1K Papers found (0.017 seconds)

Anita U Rao et al. (2007) International Worm Meeting "Genetic Dissection of Heme Pathways in C. elegans."

[

International Worm Meeting,

2007]

Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways has been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at 1.5 mM and at 800 mM heme. These results suggest that although worms are obligate heme auxotrophs they are likely to have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans, we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 mM heme in liquid axenic medium. Based on the mutants growth profile in medium containing low and high heme, we categorized the mutants into three broad phenoclusters: class A, class B and class C. Class A mutants grew robustly under low and high (800 and 1000mM) heme, Class B mutants grew exceptionally well under low heme, moderately well at 800 mM heme, and not at all at 1000 mM heme, and Class C mutants grow moderately well under high heme (800mM), but exhibit normal growth under low heme. The mutants were further sub-clustered by using gallium protoporphyrin (GaPP), a toxic heme analog. Complementation analyses revealed that these 13 mutants fall into five complementation groups. Genetic mapping by recombination localized the mutants from each complementation group to chromosome III. We are now producing a high resolution map to define a genetic interval and pinpoint the exact nature of the molecular lesion in these mutants.

Denise Chun et al. (2007) International Worm Meeting "Analyzing the Role of UNC-101/ m1 and UNC-108/Rab-2 in Trafficking of the GLR-1 Glutamate Receptor."

Joshua Kaplan, Denise Chun, Jason McEwen, Michelle Burbea

[

International Worm Meeting,

2007]

Glutamate receptor localization has been suggested to play an important role in long-lasting changes in synaptic transmission, which are thought to form the basis of learning and memory. We study a fluorescently-tagged AMPA-type glutamate receptor GLR-1 in that localizes to punctate spots, representing synapses, in the nerve cord. Mutants lacking UNC-101, an AP-1 m1 clathrin adaptin subunit, were identified in a screen for increased GLR-1 abundance in the ventral cord. We are currently characterizing the role that UNC-101/ m1 plays in the trafficking of GLR-1. We also isolated an unc-108 loss-of-function allele in our screen for increased GLR-1 in the ventral cord. The unc-108 gene encodes an ortholog of mammalian Rab-2 GTPase. Previous work in mammals has shown that Rab-2 primarily functions in retrograde trafficking of proteins from the golgi to the ER. We demonstrate that UNC-108/Rab-2 is expressed primarily in C. elegans neurons. We have found that the early/recycling endosome marker, Syntaxin-13, is mislocalized in unc-108 mutants, suggesting that UNC-108/Rab-2 may play a role in endosomal trafficking. We are further characterizing the function of UNC-108/Rab-2 in C. elegans neurons, and its role in GLR-1 trafficking and localization.

Chun-Liang Pan et al. (2007) International Worm Meeting "DPY-23, the C. elegans ortholog of the m2 subunit of AP-2, regulates Wnt signaling and functions in the Wnt-producing cells."

Chun-Liang Pan, Gian Garriga, Paul D. Baum

[

International Worm Meeting,

2007]

The AP-2 adaptin complex has been shown to mediate endocytosis from the plasma membrane by linking cell surface receptors to the clathrin triskelion. Here we report that DPY-23, the C. elegans ortholog of the m2 subunit of AP-2, controls directed neuronal migrations and orients the polarity of axon outgrowth. During embryogenesis, the HSN neurons migrate anteriorly. In the L1, the left Q neuroblast (QL) and its descendents migrate posteriorly, while the right Q neuroblast (QR) and its descendents migrate anteriorly. Mutations in dpy-23 caused the HSN and QR neuroblast to terminate their anterior migrations prematurely, and the QL neuroblast to reverse its normal posterior migration and migrate anteriorly. In dpy-23 mutants, the ALM neurons, which normally extend a single anterior process, extended both anterior and posterior processes. These phenotypes are similar to those found in egl-20 and cwn-1 Wnt mutants, suggesting that DPY-23 may regulate Wnt signaling. Our genetic experiments indicate that DPY-23 primarily regulates EGL-20 functions, but may also control other Wnts as well. EGL-20 controls the posterior migration of QL and its descendents by transcriptionally activating the Hox gene mab-5. We found that mab-5 expression in QL and its descendents was attenuated in dpy-23 mutants and that a gain-of-function mab-5 mutation completely suppressed the QL migration defects of dpy-23 mutants, suggesting that dpy-23 acts upstream of mab-5. To determine where DPY-23 function is required, we expressed a dpy-23 cDNA in dpy-23 mutants from tissue-specific promoters. Expression in the HSN and Q descendents failed to rescue, while expression of DPY-23 in EGL-20 expressing cells rescued the Q defects and partially rescued the HSN defects, suggesting that DPY-23 primarily functions cell non-autonomously in Wnt-producing cells. We are now designing experiments to test whether DP-23 regulates Wnt secretion by recycling MOM-3, a transmembrane protein necessary for Wnt secretion.

