Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

Background: Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. Results: In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPR mt ). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPR mt and growth. Conclusions: The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user's requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.European Research Council ERC-2011-StG-281691Ministerio de Economía y Competitividad BFU2012–3550

A Second-Generation Device for Automated Training and Quantitative Behavior Analyses of Molecularly-Tractable Model Organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science

E. coli folate synthesis and C. elegans ageing: Investigating the effect of sulfamethoxazole on bacterial lawn morphology and metabolism

The gut microbiota is essential for host nutrition and may influence ageing. The nematode worm C. elegans provides a useful simplified model for investigating bacterial-host interactions. E. coli is used as a food source for C. elegans. Previous research has shown a decrease in E. coli folate synthesis results in extension of C. elegans lifespan. Potential detrimental effects of bacteria when producing normal amounts of folate are unlikely to be mediated by bacterial growth rate or direct effects of folate on the nematode. Potential toxicity of metabolic chemicals produced by wild type E. coli could explain the shorter lifespan of C. elegans. This thesis aims to understand the interaction between E. coli and C. elegans by investigating components that may be affected by folate synthesis and influencing lifespan. Toxicity from bacterial formaldehyde synthesis was explored with a formaldehyde sensing lacZ reporter. A novel method was developed to quantify reporter output in a bacterial lawn. The lifespan of C. elegans maintained on E. coli constitutively expressing the formaldehyde detoxification enzymes FrmA/B was also investigated. Formaldehyde synthesis was not found to be a source of toxicity that accelerates C. elegans ageing. The effect of sulfamethoxazole on bacterial lawn growth, morphology and proliferation was examined and found to alter morphology, attenuate growth and impair proliferation compared to wild type and lifespan increasing mutant E. coli. A novel LC-MS/MS method was developed to analyse amino acids in agar. It revealed sulfamethoxazole alters bacterial amino acid metabolism associated with the serine-glycine pathway and growth. The absence of glycine in the media was also examined. It revealed changes to the exometabolome in both sulfamethoxazole treated and untreated conditions that may slow C. elegans ageing without altering bacterial growth. This work developed novel methods for exploring the bacterial lawn and metabolism, providing insight into the mechanism of how sulfamethoxazole disruption of bacterial folate synthesis may influence C. elegans ageing

Biological applications of multimodal imaging involving Raman and 4Pi Raman microscopy

Raman microscopy is becoming an increasingly important label-free imaging technique. It proved to be a viable tool for life science applications allowing to analyze bacteria, cells, and tissues at the molecular level. Combining Raman microscopy with complementary imaging modalities and techniques is explored here to: (1) analyze mild traumatic brain injury (mTBI) in a combination with magnetic resonance imaging (MRI) for detecting mild, and invisible to medical imaging techniques, brain tissue damage; (2) reveal complementarity of Raman and fluorescence microscopy approaches for investigating and tracking bovine lactoferrin inside calf rectal epithelial cells in the presence of enterohemorrhagic Escherichia coli (EHEC); (3) apply Raman microscopy along-side the molecular analysis approaches (such as scanning transmission electron microscopy-energy dispersive X-ray (STEM-EDX), low energy X-ray fluorescence (LEXRF), nanoscale secondary ion mass spectrometry (Nano-SIMS)) to uncover the origin of the long-range conductance in cable bacteria; (4) develop multifunctional surface enhanced Raman scattering (SERS) platform based on calcium carbonate particles for enhancing a weak Raman scattering signal of biomolecules as well as to apply Raman microscopy for particle detection in vivo in Caenorhabditis elegans (C. elegans) worms; and (5) combine Raman microscopy and atomic force microscopy (AFM) to track Chlamydia psittaci in cells. Analysis of described above samples and phenomena is based on Raman molecular fingerprint images, where, similarly to fluorescence light microscopy, the resolution is limited by diffraction of light. Therefore, efforts are also put to enhance the resolution of Raman microscopy-based imaging by adding a 4Pi configuration to a confocal Raman microscope. As a result, a possibility to enhance the axial (also called longitudinal) resolution is investigated by constructing a 4Pi confocal Raman microscope, which is also applied to study bacteria inside cells. Results presented in this work emphasize the added value of multimodal microscopy approaches, particularly involving Raman microscopy, in a broad range of applications in bioengineering, biomedicine, and biology

Characterisation of quiescin-sulfhydryl oxidase and nematode astacin mutants using functional studies in caenorhabditis elegans.

