Toshiaki Suzuki et al. (2003) International Worm Meeting "Analysis of Ce-PARKIN in C. elegans"

Nobutaka Hattori, Toshiaki Suzuki, Keiji Tanaka, Yoshikuni Mizuno, Shohei MItani, Noriyuki Matsuda, Hideki Yashiroda, Toshiaki Kitami, Keiko Gengyo-Ando

[International Worm Meeting, 2003]

Autosomal recessive juvenile parkinsonism (AR-JP) is one of the most common forms of familial parkinsons disease characterized by selective loss of dopaminergic neurons in substantia nigra and the locus coeruleus. parkin is the causative gene of AR-JP. The human parkin gene encodes 465 amino acids with a ubiquitin-like domain in the amino-terminus and two RING finger motifs in the carboxy terminus. So far, various deletion mutations and point mutations have been discovered in patients of AR-JP, suggesting that the loss of function of Parkin is the cause of AR-JP. Recently we and others showed that Parkin has a ubiquitin-protein ligase activity and therefore suggested that the defect of protein degradation in the neurons of AR-JP patients (Shimura H. et al. Nat. Genet. 25, 302-5, 2000). To investigate the function of Parkin in vivo, we began to analyze the Ce-PARKIN of C. elegans. Two deletion mutations in parkin genes show no defect in their viabilities. The expression of Ce-PARKIN seems to be specific to subset of neurons. Therefore, we focused on the function of Ce-PARKIN in the neurons and the analysis is underway.

Kuchma SL et al. (1996) Midwest Worm Meeting "PROGRESS TOWARDS THE CLONING OF smg-3."

Kuchma SL, Anderson P, Cali BM

[Midwest Worm Meeting, 1996]

We are using a positional cloning approach to clone smg-3, one of 7 smg genes required for the degradation of nonsense mutant mRNAs in C. elegans. Several lines of evidence indicate that smg-3 maps to a cosmid gap on LGIV. Our current strategy for cloning smg-3 employs a genomic walk into the cosmid gap described below. smg-3 was localized to a gap between the cosmids T13A10 and F15B10 in the unc-44 to deb-1 physical map interval using an RFLP mapping approach (see Cali and Anderson, abstract #71, 1993 International Worm Meeting). The T13A10/F15B10 cosmid gap is bridged by a single 600 kb YAC, Y73B6. Positioning of smg-3 to the T13A10/F15B10 cosmid gap was confirmed by transformation rescue experiments. Y73B6, which spans and extends to the right of the T13A10/F15B10 gap has smg-3(+) activity. A circularized derivative of Y73B6 containing only the C. elegans genomic DNA from T13A10 to F15B10 was isolated. This derivative, TR#242, is approximately 250 kb and has smg-3(+) activity. Y48F8, which extends into but not across the cosmid gap does not rescue smg-3 mutants. Thus, smg-3 is not wholly contained on Y48F8. Since smg-3 is in the cosmid gap to the right of the Y48F8 right endpoint, a chromosome walk from the right end of Y48F8 was initiated. For this walk, we are using an unamplified lambda library constructed from yeast strains carrying Y48F8 and TR#242 (A. Davies, pers. comm.). Genomic clones obtained from our walk were shown by restriction and Southern blot analyses to overlap with a lambda clone contig isolated by Andrew Davies as part of a genomic walk from T13A10 rightward. Thus, these clones define the right end of a new lambda contig which extends from T13A10 rightward for approximately 55 kb (8 kb beyond the right end of Y48F8). Both gut-4 and bli-6 , which are separated by 0.08 map units (A. Davies and J. Shaw, abstract #177, 1995 International Worm Meeting) are contained on this 55 kb lambda contig (A. Davies, pers. comm.). 3-factor mapping data suggests that smg-3 is tightly linked to bli-6 (of 13 recombinants in the bli-6 to unc-24 interval, no recombinants between smg-3 and bli-6 were isolated). These data suggest that smg-3 is close to the right end of Y48F8. We are testing clones isolated from this walk in transformation rescue experiments as well as in Southern blot analyses to detect potential allele-specific polymorphisms of smg-3 mutants.