Windy A. Boyd et al. (2007) International Worm Meeting "Using a C. elegans feeding response assay in high-throughput chemical and genetic screening."

Windy A. Boyd, Jonathan H. Freedman, Sandra J. McBride

[

International Worm Meeting,

2007]

Feeding in Caenorhabditis elegans is a complex behavior controlled by an integrated and well-regulated neuromuscular system. C. elegans feed via constant pharyngeal pumping while moving through the environment. Exposures to neuroactive compunds have been shown to affect feeding in C. elegans making this response a sensitive endpoint in toxicological testing. Previous feeding assays required microscopic examinations of individual nematodes, limiting the number of samples that could be observed. We present a method that uses COPAS Biosort flow cytometry technology to measure the size and fluorescence characteristics of hundreds of nematodes per minute. After chemical exposures, C. elegans are allowed to feed on red fluorescent microspheres such that accumulated red fluorescence is used as a measure of feeding response when compared to non-exposed control nematodes. To validate our system, we first compared the feeding responses in the C. elegans wild type N2 strain with 18 different Eat mutant strains that are known to express abnormal pharyngeal pumping. The Eat mutations that affected feeding most severely exhibited the lowest feeding responses with our assay. Next, we assayed two chemicals that either stimulate (serotonin) or uncouple pharyngeal pumping (arecoline) in the presence or absence of a food source. Concentration-dependent increases in feeding responses after serotonin exposures were dependent on food availability while feeding responses decreased in arecoline-exposed nematodes regardless of the presence of food. Using our feeding assay, the effects of more than 50 chemicals on the feeding response of C. elegans have been assessed. Cadmium and chlorpyrifos will be used to illustrate the effects of toxicants on C. elegans feeding. Cadmium chloride exposures led to a sharp drop in the feeding response above 200 mM. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 mM.

Juan Cueva et al. (2007) International Worm Meeting "The Nanoscale Relationship Between the MEC-4 Transduction Channel and the Extracellular Matrix Molecule, MEC-5."

Miriam Goodman, Juan Cueva

[

International Worm Meeting,

2007]

Mechanotransduction is the first step in hearing, pain, and touch. In C. elegans, displacement of the body wall opens MEC-4/degenerin transduction channels in touch receptor neurons (TRN). Touch sensation also requires components of a characteristic 15-protofilament (15-pf) microtubule bundle and extracellular matrix (ECM). Genetic interactions between the genes encoding the MEC-4 channel, 15-pf microtubules, and ECM led to the hypothesis that MEC-4 channel gating occurs in a tethered mode. Previously, we used novel antibodies against the MEC-4 channel subunits, MEC-2 and MEC-4, to establish that the MEC-4 channel is present intracellularly on the 15-pf microtubules and on the plasma membrane.1 The membrane-associated MEC-4 channels are distributed around the circumference of the TRNs and separated by >100 nm from the nearest points of contact between the 15-pf microtubules and the plasma membrane. Thus, the MEC-4 channels are not consistently associated with the 15-pf microtubules and unlikely to be tethered by them. However, the spatial relationship between membrane-associated MEC-4 channels and the ECM remains unclear. We tested the hypothesis that the MEC-4 channel is associated with the ECM molecule MEC-5 using post-embedding immunoelectron microscopy. First, we developed antibodies against the collagen-like molecule MEC-5. At the light level, MEC-5 antibodies label a single row of uniform puncta on the TRN. On average, MEC-5 puncta are 0.27 ± 0.07 mm (n = 142 puncta) long or less than half the size of the puncta labeled by antibodies against MEC-2 (0.75 ± 0.36 mm, n = 625 puncta) and MEC-4 (0.74 ± 0.32 mm, n = 399 puncta). In addition, the MEC-5 puncta are more numerous and more dense than either the MEC-2 or MEC-4 puncta. The labeling with the MEC-5 antibodies is specific since TRN neurites were not labeled in mec-5 null mutants. Second, we used the MEC-5 antibodies to label TRNs in 50 nm thin sections. MEC-5 antibodies were detected within the specialized electron-dense ECM between the TRN neurites and the surrounding hypodermal cell. Consistent with published genetic and molecular observations, the MEC-5 label, like the electron-dense ECM, was concentrated at the apical (cuticle) side of the neurite. These data argue that the MEC-4 channels and MEC-5 are co-expressed in the TRN neurite but may not interact physically. Thus, previously reported genetic interactions between mec-5 and mec-7 b-tubulin are unlikely to be mediated by a common complex incorporating MEC-5, the MEC-4 transduction channel, and the 15-pf microtubules. 1Cueva & Goodman, (2006) Neuronal Development, Synaptic Function, and Behavior, Topic Meeting #2, Madison, WI. Online.