Nematodes, both free-living and parasitic, are dependant upon their Extra Cellular Matrix (ECM) for multiple aspects of functionality. Two distinct ECMs are present in Caenorhabditis elegans, the basement membrane and the cuticle. The cuticle of C. elegans, like other nematodes is composed largely of collagen-like proteins, with the trimeric collagenous proteins forming approximately 80% of the cuticle. Cuticle collagens are believed to be highly processed in a manner similar to vertebrate collagen maturation, with collagens being; co-translationanly modified, folded into triple helices and proteolytically cleaved at the C- and N- termini. Cross-linking of mature triple helical collagens into higher order structures leads to the generation of a flexible yet robust cuticle. Disulphide bonding is crucial in the formation of the cuticle, with cysteine cross-linking mutants having been shown to produce severely disrupted cuticles and associated lethal phenotypes. During the life cycle, C. elegans progresses through four moults during which a new cuticle is synthesised and the old cuticle is shed. Moulting occurs by proteolytic digestion and shedding of an anterior cuticular cap which provides an opening for the nematode to escape the previous stage cuticle. Both free-living and parasitic nematodes shed and exsheath their cuticles in this manner. Two distinct phases of cuticle processing become apparent: cuticle synthesis and cuticle degradation. Of the enzymes involved with processing of cuticular collagens, the quiescin sulfhydryl oxidases (QSOX), and the nematode astacins (NAS) are of particular interest with regard to cuticle synthesis and proteolytic cleavage of cuticular collagens respectively. QSOX have been shown to be linked directly to the generation of disulphide bonds, and have also been shown to associate with other essential proteins of cuticle formation, namely the protein disulphide isomerases. There are three distinct QSOX family members found within the C. elegans genome, which have been shown to temporally coincide with lethargus (cuticle synthesis) and have been proven to spatially localise to the C. elegans hypodermis, the tissue responsible for cuticle secretion. Characterisation of qsox mutants reveals weak cuticular phenotypes when disrupted singly; but, in combination, silencing of qsox-1 and qsox-2 resulted in blistered cuticles and lethality, by RNA mediated interference and double knockouts respectively. This demonstrates the essential nature of the cuticle associated QSOX enzymes, and to my knowledge represents the first loss-of-function mutant in a QSOX enzyme. xv Investigation of the NAS enzymes focused on the group V astacins, members of which exhibit the only notable defects associated with disruption of C. elegans nas genes, namely: dumpy body shape, nas-35/dpy-31; hatching, nas-34/hch-1; and moult defects, nas-36 and 37. With regard to proteolytic degradation of cuticular components, NAS-36 and NAS-37 were of specific interest as mutants resulted in moult defective nematodes unable to digest and fully escape their previous stage cuticles; in addition, spatial expression illustrated an association of these gene products with regions of cuticle attachment and degradation. C. elegans NAS-36 and NAS-37 were also shown to digest isolated L3(2M) trichostrongylid cuticles of parasites of veterinary importance, suggesting that the metalloprotease and cuticle substrates involved in exsheathment is conserved between trichostrongylid and free-living nematodes. Conservation is poor between ecdysozoan and non-moulting organisms, meaning that proteins such as NAS-36 and 37 could become specific novel targets for anti-nematode drug development

Membrane dynamics and advective transport of the PAR polarity proteins

The establishment of cell architecture, whether in a migrating cell, a polarized epithelial cell, or an asymmetrically dividing stem cell, requires proper intracellular patterning. One mechanism by which cells are able to locally concentrate molecules in space is through directed transport, typically through the action of cytoskeletal-motor networks. Using polarization of the C. elegans embryo as a model system, I sought to understand how actomyosin cortical flows drive efficient segregation of polarity proteins to one side of the cell. Prior data established that anterior PAR proteins are segregated into the anterior by cortical actomyosin flows, yet the mechanisms underlying this transport are unclear. More recent work suggested that oligomerization of PAR-3 and its ability to recruit other aPAR proteins is specifically required for efficient segregation. This data raised additional questions: Do all membrane-associated molecules sense flows? Do particular features of molecules such as clustering enable their segregation by advection? How is this regulated to enable correct spatiotemporal control of protein targeting? We combined single molecule tracking methods with perturbation of PAR-3 cluster dynamics to directly assess the ability of polarity molecules to be advected by flows and how this may be affected by cluster regulation. My results suggest that a variety of polarity molecules are advected by cortical flows, allowing flow to shape molecular distributions. At the same time, not all molecules are advected, or at least not advected efficiently, indicating that specific molecular features of protein complexes facilitate advection. Surprisingly, despite being required for efficient segregation, clustering of PAR-3 is not required to sense flows. Moreover, although clustering alters diffusivity, the observed changes are minimal and would not be expected to substantially alter their ability to be segregated. Rather, clustering is most likely required to shape the pattern of membrane association, potentially through positive feedback, though this remains to be definitively explored

The influence of cell size on cytokinesis in situ and genomic interrogation of human cell size regulation