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.

Jungsoo Lee et al. (2007) International Worm Meeting "Chronic effects of Ethanol in C. elegans."

Steven L. McIntire, Jungsoo Lee

[International Worm Meeting, 2007]

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Several mutants are also resistant to acute effects of ethanol on locomotion and egg laying. Some of the mutants have a defect in the acute tolerance that occurs during a continuous ethanol exposure. None of the mutants have an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We have mapped two mutants to the center of chromosome X and are currently in the process of cloning these genes. Reference: Diamond I and Gordon AS (1997). Cellular and molecular neuroscience of alcoholism. Physiol Rev. 77(1):1-20. Review Davies AG et al. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115(6):655-66.

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.

John J Yochem et al. (2003) International Worm Meeting "The molecular identities of sym-3 and sym-4, two genes that are synthetically lethal with mec-8"

John J Yochem, Leslie R Bell, Robert K Herman, Jocelyn E Shaw

[International Worm Meeting, 2003]

Loss-of-function mutations in mec-8, which encodes a putative RNA-binding protein that is required for alternative RNA splicing of the unc-52 gene, confer mechanosensory and chemosensory defects but do not cause extensive lethality. The combination of a mec-8 loss-of-function mutation, however, with a viable mutation in any of five sym (synthetic lethal with mec-8) genes (or with a viable allele of unc-52, the prototype analysis for this study) results in highly penetrant embryonic or early larval lethality. The molecular identities of sym-1 and sym-2 have been previously described [Davies et al. (1999) Genetics 153: 117; Yochem et al. (2001) IWM abstract #1090].The identities of sym-3 and sym-4 are now known. sym-3 corresponds to C54H2.1, which encodes a protein of unknown function. The amino-terminal half is highly similar to a protein predicted from the Drosophila and human genome projects. The sym-3(mn618) mutation is in the final splice site. sym-4 corresponds to R03E1.1, which encodes a large protein with WD repeats. The sym-4(mn619) mutation changes a leucine to a glutamine in the third WD repeat. The carboxyl-terminal half of SYM-4 contains the WD repeats and resembles a WD protein of unknown function in Drosophila and humans. The third WD repeats of these proteins, including the leucine that is altered by mn619, are especially well conserved. The phenotype of the mec-8;sym-3 double mutant closely resembles that of the mec-8;sym-4 double. The mutants arrest growth as either late-stage embryos or L1 larvae. In either case, the pharynx, which is overtly normal in morphology, is not attached to the outer cuticle, and the larvae therefore cannot feed. The similarity in phenotype raises the possibility that SYM-3 and SYM-4 act in a pathway that is redundant with a pathway that requires MEC-8 for proper splicing of one of its components. Consistent with this hypothesis, the sym-3(mn618) sym-4(mn619) double mutant is indistinguishable from either single mutant [Davies et al. (1999)].

Mancuso, Vincent P. et al. (2011) International Worm Meeting "Extracellular leucine-rich repeat proteins and maintenance of epithelial integrity."

Poggioli, Corey, Hall, David, Storer, Luke, Parry, Jean, Sundaram, Meera, Mancuso, Vincent P.

[International Worm Meeting, 2011]

