Ce projet vise à cartographier la machinerie de régulation des Rho GTPases chez C. elegans afin de mieux comprendre les mécanismes d'intégration des voies de signalisation au cours du développement des organismes multicellulaires. La première partie du projet consistait à caractériser la spécificité catalytique des Rho GAP envers les Rho GTPases in silico et in vitro. En adaptant un programme de prédiction de structures tridimensionnelles, Combinatorial Extension (CE), nous avons pu faire corréler une similitude structurelle avec la spécificité catalytique des GAP. Ce prédicteur amélioré a validé des interactions GAP-GTPase connues: cinq chez les mammifères et quatre avec des GAP homologues chez C. elegans. Les résultats de conservation des interactions que nous avons obtenus, permettent de justifier l'utilisation de ce nématode comme modèle d'étude pour l'humain. Par la suite, nous avons optimisé les essais expérimentaux pour tester la spécificité catalytique des Rho GAP de C. elegans en vue de valider le prédicteur. Dans la deuxième partie nous avons étudié les interactions fonctionnelles des Rho GAP au cours du développement embryonnaire du C. elegans in vivo. Les résultats obtenus nous ont aidés à mieux comprendre le réseau de régulation de la GTPase rho-1 par les GAP rga-5, gei-1 et rga-13 au cours de l'élongation embryonnaire. De plus, nous savons désormais qu'ocrl-1 est dans une voie antagoniste à rga-5. Enfin, dans la troisième partie de notre travail, nous avons mis au point une méthode d'analyse haut débit des interactions génétiques des Rho GAP sur le cytomètre en flux pour vers (le COPAS Biosort) avec des transgéniques histone::GFP. Puis cette méthode a été adaptée pour permettre d'utiliser des souches non fluorescentes, en colorant les noyaux des cellules des embryons fixés. Ce travail a apporté un nouvel outil de prédictions et a permis de simplifier les méthodes de travail pour l'étude des machineries de régulation de GTPases Rho. La technique haut-débit que nous avons développée pourrait aussi s'adapter à l'étude d'autres régulateurs (GEF, etc). ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Caenorhabditis elegans, Rho GAP, prédicteur, développement embryonnaire, haut débit

<p>Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the <italic>Drosophila melanogaster</italic> </p><p>Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode <italic>Caenorhabditis elegans</italic>. Immunity in <italic>C. elegans</italic> has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin <italic>clec-67</italic>, a marker of immunity. </p><p>We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing <italic>C. elegans</italic> immunity besides its involvement in germline development.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 34-38).

The ability to switch between different forms of locomotion is critical to many aspects of survival, whether it is switching from walking to running to evade predators, or switching to a slower gait to obtain food. Uncovering the mechanisms behind gait transitions has implications for many fields, from treating Parkinson Disease to understanding the impact of drugs of abuse on movement. However, the mechanisms of gait transitions are not well understood. The experiments outlined in this thesis sought to understand the neuronal basis for gait switching. This work employed the nematode Caenorhabditis elegans, a unique model organism chosen for its genetic tractability and fully characterized nervous system. C. elegans displays different forms of motion: crawling on land and swimming in liquid. First, I sought to determine the mechanisms for switching between these forms of motion in collaboration with Dr. Andres Vidal-Gadea. In the process, we discovered that crawling and swimming actually represent distinct gaits in contrast to recent reports that suggested they were merely a single gait. We further elucidated mechanisms for gait transition in C. elegans. For instance, we found that the transition to crawling required viii the D1-like dopamine receptors DOP-1 and DOP-4; and activation of dopamine neurons via the light-activated cation channel Channelrhodopsin2 was sufficient to induce crawling behavior in worms immersed in liquid. Conversely, photoactivation of serotonergic neurons expressing Channelrhodopsin2 induced swim-like behavior on land. Finally, laser microablation of dopaminergic or serotonergic neurons was sufficient to impair the transition to crawl or swim, respectively. Together these results show that transitions to crawling and swimming are controlled by dopamine and serotonin respectively. Next I wanted to better understand how gait transitions are impaired by a drug of abuse, alcohol. I found that, as in other organisms, ethanol disrupts gait transitions, causing worms in water to inappropriately transition from swim to crawl and to display other land-specific behaviors. Animals lacking the D1-like dopamine receptor DOP-1 were resistant to the ethanol-induced transition to crawl. Finally, I found that several interneurons required for the transition to crawl. Specifically, laser microablation of the DOP-4 receptor-expressing neuron RID or the DOP-1-expressing neurons PQR or RIS resulted in a significant impairment in the time to crawl onset. Overall, the findings presented in this thesis represent the first evidence that C. elegans uses an evolutionarily conserved mechanism to transition between gaits and provides the beginning of a molecular description of gait transitions. / text

