Jill C Bettinger et al. (2004) West Coast Worm Meeting "The genetics of acute adaptation to ethanol intoxication in worms."

Steven L McIntire, Jill C Bettinger, Armando R De La Torre, Andrew D Goldsmith

[West Coast Worm Meeting, 2004]

We have been working to unravel the molecular basis of variation in ethanol sensitivity in C. elegans . Natural variation in ethanol sensitivity in humans is strongly correlated with the propensity to abuse alcohol; people with relatively higher innate tolerance are relatively more likely to become alcoholic (Schuckit, 2002). Ethanol is intoxicating to worms and humans at similar tissue concentrations. Drunk worms become progressively more uncoordinated, slow and Egl as ethanol concentration increases (Davies et al. , 2003). This response is not static; over time worms undergo an acute adaptation (acute tolerance) to the presence of ethanol that does not reflect a decrease in internal ethanol concentration. We noted that there is substantial natural variation in ethanol response, particularly in the rate of development of acute tolerance, between the wild strains N2 and CB4856. This difference is due to the previously described allelic variation at the npr-1 locus (de Bono and Bargmann, 1998; Davies et al. , 2004). Genetic analysis indicates that NPR-1 activity antagonizes the development of acute tolerance. We have performed a genetic screen for molecules involved in the development of acute tolerance by screening for suppressors of the npr-1(ky13) rapid development of acute tolerance phenotype. We have screened approximately 4000 haploid genomes and have isolated 10 suppressors, identifying at least 6 complementation groups. Only a small subset of these mutations (two) suppresses npr-1 -mediated clumping. Thus, most of our mutants presumably identify components that are more specific to the acute ethanol tolerance pathway, rather than general suppressors of npr-1 . None of the mutations has an obvious phenotype in the absence of ethanol. We are pursuing molecular characterization of two of these suppressors, eg613 and eg614 . Both are completely recessive, and neither has a detectable ethanol-sensitivity phenotype in the absence of the npr-1 mutation.

Jill C Bettinger et al. (2005) International Worm Meeting "The genetics of acute adaptation to ethanol in worms."

Steven L McIntire, Jill C Bettinger, Andrew D Goldsmith, Alfred P Kaye, Armando R De La Torre

[International Worm Meeting, 2005]

We are interested the molecular bases of ethanol response in C. elegans. Natural variation in the acute ethanol response in humans is strongly correlated with the propensity to abuse alcohol such that people with relatively higher innate tolerance are more likely to become alcoholic (Schuckit, 2002). Worms get drunk at similar concentrations of ethanol to those that intoxicate humans, suggesting that there is a conserved mechanism for the effects of ethanol on the nervous system, and that worms can be a good model for studying ethanol responses. Worms become progressively more uncoordinated, slow and Egl as the ethanol dose increases (Davies et al., 2003). In response to exposure to a constant ethanol concentration, worms undergo an acute adaptation (acute tolerance) to the presence of ethanol that does not reflect a decrease in tissue ethanol concentration. There is substantial natural variation in the rate of development of acute tolerance between the wild strains N2 and CB4856. This difference between the behavior of the wild strains is due to the previously described allelic variation at the npr-1 locus (de Bono and Bargmann, 1998; Davies et al., 2004). Genetic analysis indicates that NPR-1 activity antagonizes the development of acute tolerance. To better understand the molecular mechanisms of this development of acute tolerance, we have performed a screen for suppressors of the npr-1(ky13) rapid development of acute tolerance phenotype. To date, we have screened approximately 4000 haploid genomes and have recovered 10 suppressors from at least 6 complementation groups. Most of our mutations do not suppress npr-1-mediated clumping, indicating that they identify components that are specific to the acute ethanol tolerance pathway, rather than general suppressors of npr-1. None of the mutations has an obvious phenotype in the absence of ethanol. We have mapped and cloned the first of these suppressors, eg613, which is a null allele of the gene F49E10.5. Analysis of a transcriptional reporter construct reveals that F49E10.5 is expressed in a small subset of neurons. We are pursuing identification of these neurons and exploring the molecular mechanisms by which this molecule affects development of acute ethanol tolerance.

