Guimaraes de A Barros A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Addressing methodological controversies in the study of C. elegans fat regulation"

Ashrafi K, Guimaraes de A Barros A, Mullaney B, Liu J, Cunningham K, Lemieux G

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The vital dyes Nile Red and bodipy labeled fatty acids been used extensively to visualize fat depots in organisms as diverse as yeast, flies, and mammals. Application of these dyes to C. elegans combined with various genetic manipulations have led to elucidation of multiple fat regulatory pathways in C. elegans, many of which are counterparts of evolutionary conserved mechanisms already well studies in other species. However, a number of recent publications have called into question the validity of these vital dyes for analyzing C. elegans fat pathways and proposed fixed staining methods such as Oil-red-O as viable alternatives. We will present biochemical, cell biological, and spectral fluorescence data that validate the use of vital dyes for studying C. elegans fat depots. We will also present data showing that the current biochemical methods used for assessing total C. elegans fat content have relatively limited sensitivity accounting for some of the noted discrepancies and that some of the claims derived from stainings of fixed animals are likely experimental artefacts. We will present on-going efforts aimed at improving existing methodologies and discuss important considerations required for evaluating various methodologies that are in use for assessment of C. elegans fat.

Chen, P. et al. (2009) International Worm Meeting "The Early-Onset Torsion Dystonia Gene Product, torsinA, is a Mediator of ER Stress and Protein Homeostasis."

Roberts, N., Porter, J.C., Burdette, A.J., Berkowitz, L.A, Ricketts, J.C., Caldwell, K.A., Caldwell, G.A., Chen, P., Fox, S.A.

[International Worm Meeting, 2009]

The capacity of cells to carry out their various functions is wholly dependent upon efficient protein synthesis, processing, trafficking, and degradation. In this context, dysfunction or deficit of proteins that monitor and ensure the proper maintenance of cellular homeostasis can have serious consequences in terms of disease onset, penetrance, or progression. A single codon deletion (GAG = DE) in the gene encoding human torsinA causes a non-degenerative neurological disorder termed early-onset torsion dystonia. TorsinA, an endoplasmic reticulum (ER) resident protein, belongs to the diverse AAA+ (ATPases Associated with a variety of cellular Activities) family. Here we use a well established ER stress model in the nematode C. elegans (hsp-4::GFP) to investigate the role of torsinA in maintaining protein homeostasis in the ER. Using GFP as an ER stress reporter, we found that transgenic nematodes expressing wild type (WT) human torsinA exhibited a striking reduction in ER stress caused by tunicamycin, while animals with either DE or a mixture of WT/DE torsinA failed to do so. Interestingly, DE and WT/DE torsinA worms exhibited higher ER stress than WT torsinA animals, even in the absence of tunicamycin treatment. In addition, mutations within torsinA ER signal sequence and N-terminal hydrophobic region abolished its capacity to maintain an ER stress threshold against cellular stressors, indicating the importance of its localization to the ER for this activity. Furthermore, although torsinA possesses very low ATPase activity, the ATPase domain appeared important for ER stress suppression, as two different mutations within the ATPase domain, K108A and E171Q, resulted in loss of stress suppression. A single nucleotide polymorphism (SNP), D216H, diminishes the penetrance of dystonia from 30-40% (WT/DE) to 3% (D216H/DE) in patients. In C. elegans, coexpression of DE torsinA with D216H torsinA significantly protected worms from ER stress caused by tunicamycin, thereby recapitulating the effect on penetrance observed with patients. In order to find genetic interacting factors of torsinA, a small scale of RNAi screen for altered ER stress levels in DE torsinA worms, was carried out. Positives from the screen included glutaredoxin, H3 histones, and 13 core components and regulators of Ras signaling. Taken together, our data indicate that torsinA serves to maintain protein homeostasis in the ER, shedding new light on the pathophysiology of torsion dystonia.

Wang, Wenqing et al. (2013) International Worm Meeting "New path to NAD+: In addition to salvage biosynthesis, NRK and de novo NAD+ synthesis contribute to NAD+ recycling in C. elegans."