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clk-1 encodes a hydroxylase that is necessary for ubiquinone (UQ) biosynthesis. clk-1 mutants do not synthesize UQ, but instead accumulate the precursor demethoxyubiquinone (DMQ). When fed on bacteria that synthesize UQ the mutants are viable but display slow development, behaviours and aging. However, they arrest development when fed on UQ synthesis-deficient bacteria. I have taken a genetic suppressor approach to investigate the causes of the various phenotypes as well as of the dietary requirements of the clk-1 mutants. / We identified two classes of mutants that suppress the defecation phenotypes of clk-1. All of these "dsc" mutants suppress the lengthened cycle of clk-1. Class I mutants also restore the ability to react normally to changes in temperature whereas the Class II mutants do not. The characterization of the Class I mutants suggests that part of the phenotype of clk-1 is due to an alteration of lipid metabolism, likely the level of lipid or lipoprotein oxidation. dsc-4 encodes the worm homolog of the Microsomal Triglyceride Transfer Protein (MTP), a protein required for the formation of low density lipoproteins (LDL) in vertebrates, and whose absence in people leads to abetalipoproteinemia. dsc-3 appears to be allelic to tat-2, which encodes a type IV P-type ATPase that is related to a family of human aminophospholipid transporters that includes ATP8B1/FIC1, whose inactivation results in cholestatic liver disease. dsc-3 and dsc-4 appear to affect distinct aspects of lipid metabolism. A general link between the Class II mutants and clk-1 remains elusive. dsc-1, a Class II gene, encodes a paired-like homeodomain transcription factor that is necessary for the GABA sensitivity of enteric muscles. / We also identified 9 clk-1(e2519)-specific suppressors, which suppress most Clk phenotypes, including their requirement for dietary UQ. Our analysis of these suppressors reveals that it is the lack of UQ rather than the presence of DMQ that is responsible for most phenotypes. In addition, they allowed us to show that most Clk phenotypes can be uncoupled from each other. We cloned six suppressors and all encode missense tRNA(Glu) suppressor genes. To my knowledge, these represent the first missense tRNA suppressors identified in any metazoan.

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair.

Cell migration and extension is essential for development. The ability of a cell or cell extension to reach its target is dependent on spatial cues and receptors that translate positional information into directed plasma membrane extension. For example, the UNC-40/DCC receptor is required to direct circumferential migrations towards the source of the ligand, UNC-6/Netrin, expressed at the ventral midline. To better understand the process of cell extension, I used a specialized process called muscle arms as a model system. In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nearest nerve cord at the midline. These muscle arms harbor the postsynaptic element of the neuromuscular junction and extend in a stereotypical and regulated manner. In a screen for muscle arm development defective (Madd) mutants, I isolated madd-2, a novel component of the UNC-40 pathway. MADD-2 is a C-1 TRIM protein and functions cell-autonomously to direct numerous muscle and axon extensions to the ventral midline of worms. In a striking analogy, mutations in a human homologue of MADD-2, MID1, cause numerous ventral midline defects that culminate as Opitz Syndrome. How MID1 regulates midline development is unclear. MADD-2 enhances UNC-40 pathway activity by facilitating the physical interaction between UNC-40 and the downstream Rho-GEF, UNC-73. It is possible that MID1 may mediate the function of a DCC-like pathway at the ventral midline of humans. This work provides the first indication that C1-TRIM proteins may have a conserved biological role of regulating midline-oriented development events and may provide key insights into the role of MID1 in the pathogenesis of Opitz syndrome.