Keith Nykamp et al. (2006) Development & Evolution Meeting "The Conserved La Motif Protein, LARP-1, Negatively Regulates MAPK Signalling in the Germ Line"

Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2006]

The human La protein was first described as an autoantigen present in the sera of systemic lupus erythematosus (SLE) and Sjogren's patients. Since then, a large family of La-related proteins has been described (Wolin & Cedervall, 2002). La family members are all characterized by a highly conserved La Motif (LaM), which has RNA-binding activity. Typically, LaM family members, including human La and yeast Lhp1p, harbor additional RNA Recognition Motifs (RRMs) and localize predominately to the nucleus. Both La and Lhp1p bind RNA polymerase III transcripts (i.e. tRNAs, 5S rRNAs and snRNAs) and direct their maturation. In contrast, two atypical yeast LaM proteins, Sro9p and Slf1p, do not have any other recognizable RNA-binding domains and are predominately cytoplasmic. Sro9p and Slf1p are believed to play a role in mRNA metabolism, although it is unclear what their RNA targets and exact mechanism of control might be (Sobel and Wolin, 1999). The Caenorhabditis elegans genome encodes three LaM family members: C44E4.4, T12F5.5 and R144.7/larp-1. Of the three, LARP-1 is most closely related to yeast Sro9p and Slf1p. Initial RNAi experiments indicated that LARP-1 might be important for oogenesis. To confirm the RNAi results, we isolated larp-1(q783), a deletion mutant that eliminates expression of LARP-1 protein. We find that larp-1(q783) hermaphrodites are self-fertile, but have more developing oocytes in the proximal gonad than normal. We have also found active MAP kinase to be more abundant in larp-1(q783) germ lines than in wild-type. Our working model is that LARP-1 negatively regulates MAPK activity to ensure proper oogenesis.

Esther Zanin et al. (2005) International Worm Meeting "Studying the function of the PAR-3 complex in the early C. elegans embryo"

Monica Gotta, Esther Zanin

[International Worm Meeting, 2005]

Cell polarity is a crucial biological process that is essential to generate cell diversity. The anterior-posterior axis of the C. elegans embryo is determined by the entry of the sperm. The earliest polarity markers known are the conserved partitioning defective (PAR) proteins that localize asymmetrically on the cell cortex in the one cell embryo after fertilization. A protein complex consisting of PAR-3, PAR-6 and aPKC-3 (atypical protein kinase C 3) is enriched on the anterior cortex. Together PAR-3, PAR-6 and aPKC-3 (PAR-3 complex) determine the orientation of the first mitotic spindle along the anterior-posterior axis and the displacement of the spindle during anaphase toward the posterior of the embryo. In addition the PAR-3 complex has been suggested to regulate the cell lineage specific expression of cell fate determinants. Although there is clear support for the requirement of the PAR-3 complex in establishing and maintaining anterior-posterior polarity in the early C. elegans embryo, much less is known about its molecular functions. Therefore we want to identify phosphorylation targets of PKC-3 in C. elegans and investigate how their activity is modulated by PKC-3 in order to generate cell diversity. We performed a yeast two-hybrid screen using a C. elegans embryonic cDNA library and the kinase-dead kinase domain of PKC-3 as a bait. We identified a LA-domain containing protein as an interactor of PKC-3. The LA-domain is a putative RNA-binding domain but the molecular function of this protein is unknown. Interestingly SRO9, a S. cerevisiae LA-domain containing protein, shows a genetic interaction with tropomyosin 1 and RHO3. This observation, together with other data in S. cerevisiae, indicates that SRO9 is required for the stabilization of actin filaments. Depletion of LA by RNAi results in less than 10% embryonic lethality. However, the embryos show a strong posterior meeting of the paternal and maternal pronuclei, a reduced AB size and they are small. Interestingly we found that reducing the level of the LA by RNAi can partially rescue par-2 (it5ts) lethality. This suggests that the LA-domain protein could function either as a regulator or an effector of the PAR-3 complex. In order to understand if the reduction of the LA-domain protein influences the localization and/or levels of the PAR-3 complex we will analyze PAR-3 and PAR-6 immuno-staining in par-2 (it5ts) mutant embryos where the LA has been depleted by RNAi. Finally we aim to reveal if the LA-domain protein regulates F-actin stability via tropomyosin by testing its genetic interactions further. Current progress on the project will be presented at the meeting.

