Hawkins, E.G. et al. (2015) International Worm Meeting "Tolerance to a cholinergic activating effect of alcohol requires a specific function of the EAT-6 Na+/K+-ATPase."

Kondo, L.M., Davies, A.G., Hawkins, E.G., Martin, I., Bettinger, J.C.

[International Worm Meeting, 2015]

Alcohol (ethanol) produces activating and depressing behavioral effects in humans. We have investigated the basis for an activating effect of ethanol in C. elegans that produces body hypercontraction. Acute ethanol exposure induces a shortening of the animal that requires normal acetylcholine levels and a functional UNC-63 acetylcholine receptor (AChR) subunit. Elimination of the two receptors currently known to contain the UNC-63 subunit, the ACR-2R and the levamisole-sensitive AChR, do not alter sensitivity to ethanol. This suggests that UNC-63 may be acting in an unidentified receptor to mediate this effect of ethanol. AChRs in mammals have been implicated in mediating activating effects of ethanol, and genetic variation in AChR subunit-encoding genes has been associated with the development of alcoholism in humans. We are pursuing the tissue-specific requirements for unc-63 expression for its role in this ethanol effect.The hypercontracted animals show a recovery of pre-treatment body length despite continuous exposure to ethanol (and a gradual increase in internal ethanol concentration). This observation suggests that a physiological tolerance is developing to the drug. We identified the eat-6(eg200) mutation as a mutant that fails to develop tolerance to this ethanol effect. An examination of several existing alleles of eat-6 found that mutants with strong effects on pharyngeal pumping did not alter the response to ethanol-induced hypercontraction, whereas the ad601 allele, which resembles the eg200 mutant in having weak effects on pumping but strong effects on aldicarb sensitivity, does alter the maintenance of tolerance to ethanol. These data suggest that eg200 and ad601 alter a specific function of the Na+/K+-ATPase that is important for the development and/or maintenance of tolerance to ethanol for this cholinergic activating effect of ethanol. One possibility that we are testing, is that EAT-6 has a structural relationship with an ethanol-sensitive AChR, and that the mutations eg200 and ad601 prevent desensitization or compensatory trafficking of that receptor.

O'Neil NJ et al. (1998) West Coast Worm Meeting "ANALYSIS OF ESSENTIAL GENES IN THE sDf125 REGION"

Vatcher GP, Baillie DL, Kuervers LM, O'Neil NJ

[West Coast Worm Meeting, 1998]

We have demonstrated that transgenic rescue is an effective method for high resolution mapping of essential genes. Our lab has constructed a map of 112 essential genes isolated on LGIII left. We have divided the region between dpy-17 and dpy-19 into 12 zones that are defined by overlapping deficiencies and duplications. These zones were determined physically by PCR testing the deficiencies to determine endpoints. Into these zones, we have mapped the 112 essential gene mutations. One such zone (zone 7) is defined by sDf125 and sDp8. This region spans 380 000 base pairs and is completely covered by sequenced cosmids. Within this zone there are 101 predicted coding elements. To date, we have mapped 14 essential genes into this zone. We have used a systematic approach to rescue these essential genes with cosmid transgenics from our cosmid transgenic library. We have to date rescued 10 genes in this zone. These transgenic rescues map the genes to 40 kB (one cosmid) resolution as opposed to the 380 kB resolution of the deficiency and duplication data. This is almost a ten-fold increase in resolution and reduces the number of predicted coding elements which may be responsible for the gene function. The next step will be to subclone the cosmids with which we have rescued essential gene mutations. This will directly correlate the lethal or maternal effect lethal phenotype with the appropriate coding sequence. We have already begun the subcloning of the cosmids (see abstracts by G. P. Vatcher et al., L.M. Kuervers et al. this meeting).

Alicea, Bradly et al. (2015) International Worm Meeting "An experimental evolution model of C. Elegans adaptability."