La cellule est l’élément fondamental de la vie. Plus d’une vingtaine de trillions de cellules forment les organes et tissus de notre corps. Ces cellules sont de taille spécifique puisqu’elles ont des fonctions précises au sein de leur tissu respectif. Dans la plupart des cas, les cellules doivent proliférer en se divisant pour se renouveler et ainsi assurer le bon fonctionnement d’un organisme. La dernière étape de la division cellulaire, la cytokinèse, est exécutée par la contraction d’un anneau contractile d’actomyosine, nécessaire pour effectuer la séparation physique de la cellule en deux cellules filles. La première partie des travaux décrits dans cet ouvrage portent sur la caractérisation de la cytokinèse en utilisant, comme modèle in vivo, les cellules précurseur de la vulve (VPCs) du nématode C. elegans. Notre étude révèle que plusieurs aspects de l’anneau d’actomyosine s’ajustent en fonction de la taille de la cellule. Entre autres, la largeur de l’anneau contractile, juste avant sa constriction, s’ajuste en fonction de la longueur des VPCs. De plus, la rapidité avec laquelle l’anneau se contracte dépend de la circonférence de la cellule. Ces découvertes nous ont amené à nous demander comment la cellule régule sa taille? Les cellules en prolifération maintiennent leur taille en homéostasie en équilibrant leur taux de croissance et de division cellulaire. Afin d’interroger les gènes impliqués dans le maintien de la taille cellulaire du mammifère, nous avons utilisé la technologie CRISPR/Cas9, afin d’éliminer par délétion tous les gènes humains, à raison d’un par cellule, pour identifier ceux qui causent une augmentation ou une diminution de la taille cellulaire. Cette étude nous a permis d’identifier plusieurs gènes déjà connus régulant la croissance cellulaire. De plus, nous avons identifié un groupe de gènes, incluant TLE4 un corépresseur de la transcription que nous avons caractérisé, n’ayant jamais été associé avec une fonction de contrôle de la taille cellulaire chez les mammifères. En somme, nos travaux ont contribué à l’approfondissement des connaissances sur la division cellulaire, plus précisément la cytokinèse, et des gènes impliqués dans le maintien de la taille cellulaire. Une meilleure connaissance du fonctionnement de ces deux évènements cellulaires est essentielle puisque leur dérégulation peut entrainer plusieurs pathologies, incluant le cancer.Cells are the fundamental building blocks of life. The human body contains over twenty trillion cells that make up the different tissues and organs of our bodies. Cells within organs are of specific sizes to perform their specialized functions. In most cases, these cells must divide to proliferate and replenish the population of cells essential for proper organism function. The final stage of cellular division, termed cytokinesis, entails the assembly and constriction of a contractile ring that drives the dramatic cell shape changes required to physically partition the cell into two daughter cells. The first part of the work presented in this thesis addresses the characterization of cytokinesis in the epithelial vulval precursor cells (VPCs) of the nematode worm C. elegans. This study principally revealed that several aspects of cytokinesis scale with cell size. For instance, I observed that the breadth of the actomyosin ring scaled with VPC length. In addition, the speed of contractile ring constriction scaled with the circumference of VPCs. These scaling events raised the more general question as to how cells regulate their size. Proliferating cells attain cell size homeostasis by balancing cell growth and cell division. In order to define the molecular regulators of size in human cells a genome-wide approach was taken. Recently developed CRISPR/Cas9 technology was used to perform the first pooled knockout screens for human cell size regulators in the NALM-6 pre-B lymphocytic cell line. These screens revealed many genes that affect the size of NALM-6 cells, a number of which were previously known to be involved in growth regulation. In addition, these screens revealed the identity of many genes with no previously established functions associated with cell size regulation. Amongst the previously unknown regulators, I characterized the function of a co-repressor of transcription, TLE4, which I showed functions as a regulator of the B-cell lineage. This work contributes to the knowledge of the mechanics of cytokinesis in C. elegans epithelial cells and of the genes that coordinate cell size in humans. These results provide insights into cell growth and division in normal cells and how these processes may be perturbed in cancer and other diseases

A Metabolomic Approach to Assessing Life-History Traits in Caenorhabditis elegans

The proximate causes of ageing and the biological processes that determine lifespan are still unclear. However, many studies using model organisms have led to the identification of genes associated with longevity. While there is a clear link between changes in metabolism and changes in longevity, there has been relatively little ageing-related research that has measured metabolites directly. Metabolic profiling of low molecular weight metabolites (metabolomics) has an advantage over other 'omics' techniques, in that it directly samples the metabolic changes in an organism, and integrates information from changes at the gene, transcript and protein levels, as well as post-translational modification. This thesis demonstrates that metabolic profiling provides a new and useful phenotyping tool for studying ageing in the nematode Caenorhabditis elegans. Using both nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS), I have identified metabolites that are linked with long life. I have carried out the first characterisation of the C. elegans metabolome throughout both development and ageing. Comparing these metabolic changes in wild type worms with those seen in a long-lived mutant aid the understanding of when and how mutant worms acquire their long-lived phenotype. In addition to this, I have examined the effects on metabolism of a commonly used technique in C. elegans ageing research: the inhibition of DNA synthesis to maintain synchronous ageing populations. This provided a way to control for the effects of this technique when used in my work, but also demonstrated that its use may result in artefacts in data. I have also investigated the effect of mutation accumulation on the C. elegans metabolic profile. I have shown that metabolomics provides a way to obtain new phenotypes in this type of study, and novel information about the variation that occurs as a result of spontaneous mutation