Keith Nykamp et al. (2008) Development & Evolution Meeting "LARP-1 Localizes to Germline P Bodies and Attenuates RAS-MAPK signaling During Oogenesis"

Myon-Hee Lee, Judith Kimble, Keith Nykamp

[Development & Evolution Meeting, 2008]

RNA regulators are critical for animal development, especially in the germ line. In this work, we focus on C. elegans LARP-1, a representative of one La-related protein (Larp) cluster that is broadly conserved among eukaryotes. LARP-1 possesses a signature La motif, which is an ancient RNA-binding domain, plus another conserved motif we have dubbed the LARP1 domain. LARP-1 binds RNA in vitro via the La motif and the LARP1 domain. A larp-1 null mutant has germline defects reminiscent of hyperactive Ras-MAPK signaling; genetic interactions indicate that LARP-1 normally attenuates Ras-MAPK signaling in the germ line; and mRNAs encoding certain Ras-MAPK signaling components are elevated in larp-1(0) mutant germ lines. LARP-1 co-localizes with cytoplasmic granules called P bodies, which have been implicated in mRNA degradation. We propose that LARP-1 affects the stability of selected mRNAs via its association with germline P bodies.

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.

McReynolds, Melanie et al. (2017) International Worm Meeting "Eukaryotic de novo NAD+ biosynthesis from tryptophan in the absence of a QPRTase homolog."

Wang, Wenqing, Holleran, Lauren, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

NAD+ is found in all living cells, and is an essential coenzyme, which impacts the entire metabolome. Hence, NAD+ biosynthesis has proven to be an attractive and promising therapeutic target for influencing health-span and obesity-related phenotypes as well as tumor growth. A wide range of animals and yeast synthesize NAD+ via de novo synthesis from the degradation of tryptophan (Trp), via the kyneurine pathway. The C. elegans' genome encodes all the enzymes involved in the kynurenine pathway. However, the genome lacks the critical quinolinic acid phosphoribosyltransferase (QPRTase) that converts QA into NaMN for biosynthesis of NAD+. Because C. elegans lack an apparent QPRTase homolog, it's been assumed that this species lack active NAD+ de novo synthesis. We've uncovered evidence that suggests NAD+ de novo synthesis is active and contributing to NAD+ biosynthetic capacity in C. elegans. We previously found that phenotypes that are dependent on NAD+ levels could be reversed upon supplementation with QA and other kynurenine metabolites, suggesting that the kyneurine pathway may contribute to NAD+ biosynthesis. We have directly demonstrated that isotopic label from Trp is incorporated into NAD+. Furthermore, we show that loss of kynurenine pathway activity decreases global NAD+ levels and prevents incorporation of isotopic label supplied from Trp into NAD+, supporting the presence of de novo synthesis in C. elegans. Using genetics and isotope tracing experiments, we determined that the enzyme UMPS-1, (uridine monophosphate synthetase), is required for NAD+ biosynthesis from Trp in this organism. Finally, we connected the kyneurine pathway to normal fecundity. This evidence demonstrates that NAD+ de novo synthesis is active and contributes to NAD+ biosynthetic capacity and homeostasis in C. elegans. Intriguingly, a conserved enzyme is substituting for the missing QPRTase, raising questions about the relevance of similar underground metabolic activity in higher organisms.

Keith Nykamp et al. (2005) International Worm Meeting "The conserved RNA-binding protein, LARP-1, interacts with the atypical poly(A) polymerase GLD-2 and regulates meiotic progression"

Keith Nykamp, Liaoteng Wang, Judith Kimble

[International Worm Meeting, 2005]

The adult hermaphrodite germ line is spatially organized with proliferative stem cells localized to the distal end and fully differentiated sperm and oocytes at the proximal end. In between, meiotic nuclei progress from pre-meiotic S phase and early meiotic prophase through pachytene and diplotene/diakinesis. This developmental pattern is established, in a large part, by a combination of Notch signaling, RNA regulators and MAP kinase activity. Among RNA regulators, GLD-2 and GLD-3 work together to promote entry into the meiotic cell cycle. GLD-2 is an atypical poly(A) polymerase that lacks any apparent RNA-binding motif, and GLD-3 is a KH RNA-binding protein and GLD-2 binding partner. We have focused on a different GLD-2 binding partner to explore the idea that GLD-2 is recruited to distinct mRNAs by interacting with distinct RNA-binding proteins. In a yeast two-hybrid screen for GLD-2 interacting proteins, 42% (50/118) of the positive isolates corresponded to the larp-1 (La-related protein) gene. larp-1 encodes a large protein with a single La motif. The La motif has RNA-binding activity in vitro with a preference for 3 termini, is found in multiple proteins in eukaryotes and has been associated with a wide variety of functions. We will provide genetic, biochemical and cytological data to support the idea that GLD-2 and LARP-1 function together to control the transition from pachytene to diplotene/diakinesis of developing oocytes.

