Les canaux potassiques à deux domaines P (K2P) contrôlent l’excitabilité cellulaire et jouent un rôle central dans l’établissement et le maintien du potentiel de repos membranaire dans la majorité des cellules animales. Depuis leur identification dans les années 90, ces canaux ont été impliqués dans de nombreuses fonctions comme la modulation de l’activité neuronale, l’activité du muscle cardiaque ou encore la physiologie rénale. Malgré l’importance de ces canaux, peu de données existent sur les processus cellulaires qui contrôlent leur fonction in vivo. Au cours de ma thèse, j’ai utilisé des approches génétiques, d’imagerie et d’électrophysiologie pour comprendre comment l’expression, la distribution et l’activité du canal K2P UNC-58 sont contrôlés chez le nématode modèle C. elegans.Pour cela, j’ai effectué un crible suppresseur du phénotype locomoteur du mutant gain de fonction unc-58(e665). J’ai ainsi obtenu 133 mutants présentant une large gamme de niveaux de suppression, suggérant l’implication de plusieurs gènes dans les processus de régulation du canal. En utilisant les technologies de reséquençage complet de génome, j’ai pu cloner six nouveaux gènes requis pour la fonction d’unc 58.J’ai ensuite caractérisé en détail le rôle d’unc-44/ankyrine dans le contrôle du trafic d’unc 58. Ce travail a conduit à 4 conclusions majeures : (1) UNC-58, malgré sa structure de canal potassique, possède en réalité une sélectivité ionique altérée favorisant le passage des ions sodium, (2) l’addition à UNC 58 de protéine fluorescente par approche CRISPR/Cas9 nous a permis pour la première fois d’observer directement la distribution du canal UNC-58 in vivo, (3) l’ankyrine est nécessaire à l’adressage du canal UNC-58 à la surface des muscles et dans les axones des neurones mécanosenseurs ALM. Cette fonction fait intervenir une poche d’interaction lipidique localisée au sein du module Zu5N-Zu5C-UPA d’UNC-44, (4) ce mécanisme est hautement sélectif puisqu’il n’est pas requis pour l’adressage de 6 autres canaux potassiques musculaires. Mon crible a également identifié une interaction génétique entre unc-70/ß-spectrine et unc-44/ankyrine. Toutefois, la nature moléculaire de cette interaction reste encore à préciser / Two-pore potassium channels (K2P) control cell excitability and play a central role in the establishment and the maintenance of the resting membrane potential of almost all animal cells. Since their identification in the late 90s, these channels have been implicated in a large number of functions ranging from neuronal and cardiac activity to kidney physiology. Despite the crucial functions of these channels, comparatively little is known about the cellular processes controlling their function in vivo. During my PhD, I used a wide range of strategies including genetics, microscopy and electrophysiology to understand how the expression, the distribution and the activity of the K2P channel UNC-58 are controlled in the model nematode C. elegans. I have first performed a genetic suppressor screen targeting the locomotion phenotype of the gain of function mutant unc-58(e665). The screen yielded 133 mutants, displaying a wide range of suppression level, suggesting that several genes may be implicated in the channel regulation process. By using whole genome sequencing technologies, I’ve been able to clone six new genes required for the function of UNC-58.Then, I’ve characterized in detail the role of unc-44/ankyrin in the trafficking of UNC 58. This project led to 4 main conclusions : (1) UNC-58, despite its potassium channel structure, has an altered ionic selectivity, allowing preferably sodium ions to pass through the channel (2) the addition of a fluorescent protein to UNC-58 by CRISPR/Cas9 approaches allowed us for the first time to directly observe the addressing of the UNC-58 channel to the muscle surface and axons of ALM mechanosensory neurons. This function involves a lipid binding pocket located within the Zu5N-Zu5C-UPA module of UNC-44, (4) this mechanism is highly selective since it is not required for the addressing of 6 other muscular channels.My screen also identified a genetic interaction between unc-70/ß-spectrin and unc-44/ankyrin. However, the exact molecular nature of this interaction remains to be elucidated