Elron (extracellular Leucine-Rich Repeat only) proteins are known to have roles in synapse formation, axon guidance, and cell-cell adhesion. Here we describe distinct roles for the two transmembrane Elron proteins K07A12.2 and LET-4/SYM-5/C44H4.2 in C. elegans epithelial cells. Both K07A12.2 and let-4 single mutants have lethal defects in embryonic elongation and excretory system function. In the low-penetrance embryonic phenotype, epidermal cell morphology appears normal until embryonic elongation, at which point epidermal lesions form. This suggests a role for K07A12.2 and LET-4 in the maintenance of epidermal cell integrity during the stress of elongation. Another transmembrane Elron, SYM-1, acts redundantly with LET-4 during elongation (Davies et al., 1999). Both K07A12.2 and LET-4 GFP fusion reporters localize to the apical face of epidermal cells during elongation, suggesting an interaction with the embryonic sheath. Individuals that do not have the embryonic elongation defect develop a lethal excretory system defect. In the excretory system defect phenotype, both K07A12.2 and let-4 single mutants accumulate fluid in the excretory system and die as fluid-filled L1s. The excretory system includes three tandem unicellular epithelial tubes: the excretory canal cell, the duct cell, and the pore cell. Although both K07A12.2 and LET-4 are required for a functioning excretory system, each functions in a different aspect of the excretory system. K07A12.2 mutants lack a pore cell autojunction, though it is currently unknown whether it fails to form or is lost during development. This phenotype suggests a role for K07A12.2 in initial formation of the pore cell, or in maintenance of the tube shape. A failure of either process could explain the excretory system defect. In let-4 mutants, the pore autojunction is unaffected, but the duct cell lumen becomes distorted and discontinuous just before hatch, indicating a role for LET-4 in lumen integrity. LET-4::GFP is localized to the luminal (apical) side of the duct cell, and may interact with the extracellular matrix inside the lumen of the duct and pore cells. This work suggests roles for Elron proteins in mediating interactions between epithelia and the apical extracellular matrix to help maintain cell shape and integrity. Davies AG, et al. (1999) Genetics 153, 117-134.

Michael Butterfield et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "The Effects of Ethanol Exposure on Short-Term and Long-Term Memory in C. elegans"

Michael Butterfield, Catharine Rankin

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Ethanol has been shown to have inhibitory effects on memory in many mammalian systems. Previously, our lab has demonstrated, using a distributed training protocol, that C. elegans is capable of long-term memory for habituation (>24 hours). The current studies examined whether ethanol exposure would affect long-term memory by exposing worms to various concentrations of ethanol (0.2M, 0.4M and 0.6M; as originally described in Davies et al., 2003) during training. Worms that were given habituation training 24 hours earlier without ethanol (0.0M) showed significantly smaller responses than the control group that did not receive any habituation training (p<0.0001), indicating long-term memory. When exposed to a low dose of ethanol (0.2M) during training worms continued to show a significant difference (p<0.05) between trained and control groups 24 hrs later. However, when exposed to higher doses (0.4M, p=0.7235 and 0.6M, p=0.9657) worms showed no significant difference in responses between control and trained suggesting that at higher doses ethanol exposure may interfere with memory formation. Interestingly, within the first training block there was a dose dependent effect of ethanol on the kinetics of habituation with the higher doses inducing a slower rate of habituation. Because of this observation we examined the effects of 0.4M ethanol on short-term habituation. We found that worms that were exposed to 0.4M ethanol for an hour and then given 30 stimuli at a 10 s ISI showed normal short-tem habituation, however worms that were given stimuli at a 60 s ISI showed much less habituation than worms tested without ethanol. Using effects of ethanol on locomotion as a screen Davies et al., 2003 found that mutations in the BK potassium channel encoded by the slo-1 gene mediated the effects of ethanol on behavior. When we tested short-term habituation in slo-1 (recieved from CGC) worms we found that habituation was normal at a 10 s ISI and there was no evidence of habituation at a 60s ISI. These data suggest that the effects of ethanol on short and long term habituation in C. elegans may be mediated by slo-1.