Schroeder, Nathan, Androwski, Rebecca, Alicea, Bradly

[International Worm Meeting, 2015]

While the genetic architecture of C. elegans is well-studied, the adaptability of various strains and their reproductive fitness is less understood. To address this, an experimental-based model will allow for characterization of C. elegans adaptability amongst strains which may exhibit low reproductive potential and/or survivability under selection. Two experimental approaches representing within- and between-generation survivability (preconditioning and experimental evolution, respectively) will allow survivability to be assessed. Preconditioning [1] assesses adaptability within a generation using environmental selection, while experimental evolution [2] assesses adaptability via cross-generational artificial selection. For a given strain, populations grown from single founder individuals are repeatedly subject to genetic drift and then selected for fecundity (largest brood size). These results demonstrate how specific genotypes exhibit robustness in the face of adaptive constraints.Biological adaptability involves both survivability and reproductive fitness. Survivability is the degree to which a single founder worm can survive and produce viable clones (e.g. the number of generations constituting a single genealogy). Reproductive fitness is the mean population size for a given genealogy. In strains with high survivability, founder populations with high reproductive fitness should also generate subsequent founder populations with a high degree of survivability. Strains with high reproductive fitness but low overall progeny sizes may also exhibit high adaptability if selection results enhances adaptation to varying environmental conditions. While this model does not control for stochastic effects and demographic constraints [3], it does allow for genealogies with consistently high survivability [4].1. Calabrese, E.J. et.al, Tox App Pharm, 222(1), 122-128 (2007).2. Kawecki, T.J. et.al, Trends Ecol Evol, 27(10), 547-560 (2012).3. Szendro, I.G. et.al, PNAS, 110(2), 571-576 (2012).4. Wahl, L.M., Krakauer, D.C., Genetics, 156(3), 1437-1448 (2000).

Nakajima T et al. (2000) Japanese Worm Meeting "A possible candidate of the DAF-2 ligands: structure, expression, and physiological functions"

Kawano T, Kimura Y, Ito Y, Nakajima T, Ishiguro M, Takuwa K

[Japanese Worm Meeting, 2000]

The DAF-2 protein, a C. elegans homolog of an insulin/IGF-1 receptor, is involved in dauer formation, fat metabolism, and a long life span (1). In the previous meeting, we reported two distinct genes encoding insulin/IGF-1-like peptides designated Ceinsulin-1 and Ceinsulin-2 (2). In this meeting, we will make a short talk of the Ceinsulin-1 regarding its structure, expression, and physiological functions. The precursor protein of Ceinsulin-1 consists of 95 amino acid residues and seems to include a signal peptides, B and A chains, and a so-call C peptide that could be removed out through post-translational proteolysis like the preproinsulin. First, to detect the mature peptide, a polyclonal antibody against the putative B chain was prepared and used for analyzing C. elegans protein by Western blotting. The result showed that the mature Ceinsulin-1 should consist of one polypeptide like IGFs. However, the predicted tertiary structure of Ceinsulin-1 was more similar to the crystal structure of insulin than that of IGF-1, suggesting that the Ceinsulin-1 should be a hybrid molecule of insulin and IGF-1. Next, we analyzed expression pattern of the peptide at several developmental stages using RT-PCR and Western blotting. Interestingly, the transcriptional and translational patterns were not always coincident with each other, suggesting that the peptide biosynthesis could be regulated post-transcriptionally with some degrees. Details will be shown in the meeting. Finally, an RNA-i experiment was carried out to elicit physiological functions of the Ceinsulin-1. The RNAi led to reduction of the corresponding transcript and an extended life span. The inhibition of the Ceinsulin-1 biosynthesis exhibited no drastic effects on entry into and recovery from the dauer stage. Now, the effect on fat accumulation is examined. Anyhow, other insulin/IGF-1 peptides may be involved in the dauer formation and fat metabolism. References 1. Kimura, K.D., et al. (1997) Science 227, 942-946. 2. Kawano, T., et al. (1999) Peptide Science 1998: M. Kondo (Ed.) pp117-120.

Rogalski TM et al. (1991) International C. elegans Meeting "GENETIC APPROACHES TO C. ELEGANS RNA POLYMERASE III."

