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Differentiated cells can be forced to change identity, either to directly adopt another differentiated identity or to revert to a pluripotent state. Direct reprogramming events can also occur naturally. We recently characterized such an event in Caenorhabditis elegans, in which a rectal cell switches to a neuronal cell. Here we have used this single-cell paradigm to investigate the molecular requirements of direct cell-type conversion, with a focus on the early steps. Our genetic analyses revealed the requirement of sem-4/Sall, egl-27/Mta, and ceh-6/Oct, members of the NODE complex recently identified in embryonic stem (ES) cells, and of the OCT4 partner sox-2, for the initiation of this natural direct reprogramming event. These four factors have been shown to individually impact on ES cell pluripotency; however, whether they act together to control cellular potential during development remained an open question. We further found that, in addition to acting at the same time, these factors physically associate, suggesting that they could act together as a NODE-like complex during this in vivo process. Finally, we have elucidated the functional domains in EGL-27/MTA that mediate its reprogramming activity in this system and have found that modulation of the posterior HOX protein EGL-5 is a downstream event to allow the initiation of Y identity change. Our data reveal unique in vivo functions in a natural direct reprogramming event for these genes that impact on ES cells pluripotency and suggest that conserved nuclear events could be shared between different cell plasticity phenomena across phyla.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/fisiologia , Fator 3 de Transcrição de Octâmero/fisiologia , Fatores de Transcrição SOXB1/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Células HeLa , Humanos , Proteína Homeobox NanogRESUMO
In virtually all eukaryotes, the mitochondrial DNA (mtDNA) encodes proteins necessary for oxidative phosphorylation (OXPHOS) and RNAs required for their synthesis. The mechanisms of regulation of mtDNA copy number and expression are not completely understood but crucially ensure the correct stoichiometric assembly of OXPHOS complexes from nuclear- and mtDNA-encoded subunits. Here, we detect adenosine N6-methylation (6mA) on the mtDNA of diverse animal and plant species. This modification is regulated in C. elegans by the DNA methyltransferase DAMT-1 and demethylase ALKB-1. Misregulation of mtDNA 6mA through targeted modulation of these activities inappropriately alters mtDNA copy number and transcript levels, impairing OXPHOS function, elevating oxidative stress, and shortening lifespan. Compounding these defects, mtDNA 6mA hypomethylation promotes the cross-generational propagation of a deleterious mtDNA. Together, these results reveal that mtDNA 6mA is highly conserved among eukaryotes and regulates lifespan by influencing mtDNA copy number, expression, and heritable mutation levels in vivo.
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BACKGROUND: Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. RESULTS: Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. CONCLUSIONS: This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology.
Assuntos
Evolução Molecular , Receptores Proteína Tirosina Quinases/química , Sequência de Aminoácidos , Animais , Simulação por Computador , Sequência Conservada , Variação Genética , Genômica , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência do Ácido NucleicoRESUMO
The ability to balance conflicting functional demands is critical for ensuring organismal survival. The transcription and repair of the mitochondrial genome (mtDNA) requires separate enzymatic activities that can sterically compete1, suggesting a life-long trade-off between these two processes. Here in Caenorhabditis elegans, we find that the bZIP transcription factor ATFS-1/Atf5 (refs. 2,3) regulates this balance in favour of mtDNA repair by localizing to mitochondria and interfering with the assembly of the mitochondrial pre-initiation transcription complex between HMG-5/TFAM and RPOM-1/mtRNAP. ATFS-1-mediated transcriptional inhibition decreases age-dependent mtDNA molecular damage through the DNA glycosylase NTH-1/NTH1, as well as the helicase TWNK-1/TWNK, resulting in an enhancement in the functional longevity of cells and protection against decline in animal behaviour caused by targeted and severe mtDNA damage. Together, our findings reveal that ATFS-1 acts as a molecular focal point for the control of balance between genome expression and maintenance in the mitochondria.
Assuntos
Proteínas de Caenorhabditis elegans , DNA Mitocondrial , Animais , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Caenorhabditis elegans/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dano ao DNA , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Achieving controlled reprogramming of differentiated cells into a desired cell type would open new opportunities in stem-cell biology and regenerative medicine. Experimentation on cell reprogramming requires a model in which cell conversion can be induced and tracked individually. The tiny nematode, Caenorhabditis elegans, owing to its known cellular lineage, allows the study of direct cell type conversion with a single-cell resolution. Indeed, recent advances have shown that despite its invariant cell lineage, cellular identities can be reprogrammed, leading to cell conversion in vivo. In addition, natural transdifferentiation events occur in the worm, providing a powerful model for the study of cellular plasticity in a physiological cellular microenvironment. Here, we review pioneer studies on induced and naturally occurring reprogramming events in C. elegans and the new notions that have emerged.