Lynes, Matthew R., Wang, Wenqing, Hanna-Rose, Wendy

[International Worm Meeting, 2013]

NAD+ is a vital molecule in cellular redox reactions and acts as a cosubstrate for NAD+ consuming enzymes, which are critical to aging, stress response and energy metabolism homeostasis. NAD+ salvage biosynthesis is an important pathway to recycle the nicotinamide (NAM) liberated by NAD+ consumers to rebuild NAD+ reservoir. Mutation of PNC-1, a nicotinamidase in the NAD+ salvage pathway in C. elegans, results in a series of defects that are individually linked to NAD+ insufficiency or NAM accumulation[1, 2]. Specifically, NAD+ insufficiency in pnc-1 causes gonad developmental delay. Our recent mass-spectrometry data showed that there is a drastic 18-fold increase in NAM levels following pnc-1 loss of function. However, NAD+ levels in pnc-1 mutant only decrease by 30%. These results led us to hypothesize that there are other NAD+ synthesis pathways contributing to NAD+ synthesis in C. elegans. Here we tested two potential pathways that have been studied in yeast: the NRK pathway and de novo NAD+ synthesis. The NRK pathway uses nicotinamide riboside kinase (NRK) to convert nicotinamide riboside (NR) from the diet to nicotinamide mononucleotide, which can be processed into NAD+. Mutation of NRK-1 exacerbates gonad developmental delay in pnc-1. In addition, supplementing NR rescues gonad developmental delay in pnc-1 mutants, but not in pnc-1;nrk-1 double mutants. NAD+ can also be synthesized de novo from tryptophan in yeast. By BLAST, we found that C. elegans lacks a key enzyme in de novo NAD+ synthesis, quinolinic acid phosphoribosyl transferase (QPRTase). Surprisingly, supplementing quinolinic acid (QA), the substrate of QPRTase, to pnc-1 mutants rescues gonad developmental delay. Moreover, RNAi of tryptophan 2,3-dioxygenase, another enzyme in the de novo NAD+ synthesis pathway, exacerbates gonad developmental delay. In conclusion, our results suggest that both NRK and de novo NAD+ biosynthesis pathways actively contribute to NAD+ synthesis in C. elegans. Despite the fact that a QPRTase homologue is missing in C. elegans, it is possible that a novel mechanism converts QA into NAD+. We are working to identify this new mechanism. Reference: 1. Vrablik et al. Development 2009 2. Vrablik et al. Dev Biol. 2011.

Carissa L. Perez et al. (2007) International Worm Meeting "Using Metabolic Tracers to Quantify Fatty Acid Synthesis, Absorption, and Expenditure in C. elegans."

Marc Van Gilst, Carissa L. Perez

[International Worm Meeting, 2007]

Although C. elegans is being recognized as a potent genetic tool to study the relationship of nutrition to growth and aging, surprisingly little is known about how worms utilize dietary molecules. Therefore, we have established a stable isotope enrichment strategy to characterize the ingestion and metabolism of dietary nutrients in developing and aging animals. Here, we present a study using 13C labeled bacteria to determine the relative abundance of dietary and de novo synthesized fatty acids in worm lipids using mass spectrometry. Quantitative analysis of 13C enrichment in feeding worms revealed that most lipid species; e.g., triglycerides, phospholipids, sphingolipids, etc., are primarily composed of fatty acids directly absorbed from the bacterial food source. Therefore, it is clear that direct incorporation of dietary fats plays a significant role in the formation of fat stores and lipid membranes. Because our labeling approach is effective at quantifying both dietary and synthesized fatty acids, we are now exploiting this strategy to identify genetic factors that impact de novo fat synthesis, dietary fat absorption, and/or fat expenditure. Thus far, we have surveyed genes known to significantly affect overall fat storage in nematodes to specifically identify factors that affect fatty acid synthesis. We found that mutations in the daf-2 gene result in a dramatic increase in the de novo synthesis of fatty acids, which likely explains the high fat phenotype of these mutants. In contrast, mutations in eat-2, which cause caloric restriction, lead to a significant decrease in de novo fat synthesis. We are continuing this approach to examine other genes known to affect overall fat storage. We expect our novel labeling strategy to generate new insights into how worms incorporate and process their dietary fats and other nutrients.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Camacho, Jessica et al. (2015) International Worm Meeting "Examining environmental effects on the germline epigenome."