Golomb M, Watson N, Lancy ED, Rogalski TM, Riddle DL, Albert PS

[International C. elegans Meeting, 1991]

RNA polymerase III (Rpo m) transcribes genes encoding SS and transfer RNA's. This enzyme and its associated factors have been studied extensively in other systems, but Rpo m function has not been studied genetically in a metazoan. Developmental regulation of Rpo m activity in C. elegans has been suggested by the molecular genetic analysis of nonsense suppressors (Kondo et al., J. Mol. Biol. 215:7-19, 1990). The gene for the largest subunit of Rpo m, rpc-l, has been cloned (Bird and Riddle, Mol. Cell. Biol. 9:4119-4130, 1989) and positioned on the physical map between unc44 and unc-24 on chromosome IV. The availability of cloned genes and an in vitro transcription system (Honda et al., Nucl. Acid Res. 14B:869-881, 1986) provides potential tools for a detailed analysis of Rpo III structure and function using mutants, if appropriate mutants can be identified. Two approaches to genetic analysis of rpc-l have been employed. The first approach utilized resistance to the fungal toxin, ct-amanitin. A unique strain with an extremely amanitin-resistant Rpo II (Rogalski et al., Genetics 126:889-898, 1990) was used as a parent to select even higher levels of resistance. One such mutation was mapped on chromosome IV, and preliminary nuclear run-on assays indicate that Rpo III is abnormally resistant to amanitin, in appropriate recombinants derived from this mutant strain. The mutant Rpo III transcribes SS RNA in the presence of 200 g/ml amanitin, whereas wild-type Rpo m is inhibited by 25 g/ml. These results suggest that the resistance mutation affects Rpo III, and it may be within the rpc-l gene. A second approach to rpc-l genetics utilized a collection of EMSinduced lethal mutations generated by Denise Clark. We mapped fourteen of her lethal mutations to the interval between unc44 and unc-24, a region estimated to contain approximately 30 essential genes. These lethal mutations are now being assigned to complementation groups. Lethal derivatives of the putative amanitin-resistant rpc-l allele will also be sought, and selected lethal strains will be injected with the cloned rpc-l gene in an effort to identify the genetic correlate to rpc-l by DNA transformation. Genetic, phenotypic, and biochemical analysis of these mutants may provide a useful entree into structure- function relationships in this important enzyme.

Jha, Sweta et al. (2021) International Worm Meeting "UPS modulation and autophagy."

Jha, Sweta, Holmberg, Carina

[International Worm Meeting, 2021]

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Autophagy is responsible for degrading defective cellular organelles and long-lived proteins in the cytosol and is involved in cell growth, survival, development and death. UPS mostly degrades soluble and short-lived proteins in the nucleus and cytosol and affects various cellular processes. These two proteolytic systems are essential components of the cellular protein quality control system. Several studies have reported an interplay between the UPS and ALP, however it still remains largely unknown how these systems communicate in a multicellular organism. We have recently shown that downregulation of autophagy genes elicits tissue-specific effects on UPS function in C. elegans1. Here, we address how genetic or pharmacological modulation of the proteasome and the proteasome-associated DUBs affects autophagy. We use transgenic C. elegans expressing the autophagy reporters GFP::LGG-12 or mCherry::GFP::LGG-13 and analyze the accumulation of LGG-1 puncta and autophagic flux. Our preliminary data show that downregulation of the proteasome-associated DUBs ubh-4, usp-14 and rpn-11 as well as some proteasomal subunits affect the number of puncta positive for autophagosomes in hypodermal seam cells, intestinal cells as well as in pharynx. To nvestigate a conserved function of the UPS on autophagy in human cells, we are utilizing a HeLa cell line expressing the fluorescence probe GFP::LC3::RFP::LC3G4. A better understanding of the multilayered crosstalk between UPS and ALP in vivo may facilitate development of therapeutic options for various disorders linked to dysfunction in proteostasis. References Jha, S., Holmberg, C.I. Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans. Cells 2020, 9, 1858. Melendez A, Talloczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. Autophagy genes are essential for dauer develop-ment and life-span extension in C. elegans. Science 2003. 301:1387-91 Chang J.T., Kumsta C., Hellman A.B., Adams L.M., Hansen M. (2017). Spatiotemporal regulation of autophagy during caenorhabditis elegans aging. Elife. pii: e18459 Kaizuka T., Morishita H., Hama Y., Tsukamoto S., Matsui T., Toyota Y., Kodama A., Ishihara T., Mizushima T., Mizushima N. (2016). An autophagic flux probe that releases an internal control. Mol Cell. 64(4):835-849.

