Molecular and cellular characterisation of dystrophin-dependant muscle degeneration in the nematode Cænorhabditis elegans

La Dystrophie Musculaire de Duchenne (DMD) est la plus fréquente et la plus sévère des maladies dégénératives du muscle. Elle se caractérise par une dégénérescence progressive des fibres musculaires due à l’absence de dystrophine fonctionnelle dans les muscles. Actuellement, le rôle physiologique de la dystrophine n’est pas clairement établi et il n’existe pas encore de traitement curatif pour cette maladie. La difficulté de mettre en évidence la fonction de la dystrophine et la physiopathologie de la DMD est en partie expliquée par la complexité moléculaire et cellulaire du muscle des modèles vertébrés utilisés dans les études actuelles. Notre équipe de recherche a développé un modèle de DMD chez le nématode Caenorhabditis elegans. Dans ce modèle, la mutation du gène de la dystrophine, provoque une dégénérescence progressive des muscles conduisant à une paralysie des animaux adultes. Nous utilisons ce modèle afin d’étudier la fonction de la dystrophine et les mécanismes impliqués dans la dégénérescence musculaire chez le nématode. Ce travail de thèse porte sur deux nouveaux acteurs de la dégénérescence musculaire dépendante de la dystrophine : la protéine DYC‐1 et son principal partenaire ZYX‐1. Ce travail présente la caractérisation de ces deux protéines et étudie leurs fonctions dans le muscle. Par ailleurs, ce travail de thèse présente les premiers résultats d’un projet de microscopie électronique ayant pour but de caractériser en détail les évènements subcellulaires du processus dégénératif au cours du cycle de vie du nématode dystrophique. À plus long terme, les études chez le nématode permettront de proposer de nouvelles hypothèses quant aux mécanismes moléculaires et cellulaires de la dégénérescence musculaireDuchenne Muscular Dystrophy (DMD) is the most prevalent and one of the most severe muscular dystrophy. DMD is due to the absence of functional dystrophin in cardiac and skeletal muscle cells, this lack leads to a progressive muscle degeneration of contractile fibres. Currently, the physiological role of dystrophin is not yet clearly established and curative treatments for DMD are not yet available. The lack of knowledge about dystrophin function and DMD physiopathology can be partly attributed to the complexity of vertebrate muscle, and the absence of a simple model that emulates the human pathology. Our research team developed a model of muscle degeneration in the nematode Caenorhabditis elegans. In this model, the mutation of the dystrophin gene produces a progressive muscle degeneration leading to the paralysis of the adult worms. We use this model for investigating the role of dystrophin and the mechanisms of muscle degeneration in C. elegans. This PhD work concerns two new actors of dystrophin‐dependant muscle degeneration: The DYC‐1 protein and its main interactor ZYX‐1. This study aims to characterise these proteins and to study their muscle functions. Moreover, this PhD work presents preliminary results of an in depth characterisation of subcellular processes of muscle degeneration in dystrophic worms by electron microscopy. Our aim is to visualise first events and to observe the progression of degeneration until the death of muscle cell. These molecular and cellular approaches aims to get new insights in the mechanisms underlying muscle degeneration in order to propose new hypotheses for the understanding of DM

The C. elegans dense body: anchoring and signaling structure of the muscle.

During evolution, both the architecture and the cellular physiology of muscles have been remarkably maintained. Striated muscles of invertebrates, although less complex, strongly resemble vertebrate skeletal muscles. In particular, the basic contractile unit called the sarcomere is almost identical between vertebrates and invertebrates. In vertebrate muscles, sarcomeric actin filaments are anchored to attachment points called Z-disks, which are linked to the extra-cellular matrix (ECM) by a muscle specific focal adhesion site called the costamere. In this review, we focus on the dense body of the animal model Caenorhabditis elegans. The C. elegans dense body is a structure that performs two in one roles at the same time, that of the Z-disk and of the costamere. The dense body is anchored in the muscle membrane and provides rigidity to the muscle by mechanically linking actin filaments to the ECM. In the last few years, it has become increasingly evident that, in addition to its structural role, the dense body also performs a signaling function in muscle cells. In this paper, we review recent advances in the understanding of the C. elegans dense body composition and function

Identification, Evolution and Expression of an Insulin-Like Peptide in the Cephalochordate Branchiostoma lanceolatum

International audienceInsulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF-and insulin-like functions in amphioxus supporting the idea of a common ancestral protein

Backbone NMR assignment and secondary structure of the 19 kDa hemophore HasA.

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Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype

International audienceCRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison

Sequence of the primers used in the qPCR analysis.

<p>Sequence of the primers used in the qPCR analysis.</p

Expression pattern of amphioxus <i>ilp</i> during early embryogenesis.

<p>During the first developmental stages, (A) the eight cells embryos and (B) blastula stages, <i>ilp</i> is not expressed. (C) Dorsal view of a gastrula stage embryo, black arrows indicate <i>ilp</i> expression in the presumptive paraxial mesoderm. Dorsal (D), transversal (E) and left side (F) views of an early neurula embryo. Arrows show <i>ilp</i> signal in the paraxial mesoderm, the arrowhead shows the expression in the posterior endoderm. (G) Left side of a mid-neurula stage embryo, arrowhead shows <i>ilp</i> expression in the developing endoderm. (H) Left side of a late neurula stage embryo before the mouth opening. <i>ilp</i> is expressed in the anterior and central part of the endoderm, arrowhead shows the strongest signal in the mid-gut. Scale bars are 100μm. Anterior is to the left in C, D, F, G and H.</p

Expression pattern of amphioxus <i>ilp</i> in the larvae.

<p>(A) Left side of the larvae. (B) Enlargement of the pharyngeal region of the larvae shown in A. (C) Ventral view of the same larva. Arrowheads show <i>ilp</i> expression in the club shaped gland and arrows show <i>ilp</i> expression in the anterior pharyngeal endoderm. Scale bars are 100μm.</p

Sequence IDs of members of the insulin-relaxin superfamily used to build the phylogeny depicted in Fig. 1C.

<p>Sequence IDs of members of the insulin-relaxin superfamily used to build the phylogeny depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119461#pone.0119461.g001" target="_blank">Fig. 1C</a>.</p

Structural comparison and phylogenetic relationships between amphioxus ILP and insulin-relaxin superfamily proteins in vertebrates.

<p>A. Comparison of amphioxus ILP domain structure with human insulin, IGFs and relaxin. B. Sequence alignment of the conserved A and B domains of ILP sequences from different amphioxus species and insulin, IGF and relaxin sequences from vertebrates and sea urchin. Black arrows indicate the conserved cysteine residues necessary for the formation of disulfide bonds during the processing of precursor proteins. C. Phylogenetic maximum likelihood analysis of chordate insulin/IGF subfamily members. The red square highlights amphioxus ILP sequences, the blue square highlights vertebrates insulin sequences and the yellow square highlights vertebrates IGF sequences; the grey square indicates the outgroup made with relaxin sequences. <i>B</i>. <i>lanceolatum</i> ILP sequence identified in this study is pointed with the red arrow. Bootstrap values derived from 1000 runs are shown. Branches with bootstraps lower than 500 were collapsed. The scale bar indicates the average number of amino acid substitutions per site.</p