aspects, 3. Good health, Cell biology, 030104 developmental biology, Structural biology, biology.protein, lcsh:Q, 0210 nano-technology, Cytokinesis, Function (biology)
Abstract

Summary CEP55 regulates the final critical step of cell division termed cytokinetic abscission. We report herein that CEP55 contains two NEMO-like ubiquitin-binding domains (UBDs), NOA and ZF, which regulate its function in a different manner. In vitro studies of isolated domains showed that NOA adopts a dimeric coiled-coil structure, whereas ZF is based on a UBZ scaffold. Strikingly, CEP55 knocked-down HeLa cells reconstituted with the full-length CEP55 ubiquitin-binding defective mutants, containing structure-guided mutations either in NOACEP55 or ZFCEP55 domains, display severe abscission defects. In addition, the ZFCEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZFCEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscission and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome., Graphical Abstract, Highlights • CEP55 contains two NEMO-like NOA and UBZ domains • CEP55 NOA and UBZ are crucial for the CEP55 function in cytokinetic coordination • UBZCEP55functions as cargo receptor to the midbody in a ubiquitin-dependent manner • UBZCEP55 preferentially binds non-degradative linear and K63 polyubiquitin chains, Biochemistry; Cell Biology; Structural Biology; Protein Structure Aspects

Published
2019

7. The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease

Author

Brigitte Buendia, Alain Lilienbaum, Ana Ferreiro, Julien Fauré, Corinne Dill, C. Julien, Eva Cabet, Agnès Guichet, John Rendu, C. Chauveau, Pascale Marcorelles, Nathalie Vadrot, Marie Christine Minot, Odile Dubourg, Isabelle Duband-Goulet, L. Lazaro, Jean Paul Leroy, Laurianne Davignon, Valérie Allamand, Sylvie Odent, Groupe Myologie, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), INSERM U836, équipe 4, Muscles et pathologies, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de biochimie et génétique moléculaire, CHU Grenoble, CHU Pontchaillou [Rennes], Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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 ), Service de génétique clinique [Rennes], Université de Rennes (UR)-CHU Pontchaillou [Rennes]-hôpital Sud, Service de pédiatrie, Centre Hospitalier de la Côte Basque (CHCB), Anatomopathologie Pathologique, CHRU Hôpital Morvan, Laboratoire de Neuropathologie Raymond Escourolle [CHU Pitié-Salpétriêre], Université Pierre et Marie Curie - Paris 6 (UPMC)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Service de Pédiatrie, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Raymond Poincaré [AP-HP]-Centre de Référence Maladies Neuromusculaires (GNMH), the Association Institut de Myologie, the Institut National de la Santé et la Recherche Médicale (Inserm), the Université Pierre et Marie Curie and the Université Paris Diderot, France, the International Graduate Program for Muscle Sciences, GK1631 ‘Myograd’, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Joseph Fourier - Grenoble 1 (UJF), 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), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-CHU Pontchaillou [Rennes]-hôpital Sud, Le CHCB, Centre Hospitalier de la Côte Basque, Laboratoire de Neuropathologie Raymond Escourolle, Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Pierre et Marie Curie - Paris 6 (UPMC), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Université Pierre et Marie Curie - Paris 6 (UPMC), 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 ), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-CHU Pontchaillou [Rennes]-Hôpital Sud, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Pitié-Salpêtrière [APHP], CHU Pitié-Salpêtrière [APHP], and Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-AP-HP Hôpital Raymond Poincaré [Garches]-Centre de Référence Maladies Neuromusculaires (GNMH)

Subjects
Male, 0301 basic medicine, medicine.medical_specialty, Pathology, Adolescent, RNA Stability, Cellular differentiation, [SDV]Life Sciences [q-bio], Nonsense mutation, Biology, Muscle Development, Cell Line, Mice, 03 medical and health sciences, 0302 clinical medicine, Muscular Diseases, Internal medicine, Genetics, medicine, Animals, Humans, Myocyte, Genetic Predisposition to Disease, Child, Muscle, Skeletal, Myopathy, Molecular Biology, Genetics (clinical), Muscle contracture, Myogenesis, Gene Expression Regulation, Developmental, Infant, Muscle weakness, Skeletal muscle, Cell Differentiation, Sequence Analysis, DNA, General Medicine, Pedigree, 030104 developmental biology, Endocrinology, medicine.anatomical_structure, Codon, Nonsense, Female, medicine.symptom, 030217 neurology & neurosurgery, Genome-Wide Association Study, Transcription Factors
Abstract

International audience; Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation inTRIP4encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted inTRIP4mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and inTrip4knocked-down C2C12 led to a significant reduction in myotube diameterex vivoandin vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the firstTRIP4mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism

Published
2016

8. Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Author

Arnaud Ferry, Laurianne Davignon, Jean-Louis Mandel, Ichizo Nishino, Jocelyn Laporte, Leonela Amoasii, Belinda S. Cowling, Anne Toussaint, and Pascale Koebel

Subjects
Pathology, medicine.medical_specialty, Blotting, Western, Fluorescent Antibody Technique, Biology, Sarcomere, Pathology and Forensic Medicine, Dynamin II, Mice, Microscopy, Electron, Transmission, Transduction, Genetic, medicine, Animals, Humans, Centronuclear myopathy, Muscle, Skeletal, Autosomal dominant centronuclear myopathy, Muscle Weakness, Muscle biopsy, medicine.diagnostic_test, Skeletal muscle, Muscle weakness, Regular Article, Triad (anatomy), medicine.disease, Cell biology, medicine.anatomical_structure, Mutation, medicine.symptom, ITGA7, Myopathies, Structural, Congenital
Abstract

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

Published
2011

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