Etude de l’exocytose à la membrane apicale des cellules épithéliales de Caenorhabditis elegans.

Les epithelia sont des tissus polarisés à l’interface entre le milieu extérieur et le milieu intérieur des métazoaires. La mise en place et le maintien de leur polarité sont essentiels à l’intégrité de l’organisme. Parmi les processus y contribuant, la sécrétion à la membrane apicale est une voie de trafic intracellulaire méconnue à laquelle je me suis intéressée au cours de ma thèse. Le modèle de sécrétion choisi pour cette étude était la sécrétion épidermique apicale de la cuticule du nématode Caenorhabditis elegans. Un premier gène impliqué dans cette sécrétion apicale et dans l’osmorégulation avait été caractérisé au laboratoire. Il s’agit de che-14, orthologue de dispatched/disp1 (chez D. melanogaster et M. musculus) qui est nécessaire au transport des protéines Hedgehog chez la drosophile et la souris. Dans un premier temps, il a été mis en évidence un lien entre osmorégulation et sécrétion dans les epithelia de C. elegans à travers la caractérisation de nouveaux mutants (dont vha-5 et rdy-2) affectés pour ces deux fonctions, isolés lors d’un crible de phénocopie du mutant che-14. Par la suite, l’utilisation des techniques d’ARN interférent et de mutagenèse dirigée sur vha-5, a permis de démontrer que le secteur V0 de l’ATPase vacuolaire à protons (V-ATPase) contenant la sous-unité « a » VHA-5 est impliqué dans l’osmorégulation et la sécrétion. Plus précisément, ce secteur V0, en association avec le secteur V1 de la V-ATPase est essentiel à l’osmorégulation au niveau du système excréteur, tandis qu’indépendamment de V1, il est spécifiquement requis pour une voie de sécrétion apicale atypique dans l’épiderme hyp7 de C. elegans. La combinaison de techniques de microscopie électronique et photonique confocale a permis de montrer que cette voie de sécrétion implique des corps multivésiculaires sécrétant des exosomes contenant des protéines de type Hedgehog, et est nécessaire au trafic apical de CHE-14 et de deux tétraspanines RDY-2 et TSP-15. Par ailleurs les mutants che-14 et vha-5 affectés pour la sécrétion apicale, sont hypersensibles aux infections par le champignon nématophage Drechmeria coniospora. J’ai ensuite initié une caractérisation plus complète de cette voie de sécrétion en développant des marqueurs des compartiments intracellulaires de l’épiderme, et en réalisant un crible ARNi ciblé sur des gènes potentiellement impliqués dans la régulation de cette voie et des protéines qui l’empruntent. A l’issue de ce travail j’ai proposé un modèle de sécrétion cuticulaire faisant intervenir deux voies de sécrétion apicales préalablement inconnues, et une réorganisation de l’actine corticale permettant le positionnement de la V-ATPase au cours de ce processus. J’ai également discuté l’existence et le rôle d’une signalisation de type Hedgehog chez C. elegans. Enfin, j’ai suggéré un lien entre la réponse immunitaire innée chez C. elegans et la sécrétion épidermique d’exosomes.Epithelia are polarized tissues in-between extra- and intracellular media. The establishment and the maintenance of their polarity are essential to metazoans integrity. Among processes of main importance for these phenomena, I focused on apical secretion at the plasma membrane which is still poorly characterized. The model I chose for this study was the apical secretion of cuticle by the epidermis of the nematode Caenorhabditis elegans. Previously in the lab, the che-14 gene was proved to be involved in osmoregulation and apical secretion. che-14 is dispatched/disp1 orthologue which is involved in Hedgehog proteins transport in Drosophila and mouse. At first, the link between osmoregulation and secretion was further established by the characterization of new mutants (among which vha-5 and rdy-2) obtained in a phenocopy screen of che-14 mutant. By the mean of RNAi and targeted mutagenesis on vha-5, it was proven that the VHA-5 “a” subunit-containing V0 sector of the vacuolar-H+-ATPase (V-ATPase) is involved in both osmoregulation and secretion. More precisely, V0 in association with the V1 sector of the V-ATPase is essential for osmoregulation in the excretory system, whereas V0 alone is specifically required for an unconventional new apical secretion pathway in C. elegans hyp7 epidermis. Combining electron- and confocal photon-microscopy, it is shown that this new secretion pathway involves multivesicular bodies releasing exosomes containing Hedgehog-related proteins, and is necessary for the proper apical trafficking of CHE-14 and the two tetraspan proteins RDY-2 and TSP-15. Additionally, che-14 and the vha-5 secretion-defective mutants are hypersensitive to infection by the nematode-specific fungus Drechmeria coniospora. I then started to investigate further into this secretion pathway by developing markers for intracellular compartments of the epidermis, and by targeting with RNAi genes possibly involved in the regulation of this process or expressing proteins trafficking through this secretion pathway. Finally I proposed a model for the secretion of C. elegans cuticle involving two apical secretory pathways and the reorganization of the cortical actin allowing proper positioning of the V-ATPase along this process. I also discussed the existence of a Hedgehog-type signaling pathway in C. elegans. Ultimately I suggested a link between the apical secretion of exosomes and C. elegans innate immune response

