Pinpointing cell identity in time and space

Copyright © 2020 Savulescu, Jacobs, Negishi, Davignon and Mhlanga. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.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.info:eu-repo/semantics/publishedVersio

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

International audience(F.A.) HIGHLIGHTS CEP55 contains two NEMO-like NOA and UBZ domains CEP55 NOA and UBZ are crucial for the CEP55 function in cytokinetic coordination UBZ CEP55 functions as cargo receptor to the midbody in a ubiquitin-dependent manner UBZ CEP55 preferentially binds non-degradative linear and K63 polyubiquitin chains Said Halidi et al., iScience 20, 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 NOA CEP55 or ZF CEP55 domains, display severe abscission defects. In addition, the ZF CEP55 can be functionally replaced by some ZF-based UBDs belonging to the UBZ family, indicating that the essential function of ZF CEP55 is to act as ubiquitin receptor. Our work reveals an unexpected role of CEP55 in non-degradative ubiquitin signaling during cytokinetic abscis-sion and provides a molecular basis as to how CEP55 mutations can lead to neurological disorders such as the MARCH syndrome

The lncRNA Connection Between Cellular Metabolism and Epigenetics in Trained Immunity

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

Identification et caractérisation de nouveaux gènes associés à la myopathie à multiminicores

Congenital myopathies are rare genetic disorders characterized by neonatal hypotonia, delayed motor development and muscle weakness. Our laboratory is particularly interested in the study of multi-minicore disease (MmD), which is characterised by multiple foci of mitochondria depletion and short sarcomere disorganisation areas (cores) within muscle fibers. Our group identified most of the genes associated to this genetically and phenotypically heterogeneous condition. However, at least 30% of multiminicore disease cases are not associated with the known genes and remain genetically uncharacterized. The study of a large consanguineous family by homozygosity mapping allowed the identification of a homozygous nonsense mutation in the coding sequence of a transcriptional coactivator (named thereafter TCA), which had never been associated with a muscle condition. qPCR and western blotting showed absence of messenger and protein on patient samples. A microarray performed on a transient TCA silencing model, which disclosed a tendency to downregulation of muscle and contractile proteins (in differentiation conditions), and an upregulation of cell cycle proteins (in proliferative conditions), suggesting a role of TCA in regulating the proliferation/differentiation balance in muscle. Thus, we report a novel congenital muscle condition with a unique histological pattern, stressing the histological overlap of different forms of congenital myopathies and muscular dystrophies. We characterize a new gene in human genetic conditions and a novel regulator of the proliferation/differentiation balance in muscle.Les myopathies congénitales sont des pathologies génétiques rares caractérisées par une hypotonie néonatale, un retard moteur et une faiblesse musculaire. Notre laboratoire s'intéresse à la myopathie à multiminicore (MmD) qui se caractérise par une réduction de l'activité mitochondriale en de multiples points focaux et une désorganisation des sarcomères au sein de la fibre musculaire. Notre groupe est à l'origine de l'identification de la plupart des gènes mutés dans cette pathologie. Néanmoins, 30% des cas restent à ce jour sans diagnostic moléculaire. L'étude d'une famille consanguine a permis d'identifier une mutation homozygote tronquante dans la région codante d'un coactivateur transcriptionnel (appelé TCA) dont la fonction n'a jamais été associée au muscle. L'analyse des cellules de patients a révélé l'absence d'ARN messager ainsi que l'absence de protéine. Par des analyses transcriptomiques sur un modèle d'extinction transitoire de TCA, j'ai pu mettre en évidence une diminution de l'expression des protéines musculaires contractiles (en différenciation) et une augmentation des protéines du cycle cellulaire (en prolifération) suggérant que TCA joue un rôle dans la balance prolifération/différenciation au sein du tissu musculaire. Nous présentons ici une nouvelle forme de myopathie congénitale avec un profil histologique original qui met en évidence l'existence de nombreux points communs entre les différentes formes de myopathies mais également avec les dystrophies musculaires. Nous avons identifié un nouveau gène impliqué dans les maladies génétiques humaines qui se trouve être un acteur de la balance prolifération/différenciation au sein du muscle

Identifizierung und Charakterisierung neuer Gene assoziiert mit der Multiminocore-Krankheit