Vera M. Hapiak et al. (2007) International Worm Meeting "TYRA-3 (M03F4.3) belongs to a novel family of invertebrate tyramine receptors."

Sarah B. Miller, Gareth P. Harris, Richard W. Komuniecki, Robert J. Hobson, Rachel T. Wragg, Vera M. Hapiak

[

International Worm Meeting,

2007]

Tyramine (TA) regulates a number of key processes in nematodes, including pharyngeal pumping, egg-laying, and locomotion, suggesting that TA-mediated signaling may provide useful targets for anthelminthic development, as described previously for insect OA/TA receptors. Previously, we characterized two C. elegans Gao-coupled TA receptors, SER-2 and TYRA-2. In the present study, we have identified a third C. elegans gene, tyra-3 (m03f4.3) that also encodes a TA receptor. TYRA-3 clusters most closely within a novel, recently described, family of TA-specific insect receptors that activate Ca++ dependent chloride channels after expression in Xenopus oocytes (Cazzamali et al., 2005). Membranes from COS-7 cells expressing TYRA-3 exhibit saturable [3H]LSD binding (Kd = 31.1 ± 4.7 nM) and, in inhibition binding assays, TA inhibits [3H]LSD binding with much higher affinity than other classical biogenic amines (IC50, 0.8 ± 0.01 mM). TYRA-3 exhibits a unique pharmacological profile and, in contrast to SER-2 and TYRA-2, couples to TA-dependent increases (3-5 fold) in cAMP levels after transient expression in COS-7 cells (EC50, 0.62 ± 0.12 mM). In contrast, OA has no effect on cAMP levels. tyra-3::gfp gene fusions exhibit an expression pattern that is markedly different from those reported for either tyra-2 or ser-2. tyra-3::gfp is robustly expressed in the CEP/ADE dopaminergic neurons and additional neurons in the head and tail, as well as the gonad and intestine. Interestingly, TYRA-3, but not SER-2 or TYRA-2, is essential for the TA-dependent abolition of 5-HT mediated increases in sensitivity to dilute octanol, implying a key role for TYRA-3 in olfactory modulation. However, the neuronal circuit and downstream signaling involved in TYRA-3-mediated modulation remains to be identified. These studies are continuing to examine the role of TYRA-3 in other TA-dependent phenotypes.

Diana E. Lamendola et al. (2007) International Worm Meeting "New roles for cation diffusion facilitator genes in the regulation of zinc metabolism in Caenorhabditis elegans."

Kerry Kornfeld, Diana E. Lamendola

[

International Worm Meeting,

2007]

Zinc is an essential trace element, and some organisms have been reported to be sensitive to both zinc deficiency and zinc excess. Mechanisms that regulate zinc metabolism and facilitate zinc homeostasis are not well defined in muticellular animals, and C. elegans is a promising model for the genetic dissection of this process. To characterize the response of C. elegans to changes in dietary zinc, we are using a completely defined liquid media (CeMM). Wild-type worms cultured in CeMM displayed a developmental delay in response to both low and high dietary zinc, demonstrating that C. elegans is sensitive to both zinc deficiency and zinc excess. Wild-type worms grew efficiently in a wide range of zinc concentrations (15-mM to 1-mM), suggesting that these animals have mechanisms for zinc homeostasis under these conditions. To investigate zinc homeostasis, we developed methods to measure the zinc content of worms. In response to dietary zinc concentrations ranging from 15-mM to 3-mM (200-fold difference), total zinc content of C. elegans varied over 125-fold. These results indicate that C. elegans exerts only a small degree of homeostatic control over total zinc content and suggests the possibility that additional homeostatic mechanisms may act at the level of zinc distribution within the animal. C. elegans has two genetically characterized genes (cdf-1 and sur-7) and two predicted genes (T18D3.3 and Y105E8A.3) that encode cation diffusion facilitator (CDF) zinc transporters. Loss of function mutations in cdf-1 and sur-7 were shown previously to influence growth in standard media supplemented with zinc. We demonstrated that these mutations caused hypersensitivity to high zinc when animals are cultured in CeMM, but these mutations did not affect growth in low zinc conditions. A deletion allele of the predicted T18D3.3 CDF gene also caused hypersensitivity to high zinc, demonstrating that a third CDF protein is necessary for zinc metabolism in C. elegans. To investigate the possibility that the three CDF genes have redundant functions, we constructed and analyzed double mutants. cdf-1(lf) sur-7(lf) double mutants displayed hypersensitivity to high dietary zinc that was more pronounced than either single mutant. T18D3.3(lf) sur-7(lf) double mutants also displayed enhanced hypersensitivity to high dietary zinc. In addition, this double mutant displayed hypersensitivity to low dietary zinc, a phenotype that was not displayed by either single mutant. Our findings indicate that three CDF genes are necessary for normal zinc metabolism, and these genes have partially redundant functions in both acquiring and excreting zinc from the animal.