Tammy F Wu et al. (2001) International C. elegans Meeting "The enigma of (y2): an XO-specific lethal mutation"

John D Plenefisch, Barbara J Meyer, Tammy F Wu

[International C. elegans Meeting, 2001]

During embryogenesis, a C. elegans embryo must first assess its genetic dosage of X chromosomes, and then develop accordingly as male (XO) or hermaphrodite (XX). This development involves not only morphological sexual differentiation, but also implementation of X-chromosome dosage compensation, an essential process. Dosage compensation in worms is achieved by repressing the level of X expression by half in XX animals. Genetic screens for sex-specific lethality have been a successful approach to dissecting the pathway involved in counting the number of X chromosomes (X-signal elements fox-1 and sex-1 ), and in implementing the consequences of that counting process (the sdc genes, and dosage compensation-specific genes). The target of the counting process is the xol-1 gene, which coordinates sexual morphology with dosage compensation. xol-1 XO mutants inappropriately activate the hermaphrodite mode of development--they are feminized, and die due to underexpression of X-linked genes. Interestingly, in addition to its essential role in XO animals, xol-1 has a secondary role in the feminization of XX animals. 1 The maternal-effect autosomal mutation y2 was isolated in a screen for sex-specific phenotypes and exhibits XO-specific embryonic lethality. Unlike xol-1 mutants, y2 mutants exhibit reduced expression from the X chromosome in both XX and XO karyotypes. XX hermaphrodites have phenotypes that are consistent with perturbed expression from X. Therefore, y2 XO animals could suffer from reduced gene expression below a critical threshold, and die as a result. However, overexpression of xol-1 from a ubiquitously expressed promoter can rescue y2 XO lethality, whereas the XX lethality from overexpression of xol-1 is not rescued by y2 . This result suggests that y2 does impinge on the regulatory pathway for sex determination and dosage compensation. Surprisingly, the rescued animals are feminized, a phenotype not seen when xol-1 is overexpressed in a wild-type XO background. Paradoxically, staining of dying y2 XO embryos with antibodies against the dosage compensation complex indicates that the complex is not localized to X, as it is in dying xol-1 XO embryos. At this time, no simple explanation can be found for the role of y2 , but we are intrigued by the possibilities. y2 may generally regulate gene expression, act as an autosomal signal element, be involved in the coordinate control of dosage compensation and sexual differentiation, or function with xol-1 to specify sexual differentiation. Currently, efforts are being taken to map and clone y2 , and genetic and molecular experiments are being performed to address how this mutation may act on known genes in the pathway. 1 Miller, L.M., Plenefisch, J.D., Casson, L.P., and Meyer, B.J. (1988). Cell 55, 167-183.

Annabelle Couthier et al. (2007) International Worm Meeting "Investigating the contribution of zyxin to cadherin-catenin function."

Jeff Hardin, Jonathan Pettitt, Annabelle Couthier, Aileen Flett, Theresa Grana

[International Worm Meeting, 2007]