Hosohama, Linzi, Camacho, Jessica, Allard, Patrick, Lundby, Zachary, Clark, Amander

[International Worm Meeting, 2015]

The high number of compounds to be tested, the diversity of toxicity endpoints, concentrations, and combinations of chemicals, all provide a challenge in our ability to assess the safety of chemicals. Of particular importance is the potential effect of chemicals on the germline epigenome, which can alter biological processes over several generations. While the cases of DES, vinclozolin and BPA provide evidence of epigenetic effects, there is a great need to explore the influence of environmental compounds on the epigenome and, especially, to develop methods that allow us to quickly and efficiently examine this question. We have established the use of the genetic model system, the worm Caenorhabditis elegans, as a relevant model for epigenetic and reproductive toxicity assessment. By taking advantage of the C. elegans genetic tools, we propose to comprehensively identify chemicals for their ability to disrupt the germline chromatin. To this aim, we are making use of a worm strain where GFP is specifically epigenetically silenced in the germline. We previously showed that valproic acid, a well-known mammalian histone deacetylase inhibitor, disrupts the germline epigenetic state leading to the de-silencing of the transgene in the germline. Our current experiments test this concept further by exposing the worms to environmental compounds. We tested vinclozolin and BPA to analyze a disruption in maintenance and/or establishment of epigenetic marks. Young adult worms were exposed to the compounds for 48 hours, and three subsequent generations were followed and analyzed. Results indicate that de-silencing effects last for at least three generations (up to three-fold compared to our DMSO control). Furthermore, most worms showing de-silencing in the first, second and third generation originate from worms showing de-silencing in the parental exposed generation indicating that the effect is inherited. Thus, we conclude that valproic acid, vinclozolin, and BPA disrupt the germline epigenome over several generations in a heritable fashion. Additionally, in concomitant assays, we are working with the lab of Dr. Amander Clark, using mice primordial germ cell-like cells (PGCLCs) in a mammalian validation approach. Together, we hope to establish a novel in vitro germline differentiation assay to screen for chemicals that affect germline quality.

Jiang, H. et al. (2013) International Worm Meeting "Development of a reverse genetics system for a novel nematode virus."

Wang, D., Jiang, H.

[International Worm Meeting, 2013]

Virus host interaction study in genetically tractable model organism C. elegans has been a long term interest for the scientific community. Recently, the identification of three novel viruses that can infect nematode C. elegans and C. briggsae has opened the possibility of study virus host interactions in a natural virus infection system. In virus host interaction studies, genetic manipulations of the host and the virus are equally important. C. elegans is facile to genetic manipulation and high throughput screening as of its simplicity. Thus, a reverse genetics system is demanded for the genetic modification of the novel nematode virus. Here, we report the development of a reverse genetics system for the novel nematode virus. A novel nematode virus reverse genetics was created by 1) cloning the viral cDNA into an expression vector, 2) establishing stable transgenic worms through microinjection of two viral RNA encoding plasmids along with a pRF4 phenotypic marker, 3) harvesting virus through homogenizing and filtering the transgenic worms. Viable virus production was assayed by qPCR, western blot and immunofluorescence assay on de novo transgenic homogenates infected worms. A genetic marker was further introduced into the virus genome and virus production from transgenic animals was further confirmed by Sanger sequencing of the de novo infected worms. This is the first time report of a novel virus reverse genetics created based on a whole metazoan organism. The nematode virus reverse genetics will serve as a fundamental tool in both virus characterization and virus host interaction study in model organism C. elegans.

Nancy Holroyd (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Parasitic Helminth Genomes at the Wellcome Trust Sanger Institute"

Nancy Holroyd

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Despite their importance globally, both medically and economically, parasitic helminth research has remained relatively untouched by genomics. The Wellcome Trust Sanger Institute (WTSI) is committed to producing reference genomes using de novo sequencing approaches for a cross-phyla list of parastic helminths that include hookworms, whipworms, threadworms and the filarial parasites. We aim to uncover the genomic basis for differences in the biology of these parasites. Presented here will be an overview of nematode genomic projects at WTSI and the approaches we are employing. The reference genomes are being produced using a mixture of sequencing technologies (Illumina, 454 and limited capillary sequence data). Manual improvement of the assemblies is being performed in silica and employing custom capillary sequencing. Several software packages support this process, enabling both large scale global viewing of the genomic and associated data, and in depth manipulation down to the base level. De novo sequencing relies heavily upon reliable mapping information. We are using a number of different mapping sources including genetic markers, happy map markers, large insert read pairs and fingerprint maps. We are also developing a new physical mapping approach based on second generation sequencing that could provide a powerful additional tool for de novo genome sequencing. Gene prediction is a major challenge in genome analysis. Second generation sequencing technologies are being employed to directly sequence transcriptomes and identify coding regions by alignment to the genome sequence. Comparative sequencing of related strains or species will be undertaken to identify candidate genes or other sequences relating to species-specific differences.