Autophagy promotes visceral aging in wild-type C. elegans

A plethora of studies over several decades has demonstrated the importance of autophagy in aging and age-related neurodegenerative disease. The role of autophagy in damage clearance and cell survival is well established, and supports a prevailing view that increasing autophagic activity can be broadly beneficial, and could form the basis of anti-aging interventions. However, macroautophagy/autophagy also promotes some elements of senescence. For example, in C. elegans hermaphrodites it facilitates conversion of intestinal biomass into yolk, leading to sex-specific gut atrophy and senescent steatosis

Phase transitions for the cavity approach to the clique problem on random graphs

We give a rigorous proof of two phase transitions for a disordered system designed to find large cliques inside Erdos random graphs. Such a system is associated with a conservative probabilistic cellular automaton inspired by the cavity method originally introduced in spin glass theory.Comment: 36 pages, 4 figure

Extracellular Dopamine Potentiates Mn-Induced Oxidative Stress, Lifespan Reduction, and Dopaminergic Neurodegeneration in a BLI-3–Dependent Manner in Caenorhabditis elegans

Parkinson's disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn-induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration

The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals

Control Problems for Conservation Laws with Traffic Applications

Conservation and balance laws on networks have been the subject of much research interest given their wide range of applications to real-world processes, particularly traffic flow. This open access monograph is the first to investigate different types of control problems for conservation laws that arise in the modeling of vehicular traffic. Four types of control problems are discussed - boundary, decentralized, distributed, and Lagrangian control - corresponding to, respectively, entrance points and tolls, traffic signals at junctions, variable speed limits, and the use of autonomy and communication. Because conservation laws are strictly connected to Hamilton-Jacobi equations, control of the latter is also considered. An appendix reviewing the general theory of initial-boundary value problems for balance laws is included, as well as an appendix illustrating the main concepts in the theory of conservation laws on networks

Control Problems for Conservation Laws with Traffic Applications

Conservation and balance laws on networks have been the subject of much research interest given their wide range of applications to real-world processes, particularly traffic flow. This open access monograph is the first to investigate different types of control problems for conservation laws that arise in the modeling of vehicular traffic. Four types of control problems are discussed - boundary, decentralized, distributed, and Lagrangian control - corresponding to, respectively, entrance points and tolls, traffic signals at junctions, variable speed limits, and the use of autonomy and communication. Because conservation laws are strictly connected to Hamilton-Jacobi equations, control of the latter is also considered. An appendix reviewing the general theory of initial-boundary value problems for balance laws is included, as well as an appendix illustrating the main concepts in the theory of conservation laws on networks

Introducing Commensal Bacteria from Caenorhabditis elegans to Drosophila melanogaster : Effects on Health, Ageing and Insulin-like Signalling