P. Mitchell et al. (2006) European Worm Meeting "Evidence that Ethanol Equilibriates Rapidly Across C. elegans Cuticle"

L. Holden-Dye, N.A. Hopper, K. Bull, V. O'Connor, P. Mitchell, S. Glautier

[European Worm Meeting, 2006]

P. Mitchell, K. Bull, N.A. Hopper, S. Glautier1, L. Holden-Dye, V. O''Connor Ethanol has concentration-dependent effects on neural function. At concentrations in the low mM range, equivalent to the drink-driving limit, it has an intoxicating action, which is often associated with a transient excitability, followed by inhibition. At higher concentrations, greater than 100 mM, it has an anesthetic action and elicits muscle paralysis. C. elegans has been used to define the genetic basis for behavioural responses to ethanol exposure. In these studies intact animals have been exposed to 500 mM ethanol with the prediction that the internal concentration is more than ten-fold lower and therefore equivalent to an intoxicating, rather than an anesthetic, dose (Davies and McIntire, 2004). In order to underpin further studies, we have analysed the relationship between the external and internal ethanol concentration of C. elegans. Immersion of C. elegans in a sealed chamber containing ethanol (range 100 mM to 500 mM) elicits an inhibition of thrashing rate. Inhibition reaches a steady-state value within 5 min. This effect is concentration-dependent (half-maximal effect at 300 mM). At all concentrations of ethanol a steady-state inhibition is observed within 5 min. The animals rapidly recovered when removed from ethanol. Notably, this recovery was complete within 2.5 min. This suggests that ethanol may rapidly equilibriate across the cuticle and that the internal concentration that is required to inhibit motility approximates to the external concentration. In order to directly assay the effects of ethanol on muscle we used the pharyngeal muscle preparation. In this assay the muscle is exposed and therefore the concentration of ethanol is known. This muscle is inhibited by ethanol at concentrations in excess of 100 mM. Although it could be argued that the pharyngeal muscle behaves in a different fashion to body wall muscle in terms of ethanol sensitivity, the results support the contention that concentrations in excess of 100 mM are required to inhibit muscle activity. Internal ethanol concentrations in C. elegans following exposure to ethanol have been estimated using a biochemical assay kit. However, this procedure requires that the animals are briefly washed in cold buffer prior to the measurement. Our observation that animals exposed to ethanol fully recover from the inhibition of motility within 2.5 min suggests that a significant amount of ethanol may be lost from the inside of the animals during the protocol. We tested this by determining the influence of the wash volume on the apparent estimate in internal ethanol concentration. This shows that the estimate of internal ethanol concentration increases as the volume of the wash buffer decreases. This suggests that the measurement of internal ethanol concentration using this method underestimates the true internal concentration.. Davies, A.G. and McIntire, S.L. 2004, Biol. Proced. Online 6, 113-119.. Funded by the BBSRC, UK

Hongkyun Kim et al. (2001) International C. elegans Meeting "A Genetic Analysis of the Effects of Ethanol on Egg Laying"

James Kim, Hongkyun Kim, Steven L McIntire

[International C. elegans Meeting, 2001]

Ethanol causes dose-dependent suppression of egg laying in C. elegans . Quantification of tissue ethanol concentrations from C. elegans after exposure to different exogenous doses suggests that suppression of egg laying occur at doses relevant to human intoxication. To elucidate the molecular targets of ethanol, we screened for mutants showing ethanol-resistant or -inducible egg-laying behavior. We completed a screen of 7,500 haploid genomes and identified 20 candidate ethanol resistant mutants. In this screen we isolated seven slo-1 alleles (see also abstract by Davies, Thiele, and McIntire). slo-1 encodes a calcium activated potassium channel (personal communication, Nonet and Salkoff). There is in vitro evidence for an activating effect of ethanol on vertebrate slo channels (Dopico et al. 1999 Neurochem. Int. 35:103-106). We are presently characterizing additional ethanol resistant mutants. Several of the resistant mutants show ethanol-inducible egg laying behavior at relatively low exogenous ethanol concentrations that have a weak inhibitory effect on egg laying in N2 animals. We have therefore screened for mutants laying eggs without food in the presence of low concentrations of ethanol. Thus far we have completed a screen of 4,000 haploid genomes and identified 11 candidates. This screen has generated a class of ethanol-inducible egg laying mutants as well as a class of mutants exhibiting egg-laying independent of food and ethanol. Further mapping and molecular characterization of these mutants will be presented.