Epidermal morphogenesis is responsible for the change in shape of the C. elegans embryo from an ovoid into a worm, and involves two cadherin dependent-events: ventral enclosure and elongation. We have previously shown that animals homozygous for a alpha-catenin mutation hmp-1(fe4) provide a sensitive background to identify molecules that contribute to cadherin-catenin complex function [1]. These primary results have provided the rationale for a genome-wide RNAi screen for C. elegans genes whose loss-of-function enhances the fe4 phenotype [2]. Our group has recently completed the screen of Chromosome II and III from which we have identified a number of genes that contribute to the regulation of actin dynamics. We are particularly interested in one of these genes, which encodes the C. elegans zyxin homologue. hmp-1(fe4);zyx-1(RNAi) embryos show 100%; lethality and arrest with severe elongation defects. Two mutants for zyx-1 are available: like zyx-1(RNAi), both zyx-1(um4) and zyx-1(gk442)enhance the hmp-1(fe4) phenotype. zyx-1(um4) is a deletion that removes the LIM domain from the zyxin protein. zyx-1(gk442) is a predicted null mutation, and homozygotes for the mutation display a sterile and skinny phenotype, although it is yet unclear whether the sterility is caused by the zyx-1 loss of function itself or by a linked mutation affecting another gene. Zyxin has long been known to be present at focal adhesions and it has also been shown to be present at cadherin-based junctions in cell culture experiments [3]. Our data provide the first evidence of a role for zyxin at adherens junction in an in vivo model. Zyxin has also been shown to interact with alpha-actinin and Ena/VASP suggesting a role in the regulation of actin remodelling [4]. We are currently attempting to determine the mechanistic basis of the zyxin-a-catenin interaction. 1.Pettitt, J., et al., The Caenorhabditis elegans p120 catenin homologue, JAC-1, modulates cadherin-catenin function during epidermal morphogenesis. J Cell Biol, 2003. 162(1): p. 15-22. 2.Cox, E.A. and J. Hardin. A feeding RNAi screen reveals novel regulators of cadherin-mediated adhesion during morphogenesis, including the tropomodulin homolog, TMD-1. in 15th International worm meeting. 2005. Los Angeles. 3.Hansen, M.D. and M.C. Beckerle, Opposing roles of zyxin/LPP ACTA repeats and the LIM domain region in cell-cell adhesion. J Biol Chem, 2006. 281(23): p. 16178-88. 4.Hoffman, L.M., et al., Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling. J Cell Biol, 2006. 172(5): p. 771-82.

Schutzman JL et al. (1998) East Coast Worm Meeting "Molecular identification and characterization of downstream components of FGFR signaling."

Stern MJ, Schutzman JL, Selfors LM

[East Coast Worm Meeting, 1998]

egl-15 encodes a fibroblast growth factor receptor (FGFR) that is involved in several aspects of C. elegans development1. Several lines of evidence support the hypothesis that the activity of EGL-15 is opposed by a receptor tyrosine phosphatase that is the product of the clr-1 (CLeaR) gene2. First, a construct containing a mutation in egl-15 that has the potential to cause constitutive dimerization and hence activation of EGL-15 can confer a Clr phenotype when introduced into wild-type animals by germline transformation. Second, mutations that compromise the activity of EGL-15 can suppress the Clr phenotype conferred by mutations in clr-1. Thus, the Clr phenotype of clr-1 mutants appears to result from the hyperactivity of the EGL-15 FGFR. Screens for suppressors of the Clr (Soc) phenotype of clr-1 mutants can identify other genes required for signaling through EGL-15 since mutations affecting components of this signaling pathway should reduce the hyperactivity of this pathway. Strong suppressors of the temperative-sensitive phenotype of clr-1(e1745ts) mutants identified four soc genes: egl-15, sem-5, soc-1, and soc-2. Subsequent genetic studies indicate that soc-1 and soc-2, like sem-5, are epistatic to egl-15, suggesting that they act to transduce the signal from EGL-15 to downstream targets. We are continuing the molecular and genetic characterization of soc-1 and soc-2 to elucidate the roles of these genes in FGFR-mediated signaling pathways. soc-2 has been cloned and shown to encode a leucine rich repeat protein whose human homolog, shoc-2, shares 54% amino acid identity with C. elegans SOC-23. Based on genetic and molecular criteria, alleles of soc-2 isolated in the original Soc screen seem unlikely to eliminate soc-2 function completely. The only dramatic phenotype exhibited by these soc-2 mutants is their Soc phenotype, although these animals also appear to be slightly scrawny (Scr), a characteristic observed among other soc mutants. In order to assess the effect of complete loss of SOC-2, we are performing a noncomplementation screen to identify null alleles of soc-2. From animals representing 14,500 haploid genomes, three independent mutations that fail to complement soc-2(n1774) were isolated. We are currently identifying the molecular lesions associated with these new alleles and carrying out their phenotypic analysis to address the specificity of SOC-2 for EGL-15 FGFR signaling. Similar to soc-2 mutants, soc-1 alleles are Soc and Scr. Multiple alleles of soc-1 have been isolated in both EMS and gamma-ray mutagenesis screens for clr-1(e1745ts) suppressors. Isolation of gamma-ray induced alleles suggests that some of these alleles may eliminate soc-1 function. soc-1 was genetically mapped to lie between unc-62 and unc-46 on linkage group V and has been rescued using a YAC from the region. We are currently in the process of further refining this domain by aligning cosmids in the region with respect to the rescuing YAC, Southern analysis of the gamma-ray induced alleles and germline transformation assays with individual cosmids. 1DeVore, D.L. et al. (1995). Cell 83, 611-620. 2Kokel, M. et al. (1998). Genes Dev., in press. 3Selfors, L.M. et al. (1998). Proc. Natl. Acad. Sci. USA, in press.