Lipids carry out important structural as well as signaling functions in the cell. In recent years, enzymes that metabolize lipids have been emerging as key regulators of basic cellular functions and developmental processes. In order to study metabolism of lipids, we have focused our research on a class of proteins: the cytochrome P450s (CYPs), which are involved in lipid production in many organisms. We have used C. elegans, a classical genetic model system, to investigate lipid metabolism because this nematode offers several technical advantages that render it suitable for our investigations. The aim of our project was to identify and characterize essential lipids for the development of worms. We have performed RNAi (RNA interference) against C. elegans CYP31A, and found that silencing of this enzyme leads to the arrest of embryonic development. Further characterization of this embryonic lethal phenotype revealed that it is caused by problems in establishment of polarity and failure in the extrusion of a polar body. Moreover, we found that embryos depleted of CYP31A are osmotic sensitive and their eggs are permeable to dyes (hoechst, FM 4-64 etc.). The defects described above are common to a class of mutants that received the denomination of POD (for Polarity and Osmotic Defects). Analysis by electron microscopy demonstrated that cyp31A(RNAi) embryos exhibit an improperly constructed eggshell. Further functional studies have demonstrated that the defects observed in cyp31A(RNAi) embryos can be ascribed to the malfunctioning of one of the three layers of the eggshell: the lipid-rich layer, but additional problems in the assembling of the other two layers are also present. In order to identify the product of CYP31A, we set up a bioassay in which we tested the capability of lipidic extract from wild type embryos to rescue the embryonic lethality. The bioassay provided a method to track the activity and allowed us to enrich the metabolic product of CYP31A by the fractionation of the total lipid extract. Another POD gene, emb-8, codes for an NADPH CYP reductase. This 4 protein supplies electrons to the CYPs for their metabolic reactions. A mutant of emb-8 (emb-8(hc69)), gives a similar phenotype as the knockdown CYP31A. With the aim to test if EMB-8 and CYP31A act in the same pathway we extracted lipids from emb-8TS mutants. We tested in the bioassay if extracts from emb-8(hc69) mutants, containing the metabolic product of CYP31A, can rescue cyp31A(RNAi) phenotype. The results obtained suggest that EMB-8 and CYP31A work in the same metabolic pathway. Conclusively, CYP31A and EMB-8 cooperate to produce a class of lipids that are required for the construction of a functional eggshell. A defective eggshell causes failure in polarity establishment, extrusion of the polar bodies, osmotic sensitivity and permeability and eventually it leads to the arrest of the development of C. elegans embryos.

In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)

L’acquisition d'une identité cellulaire différenciée est souvent considérée comme définitive et figée dans le temps; or un nombre croissant d’études démontre que les cellules différenciées peuvent faire preuve de plasticité sous certaines conditions. Afin de mieux comprendre ces phénomènes, notre laboratoire a établi un modèle unique chez Caenorhabditis elegans (C. elegans) permettant l’étude d’un événement de transdifférenciation dans un contexte physiologique à l'échelle de cellules uniques. Au cours du développement, une cellule épithéliale du rectum de C. elegans, nommé Y, va migrer antérieurement puis changer d’identité pour devenir un motoneurone nommé PDA. Les travaux préliminaires du laboratoire ont montré que la voie de signalisation LIN-12/Notch est le signal le plus précoce nécessaire pour le bon déroulement de la transdifférenciation de Y en PDA. Nous avons pu mettre en évidence : i) que lors de l’embryogénèse, deux ligands canoniques (apx-1 et lag-2) semblent agir de façon redondante afin d’activer la voie Notch. ii) l’activation ectopique et contrôlée de la voie Notch est suffisante pour induire la formation d’un second neurone PDA. iii) Les facteurs nucléaires que le laboratoire a identifiés comme cruciaux pour l'initiation de cet évènement de TD sont également importants pour la reprogrammation induite de cette deuxième cellule en neurone PDA par l'activation ectopique de Notch. iv) La suractivation prolongée de la voie Notch dans la cellule Y maintien l’identité épithéliale de cette dernière, ayant pour conséquence le blocage de la transdifférenciation de Y en PDA. L’ensemble de nos résultats montrent que la voie Notch est nécessaire et suffisante afin d’établir la compétence à transdifférencier et que cela ne peut être réalisé que si la voie Notch est régulée de façon très précise dans la cellule Y. / The acquisition of a differentiated cell identity is often considered as final and frozen in time. However, a growing number of studies showed that differentiated cells can exhibit plasticity under certain conditions. To better understand these cell plasticity phenomena, our laboratory has developed a unique model in Caenorhabditis elegans (C. elegans) to study a transdifferentiation event in a physiological context and at the single-cell level. During the worm development, an epithelial rectal cell, named Y, will migrate anteriorly and change its identity to become a neuron named PDA. Preliminary work performed by our laboratory showed that the LIN-12/Notch signalling pathway is the earliest signal necessary for the proper conduct of the transdifferentiation of Y into PDA. In our study, we showed that: i) during embryogenesis, two canonical ligands (apx-1 and lag-2) appear to act redundantly to activate the Notch pathway in Y. ii) ectopic and controlled activation of the Notch pathway is sufficient to induce formation of a second PDA neuron. iii) Nuclear factors indentified in our laboratory as crucial for the initiation of this event are also important for transdifferentiation of the second PDA obtained by ectopic activation of Notch. iv) A prolonged activation of the Notch pathway in the Y cell maintains its epithelial identity, which results in the inhibition of the transdifferentiation of Y into PDA. Together, our results showed that the Notch pathway is necessary and sufficient to establish the competence to transdifferentiate. This can only be achieved if the Notch pathway is regulated very precisely in the Y cell.