Brendan C. Mullaney et al. (2007) International Worm Meeting "Genetic Analysis of Mechanisms of a Fat Reducing Compound."

Brendan C. Mullaney, Kaveh Ashrafi

[

International Worm Meeting,

2007]

We have found a number of compounds that cause fat reduction when administered to C. elegans. The molecular mechanisms through which these compounds exert their effects are not known. We combined pharmacology with genetics to identify pathways that mediate fat reducing effects LY294002, a PI3 kinase inhibitor. Animals exposed to 25 mM LY294002 are generally healthy and grow at similar rates to untreated animals but display reduced fat levels. By EMS mutagenesis we identified mutants that strongly suppress the fat reduction caused by LY294002. In one these suppressors, we found a mutation in an uncharacterized transporter, most similar to the mammalian GLUT family. Expression of the full-length transporter fused to GFP rescues the drug induced fat reduction phenotype cell autonomously. The full length GFP reporter was primarily localized to vesicles within intestinal cells. Treatment of transgenic animals with LY294002 caused elevated levels of transporter accumulation in these vesicles. In a complimentary RNAi screen, we found that mutations in this transporter suppressed the fat reduction caused by RNAi of rab-7, a small GTPase required for sorting of endocytic vesicles, suggesting that the role of this transporter in fat regulation is not limited to conditions of drug treatment. Accordingly, overexpression of this transporter caused fat reduction cell autonomously. Together, our results suggest animals regulate cellular localization and expression level of this transporter to modulate their intestinal fat levels. Using cell based assays, we are currently investigating potential substrates of this transporter.

Chan-Wei Yu et al. (2007) International Worm Meeting "Characterization and regulation of the C. elegans GCS-1 by arsenite."

Chan-Wei Yu, Vivian Liao

[

International Worm Meeting,

2007]

Arsenic is a potent toxin and carcinogen. We previously demonstrated that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress (Yu and Liao, 2005). Herein we investigate the regulation of GCS-1 in response to As(III). Our results showed that As(III) caused a concentration-dependent increase in the GSH levels up to a concentration of 10 mM. Real-time RT-PCR analysis shows that gcs-1 mRNA transcript is most abundant in L1, L2 and adult stages. Whereas GCS-1 mRNA at L1 and dauer stages exhibited higher induction ratio after 1 h of As(III) exposure. Moreover, we observed that the increase in GCS-1 mRNA expression level caused by As(III) shows dose-dependence fashion, in parallel with the increase in the GSH level. RNAi feeding assay showed that worms that were fed GCS-1 dsRNA and were grown in the presence of 0.25 mM As(III) were obviously small and development arrest. Transcription of gcs-1 was observed in the pharynx, ASI chemosensory neurons, and intestine of post-embryonic C. elegans and upon exposure to As(III), As(V), or Sb(III), the intestinal expression increases dramatically; whereas Cd(II) induced significant expression in the pharynx. Together, our results indicated that GCS-1 is developmentally regulated and it plays a critical role for the protection of C. elegans against arsenic-induced oxidative stress.

Marisa Foehr et al. (2007) International Worm Meeting "Dorsoventral patterning of the postembryonic mesoderm requires both LIN-12/Notch and TGF-beta signaling."

Marisa Foehr, Jun Liu

[

International Worm Meeting,

2007]

The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among lin-12, sma-9 and dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.

load 10 more results