ACKNOWLEDGMENTS SUMMARY ABBREVIATIONS LIST OF FIGURES LIST OF TABLES PREFACE INTRODUCTION I. SKELETAL MUSCLE TISSUE I.1 Muscle tissue (s) I.2 Skeletal muscle II. SKELETAL MUSCLE CONTRACTION II.1 Muscle contraction unit: the sarcomere II.1.a Thick filaments and associated proteins II.1.b Thin filaments and associated proteins II.1.c Titin filament as a third component of the sarcomere II.2 The triadic junction III. MYOGENESIS III.1 Muscle specific transcriptional factors III.1.a Paired box (Pax) transcription factors III.1.b Muscle Regulatory Factors III.2 Myogenic determination and differentiation III.2.a Induction of MRF III.2.b Cell cycle and progenitors expansion III.2.c Cell cycle withdrawal III.2.d Induction of differentiation and fusion III.3 Embryonic myogenesis III.3.a Specification of domains III.3.b Specification of muscle primitive progenitors III.3.c Embryonic pathways regulating muscle progenitor cell fate III.3.d Foetal myogenesis and muscle growth III.4 Adult myogenesis III.4.a Quiescent satellite cells III.4.b Activation III.4.c Satellite cell heterogeneity III.4.d Satellite cells and their niche IV. CONGENITAL MUSCULAR DISORDERS IV.1 General presentation IV.2 Congenital muscular dystrophies IV.2.a General presentation IV.2.b Muscle collagenopathies IV.3 Congenital myopathies IV.3.a General presentation IV.3.b Cap disease IV.4 Specific focus: Multiminicore disease V. METHODS OF INVESTIGATIONS IN MONOGENIC DISORDERS V.1 Human genome and monogenic disorders V.2 Classical strategies used in human mutation screening V.2.a Linkage analysis V.2.b Positional cloning V.2.c Concrete case: Investigation in consanguineous families V.3 Second generation sequencing V.4 Third generation sequencing VI. AIMS OF MY PROJECT MATERIAL & METHODS I. GENETIC INVESTIGATIONS I.1. Patients DNA samples and consent I.2. Linkage analyses I.3. Next Generation Sequencing I.4. Positional cloning and Sanger sequencing II. CELL CULTURE II.1 Human material II.2 Murine myoblastic cell line II.3 RNA silencing III. TRANSCRIPTOMIC ANALYSES III.1. RT PCR & q RT PCR III.2. Microarray III.3. Luciferase assays IV. PROTEIN ANALYSES IV.1. Western Blotting IV.2. Animals tissues sampling IV.2. Immunofluorescence VI. STATISTICAL ANALYSES RESULTS I. COHORT PRESENTATION II. IDENTIFICATION AND CHARACTERISATION OF A TRANSCRIPTIONAL COACTIVATOR MUTATED IN AN UNREPORTED FORM OF CONGENITAL MYOPATHY II.1 Original phenotypical presentation II.2 Novel locus associated to a human condition II.3 Positional candidate genes II.4 Relevance of the TRIP4 mutation II.4.a A nonsense mutation leading to messenger RNA degradation II.4.b The nonsense mutation leads to the complete absence of protein II.4.c Absence of ASC-1 impairs the intracellular localisation of known protein partners II.5 ASC-1 as a novel actor in muscle physiology II.6 Function of ASC-1 in an in vitro model II.6.a Transcriptomic analysis of a transient knock down model II.6.b ASC-1 has no major impact on myoblast proliferation in vitro II.6.c ASC-1 transient knock down induces a delay in late myogenic differentiation II.7 TRIP4 mutation as a privative condition III. INVESTIGATIONS IN A SERIE OF PATIENTS WITH MmD AND SCOLIOSIS: IDENTIFICATION OF NEW CAUSATIVE GENES III.1 Strategies used III.2 Families status and candidate genes DISCUSSION Identification of TRIP4 as a novel MmD gene: towards a reassessment of the classification of congenital muscle disorders TRIP4 deficiency and muscle disease: potential genotype-phenotype correlations ASC-1 as a novel key player in muscle physiology and pathophysiology Search for new genes in MmD: efficiency and limitations of the current genetic methods of investigation CONCLUSION & PERSPECTIVES BIBLIOGRAPHY ANNEXES ABSTRACTCongenital myopathies are rare genetic disorders characterized by neonatal hypotonia, delayed motor development and muscle weakness, associated with characteristic histological changes in the structure of muscle fibres visible on the patients’ muscle biopsies. Our laboratory is particularly interested in the study of core myopathies, which are emerging as the most prevalent form of congenital myopathy, and especially of multi-minicore disease (MmD), which is characterised by multiple focal short areas of mitochondria depletion and sarcomere disorganisation (cores) within muscle fibers. Our group identified most of the genes associated to this genetically and phenotypically heterogeneous condition. However, at least 30% of multiminicore disease cases are not associated with the known genes and remain genetically uncharacterized. During my PhD, my objective was to identify and characterize new genes responsible for this condition. The study of a large consanguineous family by homozygosity mapping allowed the identification of a homozygous nonsense mutation in the coding sequence of a transcriptional coactivator (named thereafter TCA), which had never been associated with a muscle condition. The 3 affected patients presented with a novel, very severe form of congenital myopathy with an unreported histological pattern associating minicores, nuclear internalization and cap lesions. qPCR and western blotting showed absence of messenger and protein on patient samples, suggesting NMD (nonsense mediated decay). The increased TCA expression profile in murine axial skeletal muscles is consistent with the clinical presentation. Also, I found