Charles Baer (2008) Development & Evolution Meeting "Quantifying the De-canalizing Effects of Spontaneous Mutations in Rhabditid Nematodes"

Charles Baer

[Development & Evolution Meeting, 2008]

The evolution of canalization - the robustness of the phenotype to environmental or genetic perturbation - has attracted considerable recent interest. A key step toward understanding the evolution of any phenotype is to characterize the rate at which mutation introduces genetic variation for the trait (the mutational variance, VM) and the average directional effects of mutations on the trait mean (deltaM). In this study, the mutational parameters for canalization of productivity and body volume are quantified in two sets of mutation accumulation lines of nematodes in the genus Caenorhabditis and compared to the mutational parameters for the traits themselves. Four results emerge: (1) spontaneous mutations consistently de-canalize the phenotype; (2) the mutational parameters for de-canalization - VM (quantified as mutational heritability) and deltaM - are of the same order of magnitude as the same parameters for the traits themselves; (3) the mutational parameters for canalization are roughly correlated with the parameters for the traits themselves across taxa; and (4) there is no evidence that residual segregating overdominant loci contribute to the decay of canalization. These results suggest that canalization is readily evolvable, and that any evolutionary factor that causes mutations to accumulate will, on average, de-canalize the phenotype.

McReynolds, Melanie et al. (2015) International Worm Meeting "Maintaining Global NAD+ Homeostasis Reveals Separable Functional and Compensatory Roles for NAD+ Biosynthetic Pathways in C. elegans."

Wang, Wenqing, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2015]

NAD+ biosynthesis has proven to be a useful therapeutic target for influencing health-span and obseity-related phenotypes as well as tumor growth. The goal of our research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. NAD+ is synthesized via distinct routes including de novo synthesis from tryptophan, salvage synthesis from nicotinamide, which feeds into the Preiss-Handler pathway from nicotinic acid in C. elegans, and via the phosphorylation of nicotinamide or nicotinic acid ribosides using nicotinamide riboside kinase (NRK). We hypothesize that each pathway will have tissue-specific functions independent of the others and that they will also have compensatory roles in contributing to global NAD+ homeostasis when another pathway is compromised, creating a homeostatic network. We have previously shown, using pnc-1 nicotinamidase mutants, that salvage NAD+ biosynthesis is required for the appropriate temporal progression of reproductive development and for optimum muscle function. Here we examine functions of the NRK and the de novo pathways using genetics. We find that both pathways function to maintain muscle activity, but neither pathway has an independent role in gonad development. Using LC-MS we show that NAD+ is decreased when either pathway is blocked, as previously shown for PNC-1. This data supports the hypothesis that all pathways are required for maintenance of NAD+ levels and for muscle function but only the salvage pathway normally plays a role in gonad development. To examine homeostatic mechanisms, we investigated the function of the NRK and de novo pathways in a pnc-1 mutant background. Mutations in either pathway further exacerbate pnc-1-associated muscle defects, but not the reproductive development defect. We revealed using qRT-PCR that in the absence of salvage synthesis, key enzymes in de novo and NRK-mediated synthesis are up-regulated. We also observed reciprocal trends when the de novo or NRK pathways were blocked. Consistent with these results, supplementation with either nicotinamide riboside or de novo pathway metabolites rescues the gonad delay in pnc-1, indicating that boosting other pathways can restore gonad development when salvage synthesis is blocked, even though these pathways play no role in gonad development normally. We interpret these data as evidence that homeostatic mechanisms are indeed occurring between NAD+ biosynthetic pathways. Our work has implications for efforts to therapeutically target individual NAD+ biosynthetic pathways. .