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Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Diferenciação Celular , Reprogramação Celular , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Transdiferenciação Celular , Microambiente Celular , Epigenômica , Medicina Regenerativa , Células-Tronco/citologiaRESUMO
Transdifferentiation, or direct cell reprogramming, is the conversion of one fully differentiated cell type into another. Whether core mechanisms are shared between natural transdifferentiation events when occurring with or without cell division is unclear. We have previously characterized the Y-to-PDA natural transdifferentiation in Caenorhabditis elegans, which occurs without cell division and requires orthologs of vertebrate reprogramming factors. Here, we identify a rectal-to-GABAergic transdifferentiation and show that cell division is required but not sufficient for conversion. We find shared mechanisms, including erasure of the initial identity, which requires the conserved reprogramming factors SEM-4/SALL, SOX-2, CEH-6/OCT, and EGL-5/HOX. We also find three additional and parallel roles of the Wnt signaling pathway: selection of a specific daughter, removal of the initial identity, and imposition of the precise final subtype identity. Our results support a model in which levels and antagonistic activities of SOX-2 and Wnt signaling provide a timer for the acquisition of final identity.
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Proteínas de Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transdiferenciação Celular , Mitose , Via de Sinalização WntRESUMO
Cell-Specific Mitochondria Affinity Purification (CS-MAP) enables isolation and purification of intact mitochondria from individual cell types of Caenorhabditis elegans. The approach is based on the cell-specific expression of a recombinant hemagglutinin (HA)-tag fused to the TOMM-20 protein that decorates the surface of mitochondria, thereby allowing their immunomagnetic purification. This protocol describes the CS-MAP procedure performed on large populations of animals. The purified mitochondria are suitable for subsequent nucleic acid, protein, and functional analyses. For complete details on the use and execution of this protocol, please refer to Ahier et al. (2018, 2021).
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Caenorhabditis elegans/citologia , Técnicas Citológicas/métodos , Técnicas Imunológicas/métodos , Mitocôndrias , Animais , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologiaRESUMO
In multiple species, certain tissue types are prone to acquiring greater loads of mitochondrial genome (mtDNA) mutations relative to others, but the mechanisms that drive these heteroplasmy differences are unknown. We find that the conserved PTEN-induced putative kinase (PINK1/PINK-1) and the E3 ubiquitin-protein ligase parkin (PDR-1), which are required for mitochondrial autophagy (mitophagy), underlie stereotyped differences in heteroplasmy of a deleterious mitochondrial genome mutation (ΔmtDNA) between major somatic tissues types in Caenorhabditis elegans. We demonstrate that tissues prone to accumulating ΔmtDNA have lower mitophagy responses than those with low mutation levels. Moreover, we show that ΔmtDNA heteroplasmy increases when proteotoxic species that are associated with neurodegenerative disease and mitophagy inhibition are overexpressed in the nervous system. These results suggest that PINK1 and parkin drive organism-wide patterns of heteroplasmy and provide evidence of a causal link between proteotoxicity, mitophagy, and mtDNA mutation levels in neurons.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Genoma Mitocondrial , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA Mitocondrial/genética , Heteroplasmia , Mitofagia/genética , Células Musculares/metabolismo , Neurônios/metabolismoRESUMO
Strong loss-of-function or null mutants can sometimes lead to a penetrant early lethality, impairing the study of these genes' function. This is the case for the ceh-6 null mutant, which exhibits 100% penetrant lethality. Here, we describe how we used gene bashing to identify distinct regulatory regions in the ceh-6 locus. This allowed us to generate a ceh-6 null strain that is viable and still displays ceh-6 mutant Y-to-PDA transdifferentiation phenotype. Such strategy can be applied to many other mutants impacting viability.
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Although mitochondria are ubiquitous organelles, they exhibit tissue-specific morphology, dynamics and function. Here, we describe a robust approach to isolate mitochondria from specific cells of diverse tissue systems in Caenorhabditis elegans. Cell-specific mitochondrial affinity purification (CS-MAP) yields intact and functional mitochondria with exceptional purity and sensitivity (>96% enrichment, >96% purity, and single-cell and single-animal resolution), enabling comparative analyses of protein and nucleic acid composition between organelles isolated from distinct cellular lineages. In animals harbouring a mixture of mutant and wild-type mitochondrial genomes, we use CS-MAP to reveal subtle mosaic patterns of cell-type-specific heteroplasmy across large populations of animals (>10,000 individuals). We demonstrate that the germline is more prone to propagating deleterious mitochondrial genomes than somatic lineages, which we propose is caused by enhanced mtDNA replication in this tissue.