Jeanna M Wheeler et al. (2001) International C. elegans Meeting "Genetic Analysis of the Ultradian Defecation Rhythm of C. elegans"

James H Thomas, Jeanna M Wheeler

[International C. elegans Meeting, 2001]

Biological rhythms are widespread in nature and regulate processes such as vertebrate and invertebrate heartbeats, and invertebrate swimming and gut peristalsis. Such rhythms are called ultradian since they have a period of less than 24 hours. The defecation cycle of C. elegans is an ultradian rhythm which is amenable to genetic analysis. Every 45 to 50 seconds, wild type C. elegans initiates a series of muscle contractions known as the defecation motor program. The first muscle contraction is the pBoc, or posterior body-wall muscle contraction. About three seconds later, the anterior body-wall muscles contract (aBoc), immediately followed by the enteric muscle contraction, which expels gut contents. Mutants have been identified that are lacking one or more of these steps; however, none of these have a large effect on cycle period. This indicates that the mechanism controlling the timing of the contractions is distinct from the motor program itself. The defecation rhythm shares several characteristics with other defined molecular clocks. First, it exhibits temperature compensation over a range of temperatures between 19-30 degrees Celsius. Second, it can be reset by an external stimulus. Specifically, light touch with an eyelash to the head region resets the phase of the clock. Finally, the clock can keep time in the absence of the defecation motor program. When wild type worms spontaneously leave a lawn of food, the contractions of the motor program cease, and on return to food the next defecation tends to be in phase with the original cycle (Liu and Thomas, 1994). Thirteen genes have been identified that can mutate to alter the length of the cycle. One of these Dec genes, itr-1 , has been shown to encode the only inositol trisphosphate receptor in C. elegans . Loss-of-function mutations in itr-1 cause the cycle period to be lengthened or eliminated, while overexpression of itr-1 produces a short period. In addition, mosaic analysis has shown that ITR-1 function in intestinal cells is necessary and sufficient for the defecation rhythm (DalSanto et al , 1999). Four other Dec genes have been cloned: unc-43 encodes the only CaMKII in C. elegans (Reiner et al , 1999), flr-1 encodes a DEG/ENaC sodium channel, and flr-3 and flr-4 share homology with protein kinases (Take-Uchi et al , 1998). The molecular identification of other Dec genes may provide more information regarding the signaling pathways that control the timing of the clock. In addition, better characterization of the phenotypes and gene interactions among the Dec genes may allow inference of formal genetic pathways that regulate the clock. I have begun work towards identifying dec-2. dec-2 (sa89) causes a defecation cycle period of ~100 seconds. I am also analyzing Dec mutant responses to light touch and reduced concentrations of food and constructing dec-2 double mutants for gene interaction studies. Current progress on the cloning of dec-2 and the phenotypic analysis will be presented. Dal Santo P, Logan MA, Chisholm AD, Jorgensen EM. (1999) The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans . Cell 98: 757-767. Liu DW, Thomas JH. (1994) Regulation of a periodic motor program in C. elegans . J Neurosci 14: 1953-62. Reiner DJ, Newton EM, Tian H, Thomas JH. (1999) Diverse behavioural defects caused by mutations in C. elegans unc-43 CaM kinase II. Nature 402: 199-203. Take-Uchi M, Kawakami M, Ishihara T, Amano T, Kondo K, Katsura I. (1998) An ion channel of the degenerin/epithelial sodium channel superfamily controls the defecation rhythm in C. elegans . Proc Natl Acad Sci USA 95: 11775-80.