In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)

The nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut. / text

Micromanipulation is considered a challenging task which requires high precision motion and measurement at the micro scale. When micromanipulation is concerned with living organisms important considerations need to be addressed. These include the physical or chemical properties of micro-organisms, living conditions, responses to the environment and achieving suitably delicate manipulation. Bio-micromanipulation can include micro surgery or cell injection operations, or to determine interaction forces as the basis to investigate behavior and properties of living micro-organisms. In order to achieve suitable bio-micromanipulation appropriate processes and/or sensory systems need to be investigated. This thesis aims to look into the force interaction and sensing addressing two distinctive challenges in the field of bio-micromanipulation. To this end, this thesis presents two major contributions to advancing bio-micromanipulation. Firstly, a novel Haptic Microrobotic Cell Injection System is introduced which is able to assist a bio-operator through haptic interaction. The system introduces a mapping framework which provides an intuitive method for the bio-operator to maneuver the micropipette in a manner similar to handheld needle insertion. To accurately control the microrobot, a neuro-fuzzy modeling and control scheme has been developed. Volumetric, axial and planar haptic virtual fixtures are introduced to guide the bio-operator during cell injection. Aside from improving real-time operator performance using the physical system, the system is novel in facilitating virtual offline operator training. Secondly, a first-of-its-kind micro-pillar based on-chip system for dynamic force measurement of C. elegans motion is introduced. The system comprises a microfabricated PDMS device to direct C. elegans into a matrix of micropillars within a channel mimicking its dwelling environment. An image processing algorithm is able to track the interaction of the C. elegans with the pillars and estimate contact forces based on micropillar deflections. The developed micropillar system is capable of measuring the force with sub-micron resolution while providing a continuous force output spectrum.

The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

Appetite control and satiety mechanisms help animals maintain energy homeostasis; however, these mechanisms can be misregulated, leading to overweight and obesity. Caenorhabditis elegans is an excellent model system to study appetite and satiety because of its conserved behavioral aspects of satiety and conserved molecular mechanisms. ASI senses nutrition and its activity is required for the behavioral state of satiety quiescence. The purpose of this thesis project was to elucidate the function of cGMP signaling in ASI by looking at behavioral effects from the pharmacological use of sildenafil (Viagra), a PDE inhibitor, and the effects on ASI activation from mutating guanylyl cyclase DAF-11. Sildenafil treatment increases satiety quiescence and decreases fat storage in a PDE-dependent manner. The daf-11 mutation decreased overall fluorescence intensity of ASI activation and the frequency at which ASI activated by about 50% compared to wild-type worms, suggesting that DAF-11 plays an important role in ASI to promote satiety.

Axonal regeneration within the mammalian central nervous system following traumatic damage is limited and interventions to enable regrowth is a crucial goal in regenerative medicine. The nematode Caenorhabditis elegans is an excellent model to identify the intrinsic genetic programs that govern axonal regrowth. Here we demonstrate that alterations in O-linked N- beta-acetylglucosamine (O-GlcNAc) post-translational modifications of proteins can increase the regenerative potential of individual neurons. O-GlcNAc are single monosaccharide protein modifications that occur on serines/threonines in nucleocytoplasmic compartments. Changes in O-GlcNAc levels serve as a sensor of cellular nutrients and acts in part through the insulin-signaling pathway. Loss of O-GlcNAc via mutation of the O-GlcNAc Transferase (OGT), the enzyme that adds O-GlcNAc onto target proteins, enhances regeneration by 70%. Remarkably, hyper-O-GlcNAcyation via mutation of the O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from target proteins, also enhances regeneration by 40%. Our results shed light on this apparent contradiction by demonstrating that O-GlcNAc enzyme mutants differentially modulate the insulin-signaling pathway. OGT mutants act through AKT1 to modulate glycolysis. In contrast, OGA mutants act through the FOXO/DAF-16 transcription factor to improve the mitochondrial stress response. These findings reveal for the first time the importance of O-GlcNAc post-translational modifications in axon regeneration and provide evidence that regulation of metabolic programs can dictate the regenerative capacity of a neuron. / 2021-02-20T00:00:00Z