Franklin, L. et al. (2017) International Worm Meeting "Comparative approach to understanding neuromuscular deficits in de novo purine biosynthesis mutants."

Franklin, L., Moro, C. A., Hanna-Rose, W.

[International Worm Meeting, 2017]

Caenorhabditis elegans is a powerful model organism which enables us to study, in vivo, the physiological properties of the rare metabolic disorder adenylosuccinate lyase (ADSL) deficiency. This disorder is caused by the lack of the enzyme ADSL resulting in a block in the de novo purine biosynthesis pathway. Symptoms of ADSL deficiency resemble those of an autistic patient, which can include repetitive movements, mild-to-moderate cognitive impairment, and sound sensitivity. ADSL deficiency is a relevant disease to study in C. elegans because purine biosynthesis in general and adsl-1 gene activity in particular is conserved with humans. Moreover, probing the neurological effects of ADSL deficiency may shed light on a variety of neurological disorders given the similarity of ADSL deficiency disorder to aspects of autism. We have identified a motor deficit caused by neural dysfunction as a phenotype in adsl-1 mutant animals. Adenylosuccinate Synthase (ADSS) is an enzyme which is also found in the de novo purine biosynthesis pathway. C. elegans adss-1(RNAi) has a comparable phenotype to adsl-1 deficient C. elegans in terms of motor deficits. We hypothesize that using a comparative approach, between adss-1 and adsl-1 will provide insight on how the lack of a functional de novo purine biosynthesis pathway affects motor skills and sterility. We used thrashing assays as well as aldicarb and levamisole sensitivity assays to compare the motor function of adss-1(RNAi) to adsl-1(RNAi) C. elegans. Compared to controls, adss-1(RNAi) and adsl-1(RNAi) showed reduced thrashing as well as decreased sensitivity to levamisole or aldicarb. Our results suggest the ADSS-1 enzyme is a plausible candidate for helping us to understand ADSL-1 deficiency. In the future, we plan to compare the metabolic effects of adss-1(RNAi) to that of adsl-1(RNAi) to gain mechanistic insight into the cause of the neuromuscular deficit.

Tsur, A et al. (2011) International Worm Meeting "SUMO proteases in C. elegans."

Tsur, A, Broday, L

[International Worm Meeting, 2011]

The covalent attachment of SUMO to its target proteins is a highly dynamic and reversible post-translational modification due to the activity of SUMO proteases. Both de-conjugation of SUMO (deSUMOylation) and the processing of SUMO precursors prior to conjugation are carried out by the same family of proteases called Ubl specific proteases (ULP) in yeast, Drosophila and C. elegans and Sentrin-specific proteases (SENP) in vertebrates. ULP/SENPs are therefore key modulators of SUMOylation dynamics. In order to perform a comparative analysis of the five known C. elegans ULP proteins (ULP-1-5) we generated a phylogenic tree of over 70 different ULP/SENP sequences. The tree shows that ULP-3 which was considered to be a SUMO protease is an ortholog of the mammalian SENP8, a Nedd8-specific protease. ULP-1 appears to be the most closely related to SENP1 and SENP2. ULP-2 is highly related to the mammalian SENP6 and SENP7 suggesting a possible preference towards de-conjugation of SUMOylated species and not SUMO processing [1]. Expression of a translational reporter ULP-2::GFP is mainly nuclear and appears at late gastrulation. ulp-2(RNAi) resulted in ~30% embryonic lethality. Analysis of DLG-1 and AJM-1 fluorescence reporters showed severe defects in epidermal morphogenesis. Our findings suggest a role for ULP-2 in epithelial development. 1. Mukhopadhyay D, Dasso M. Modification in reverse: the SUMO protease. Trends Biochem Sci. 2007 Jun;32(6):286-95. Epub 2007 May 17.