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Caenorhabditis elegans/genética , Fracionamento Celular/métodos , Cromatografia de Afinidade , DNA Mitocondrial/genética , Mitocôndrias/genética , Mosaicismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Replicação do DNA , DNA Mitocondrial/biossíntese , Microscopia Confocal , Mitocôndrias/metabolismo , Mutação , Especificidade de ÓrgãosRESUMO
In the version of this Technical Report originally published, chromosome representations (indicated by black lines) were missing from Fig. 2a due to a technical error. The corrected version of Fig. 2a is shown below. This has now been amended in all online versions of the Technical Report.
RESUMO
Insulin signalling is a very ancient and well conserved pathway in metazoan cells, dependent on insulin receptors (IR) which are transmembrane proteins with tyrosine kinase activity. A unique IR is usually present in invertebrates whereas two IR members are found with different functions in vertebrates. This work demonstrates the existence of two distinct IR homologs (SmIR-1 and SmIR-2) in the parasite trematode Schistosoma mansoni. These two receptors display differences in several structural motifs essential for signalling and are differentially expressed in parasite tissues, suggesting that they could have distinct functions. The gene organization of SmIR-1 and SmIR-2 is similar to that of the human IR and to that of the IR homolog from Echinococcus multilocularis (EmIR), another parasitic platyhelminth. SmIR-1 and SmIR-2 were shown to interact with human pro-insulin but not with pro-insulin-like growth factor-1 in two-hybrid assays. Phylogenetic results indicated that SmIR-2 and EmIR might be functional orthologs whereas SmIR-1 would have emerged to fulfil specific functions in schistosomes.
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Proteínas de Helminto/genética , Receptor de Insulina/genética , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
Caenorhabditis elegans is a powerful in vivo model in which transgenesis is highly developed. However, while the analysis of biological phenomena often require the expression of more than one protein of interest, no reliable tool exists to ensure efficient concomitant and equivalent expression of more than two polypeptides from a single promoter. We report the use of viral 2A peptides, which trigger a "ribosomal-skip" or "STOP&GO" mechanism during translation, to express multiple proteins from a single vector in C. elegans. Although none of the viruses known to infect C. elegans contain 2A-like sequences, our results show that 2A peptides allow the production of separate functional proteins in all cell types and at all developmental stages tested in the worm. In addition, we constructed a toolkit including a 2A-based polycistronic plasmid and reagents to generate 2A-tagged fosmids. 2A peptides constitute an important tool to ensure the delivery of multiple polypeptides in specific cells, enabling several novel applications such as the reconstitution of multi-subunit complexes.
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Caenorhabditis elegans/genética , Técnicas Genéticas , Regiões Promotoras Genéticas , Animais , Caenorhabditis elegans/embriologia , Expressão Gênica , Vetores Genéticos , Peptídeos/genética , Peptídeos/metabolismo , Ribossomos/genética , Transfecção , Vírus/químicaRESUMO
Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Transdiferenciação Celular , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Neurônios Motores/citologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Desdiferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Sistema Digestório/citologia , Histona Desmetilases/química , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Metilação , Modelos Biológicos , Dados de Sequência Molecular , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. METHODS AND FINDINGS: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA(B) receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. CONCLUSION: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.
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Aminoácidos/metabolismo , Invertebrados/enzimologia , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Ativação Enzimática , Gônadas/enzimologia , Humanos , Insetos/enzimologia , Larva/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Filogenia , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/genética , Alinhamento de SequênciaRESUMO
In spite of the numerous efforts made to control their transmission, parasite schistosomes still represent a serious public health concern and a major economic problem in many developing countries. Praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis and the only one that is available for mass chemotherapy. However, its widespread use and its inefficacy on juvenile parasites raise fears that schistosomes will develop drug resistance, and make the development of alternative drugs highly desirable. Protein tyrosine kinases (PTKs) are key molecules that control cell differentiation and proliferation and they already represent important targets for molecular cancer therapy. The recent characterization in Schistosoma mansoni of several cytosolic and receptor PTKs, with properties similar but also divergent from their vertebrate counterparts, opens new perspectives for the development of novel strategies in chemotherapy of schistosomiasis, which could be based on the use of parasite-specific tyrosine phosphorylation inhibitors.