Background: With the rise of age-related diseases and challenges associated with ageing, there is a growing demand for innovative approaches to promote healthspan. The gut microbiome, essential for health and homeostasis in vertebrates and invertebrates, emerges as a promising avenue for enhancing overall health and addressing age-related disorders. However, little is known about how gut commensals affect host biological processes, let alone what constitutes a healthy gut microbiome. Purpose: Given the shared ecological environments and co-evolutionary history between nematode roundworms and fruit flies, this study introduces Caenorhabditis elegans commensal bacteria (derived from the CeMbio database) to Drosophila melanogaster to probe for bacterial impacts on ageing and interactions with evolutionarily conserved nutrient-sensing pathway—insulin/insulin-like signalling pathway (IIS). Methods: To ascertain successful bacteria colonisation of the fly gut, fly media was adapted for bacteria growth and candidate bacteria were fluorescently transformed to enable direct observation under fluorescence microscopy. Using the optimal experimental conditions and bacterial combination, the impacts of the introduced bacteria on fly health and ageing were evaluated by assaying fly lifespan, exploratory walking behaviour, gut integrity, sleep, neuromuscular function, and fecundity. To gain insights into potential crosstalk between bacteria and IIS, bacteria were also introduced to flies with impaired IIS achieved through either ablating cells that produce Drosophila insulin‐like peptides 2-3 (d2-3GAL4/UAS-rpr) or downregulating insulin receptors expression in serotonergic neurons (trhGAL4/UAS-InRDN). Results: From 16 CeMbio bacteria, 11 bacteria grow on fly media in contingent that antifungals were omitted. Thus, all subsequent experiments utilised additive-free fly media. Out of 24 transformations, 6 new fluorescent bacteria were generated and validated for their correct identity. Three fluorescent bacteria—Ochrobactrum vermis (MYb71-sfGFP), Enterobacter ludwigii (MYb174-dTomato), and Enterobacter cloacae (CEent1-mPlum)— successfully colonised the fly gut. When introduced together, these three bacteria reduced fly median lifespan but increased early-age egg laying, resulting in earlier egg exhaustion. Across life, these bacteria attenuated changes to exploratory walking and sleep behaviour induced by IIS reduction. However, fly gut permeability and neuromuscular function remained unaffected. Conclusions: MYb71-sfGFP, MYb174-dTomato, and CEent1-mPlum may benefit adult flies during early age but be detrimental later as bacterial load increases. Regardless, these bacterial-host interactions crosstalk with IIS to affect complex behaviours like exploratory walking and sleep. These findings provide evidence that the study of ecologically relevant commensal bacteria from C. elegans can be translated onto D. melanogaster to further probe for interactions affecting evolutionarily conserved biological pathways and complex behaviours

Stressed-Induced TMEM135 Protein Is Part of a Conserved Genetic Network Involved in Fat Storage and Longevity Regulation in Caenorhabditis elegans

Disorders of mitochondrial fat metabolism lead to sudden death in infants and children. Although survival is possible, the underlying molecular mechanisms which enable this outcome have not yet been clearly identified. Here we describe a conserved genetic network linking disorders of mitochondrial fat metabolism in mice to mechanisms of fat storage and survival in Caenorhabditis elegans (C. elegans). We have previously documented a mouse model of mitochondrial very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency.[1] We originally reported that the mice survived birth, but, upon exposure to cold and fasting stresses, these mice developed cardiac dysfunction, which greatly reduced survival. We used cDNA microarrays[2], [3], [4] to outline the induction of several markers of lipid metabolism in the heart at birth in surviving mice. We hypothesized that the induction of fat metabolism genes in the heart at birth is part of a regulatory feedback circuit that plays a critical role in survival.[1] The present study uses a dual approach employing both C57BL/6 mice and the nematode, C. elegans, to focus on TMEM135, a conserved protein which we have found to be upregulated 4.3 (±0.14)-fold in VLCAD-deficient mice at birth. Our studies have demonstrated that TMEM135 is highly expressed in mitochondria and in fat-loaded tissues in the mouse. Further, when fasting and cold stresses were introduced to mice, we observed 3.25 (±0.03)- and 8.2 (±0.31)- fold increases in TMEM135 expression in the heart, respectively. Additionally, we found that deletion of the tmem135 orthologue in C. elegans caused a 41.8% (±2.8%) reduction in fat stores, a reduction in mitochondrial action potential and decreased longevity of the worm. In stark contrast, C. elegans transgenic animals overexpressing TMEM-135 exhibited increased longevity upon exposure to cold stress. Based on these results, we propose that TMEM135 integrates biological processes involving fat metabolism and energy expenditure in both the worm (invertebrates) and in mammalian organisms. The data obtained from our experiments suggest that TMEM135 is part of a regulatory circuit that plays a critical role in the survival of VLCAD-deficient mice and perhaps in other mitochondrial genetic defects of fat metabolism as well