increased protein expression during in vitro C2C12 myoblastic cell line differentiation, which is compatible with a contribution to myogenesis. Subsequently, I performed microarray analysis on a transient TCA silencing model, which disclosed a tendency to downregulation of muscle and contractile proteins (in differentiation conditions), and an upregulation of cell cycle proteins (in proliferative conditions), suggesting a role of TCA in regulating the proliferation/differentiation balance in muscle. Consistently, Dual Reporter Luciferase assays performed on proliferative C2C12 identified p21 as an activated target of TCA suggesting a role of the protein in the cell cycle exit regulation more specifically. Thus, we report a novel congenital muscle condition with a unique histological pattern, stressing the histological overlap of different forms of congenital myopathies and muscular dystrophies. We characterize a new gene in human genetic conditions and a novel regulator of the proliferation/differentiation balance in muscle. In parallel, to identify other genes associated with MmD, I investigated four highly informative and consanguineous families with MmD non-associated with the known genes. By crossing homozygosity mapping data and massive parallel sequencing, I identified a candidate gene, which encodes a protein potentially implicated in cell stemness and linked to p53 activity. Confirmation of the pathogeneicity of this change and gene are in progress in our laboratory. A novel transcriptional coactivator is pivotal in regulating the balance between proliferation and differentiation of myogenic progenitors and is mutated in a novel form of congenital myopathy. Davignon et al. – in preparationIdentifikation und Charakterisierung eines neuen Genes das mit einem unbeschriebenen Phänotyp der Kongenitalen Myopathie korreliert. Kongenitale Myopathien sind seltene genetische Erkrankungen zu deren Symptomen neonatale Hypotonie, eine verspätete motorische Entwicklung und Muskelschwäche gehören. Sie sind mit charakteristischen histologischen Veränderungen in der Muskelfaserstruktur assoziiert. Unser Labor beschäftigt sich mit der Erforschung der Kongenitalen Myopathien – insbesondere der Multiminicore Krankheit (MmD), die durch multiple fokale kurze Areale mit mitrochondrialem Abbau die zur Auflösung des Sarkomers führen charakterisiert ist. Unsere Arbeitsgruppe identifizierte die meisten der mit dieser Krankheit assoziierte Gene. Dennoch sind 30% der Multiminicore Erkrankungen sind nicht mit diesen Genen assoziiert und noch nicht genetisch charakterisiert. Das Ziel meiner Promotionsarbeit war die Identifikation und Charakterisierung neuer MmD Gene. Als Vorarbeiten hatte unser Labor eine Großfamilie mit drei Patienten untersucht, die ungewöhnliche Minicores mit Kerninternalisierungen und Cap- Läsionen aufwiesen, als Zeichen einer bisher unbekannten, schweren Form der Kongenitalen Myopathie. Die zugrundeliegende homozygote Nonsens-Mutation des Transkriptionscoaktivators (TCA) wurde zuvor nicht mit der Kongenitalen Myopathie in Verbindung gebracht. Der Verlust von TCA mRNA und Protein wurde in Realtime PCR und Western Blot dokumentiert und impliziert nonsense mediated decay (NMD) als moleklulare Grundlage der Erkrankung. Das Expressionsprofil von TCA im Skeletmuskel in vivo als auch in vitro (C2C12). Letztere wiesen eine erhöhte Expression von TCA während der Differzierung auf. Dies unterstützt die Hypothese der Beteiligung von TCA an der Myogenese. Nachfolgend untersuchte ich TCA defiziente C2C12 Zelllinien mit Hilfe einer Microarray Analyse. Die Ergebnisse zeigten eine Runterregulierung von kontraktilen Muskelproteinen und gleichzeitig die Hochregulierung von Zell- Zyklus-Proteinen. Dies suggeriert die Beteiligung von TCA an der Regulierung der Proliferation und der Differenzierung im Skelettmuskel. Zudem zeigten Reporter- Luciferase-Assays in proliferierenden, TCA überexpremierenden C2C12 Zellen eine direkte und aktivierende Interaktion zu p21, was die Rolle von TCA auf das Ende des Zell-Zyklus spezifiziert. Wir beschreiben somit eine neue kongenitale Muskelkondition mit einem einzigartigen histologischen Muster, die eine histologische Überlappung zwischen verschiedenen Kongenitalen Myopathien und Muskeldystrophien aufzeigt. Zudem charakterisierten wir unter humanen genetischen Bedingungen ein neues Gen, welches die Proliferation und Differenzierung des Muskels reguliert. Weitergehende Untersuchungen des Gens werden in unserem Labor bereits durchgeführt. Parallel untersuchte ich fünf weitere Familien mit MmD. Dabei identifizierte ich ein weiteres Gen dessen Protein potentiellen Einfluss auf den Differenzierungsmechanismus hat und mit der Aktivität von p53 verknüpft zu sein scheint. Die Pathogenität der Veränderung und das Gen werden in unserem Labor weiter untersucht. Eine Veröffentlichung mit dem Titel „A novel transcriptional coactivator is pivotal in regulating the balance between proliferation and differentiation of myogenic progenitors and is mutated in a novel form of congenital myopathy.” ist in Bearbeitung. Davignon et a

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

International audienceCEP55 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

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

International audienceDespite 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 mechanis