V Raymond et al. (1999) International C. elegans Meeting "Actions of cholinergic anthelmintics and ivermectin on recombinant homomeric nicotinic acetylcholine receptors chicken a7 and C. elegans ACR-16"

NP Mongan, V Raymond, DB Sattelle

[International C. elegans Meeting, 1999]

Two homomer-forming nicotinic acetylcholine receptor subunits (nAChRs) with 47% identity in their amino acid sequences have been used to compare aspects of the pharmacology of expressed vertebrate and nematode nAChRs of defined molecular composition. Functional recombinant receptors were studied in Xenopus oocytes following nuclear injection of cDNA encoding either chicken a 7 or C. elegans acr-16 (1) nAChR subunits. Voltage-clamp electrophysiology was used to compare the actions of cholinergic anthelmintics on these recombinant homomeric nicotinic AChRs. Butamisole (an imidazothiazole), morantel (a tetrahydropyrimidine) and methyridine (a pyrimidine) were without agonist effects on either the chicken a 7 or the C. elegans ACR-16 in the range 10nM-1mM. However, for both receptors butamisole and morantel antagonised responses to acetylcholine (ACh) whereas at high concentrations (1mM), methyridine did not affect responses to ACh of either receptor. Oxantel, a tetrahydropyrimidine structurally related to morantel, was without agonist actions on ACR-16, but was an ACh antagonist. In contrast, it was found to have partial agonist actions on chicken a 7 at concentrations in the range 10microM-300microM. In agreement with a previous study (2), the macrocyclic anthelmintic compound ivermectin, which acts primarily on L-glutamate gated chloride channels, was found to enhance the amplitude of responses of chicken a 7 AChRs to ACh and nicotine. However, ivermectin did not potentiate the ACh induced responses of ACR-16 but diminished slightly ACh responses of the nematode ACR-16. This points to differences in the modulation of these similar homomeric nAChRs. This study has revealed differences in pharmacology of vertebrate and nematode recombinant nAChRs with respect to the action of cholinergic anthelmintics and ivermectin. Further studies employing chimeras and site-directed mutagenesis should increase our understanding of the molecular basis of these pharmacological differences and may eventually assist in the design of novel anthelmintics with enhanced selectivity. (1)Ballivet et al. (1996) J. Mol. Biol. 258, 261-269 (2)Krause et al. (1998) Mol. Pharmacol. 53, 283-294

Mongan NP et al. (1998) European Worm Meeting "Actions of cholinergic anthelmintics and ivermectin on an expressed homomeric nicotinic acetylcholine receptor of Caenorhabditis elegans."

Sattelle DB, Mongan NP, Raymond V

[European Worm Meeting, 1998]

Nicotinic acetylcholine receptors (nAChRs) mediate the fast actions of acetylcholine (ACh) at the neuromuscular junction and at nerve-nerve synapses in the central nervous system. The subunit composition of these pentameric receptors has been established for adult (a1(2)b1ge) and embryonic (a1(2)b1gd) skeletal muscle of vertebrates. In the nervous system, only two classes of subunit are recognized (a and b) but multiple isoforms of both exist (a2-9 and b2-4) (Hucho et al., 1996). Receptors containing a4b2 are abundant in the vertebrate brain and a3b4-containing nAChRs are present in the autonomic ganglia. A subset of vertebrate neuronal a subunits (a7-9) can form functional homomeric receptors in heterologous expression systems such as Xenopus oocytes. The a7 subunit has been exploited extensively for functional analysis of site directed mutants. The discovery in C. elegans of a homomer forming subunit, Ce21 (Ballivet et al., 1996), offers opportunities to compare function and pharmacology of nematode and vertebrate nAChRs. The C. elegans Ce21 a subunit homomer is activated by ACh and nicotine (Ballivet et al., 1996) and we have shown it is also strongly activated by the potent amphibian toxin, Epibatidine, which is a full agonist. Differences have been observed between the anthelmintic pharmacology of an expressed C. elegans heteromeric receptor (Fleming et al., 1997) and Ce21. Levamisole is an open channel blocker for Ce21, but has been shown to be an agonist for UNC-38+LEV-1+UNC-29 heteromers. Actions of several cholinergic anthelmintics including morantel, pyrantel and oxantel have been studied on both Ce21 and a7. Only oxantel was found to have an agonist effect. Of the others tested to date, all blocked responses to nicotine. Ivermectin (IVM), an anthelmintic which acts on L-glutamate gated chloride channels (L-GluRs) (Cully et al., 1994) was recently shown to act as a positive allosteric modulator of a7 homomers (Krause et al., 1998). IVM was also found to interact with Ce21. Comparison of nematode and vertebrate nicotinic receptor pharmacology may facilitate the design of novel pest control agents with enhanced selectivity. The authors are indebted to Dr. Marc Ballivet for the provision of Ce21 and a7 clones. Ballivet, M., Alliod, C., Bertrand, S., and Bertrand, D. (1996) J.Mol.Biol. 258, 261-269 Cully, D.F., Vassilatis, D.K., Liu, K.K.Paress, P.S., Van der Ploeg, L.H.T., Schaeffer, J.M. and Arena, J.P. (1994) Science 371 707-711 Hucho, F., Tsetlin, V.I. and Machold, J. (1996).Eur.J.Biochem. 239, 539-557 Fleming JT Squire MD Barnes TM Torn!e CT Matsuda K Sulston JE Barnard EA Sattelle DB & Lewis JT (1997) J Neurosci 17 5843-5857 Krause, R.M., Buisson, B., Bertrand, S., Corringer, P.J., Galzi, J.L., Changeux, J.P. and Bertrand, D. (1998) Mol.Pharmacol. 53, 283-294

Jennifer M Ross et al. (2002) Mid-west Worm Meeting "A role for the Polycomb Group in the development of the C. elegans male nervous system"

Jennifer M Ross, David A Zarkower

[Mid-west Worm Meeting, 2002]

Development of the C. elegans male-specific nervous system depends on interplay between two regulatory pathways, one involving the Hox genes mab-5 and egl-5, and the other involving the conserved sexual regulator mab-3. Males harboring Hox or mab-3 mutations lack V rays, sensory structures required for mating. The Hox proteins activate the bHLH gene lin-32 to specify the V rays, while the doublesex homolog MAB-3 synergizes with the Hox/lin-32 pathway to promote V ray differentiation. V rays can be restored in mab-3(-) males by overexpression of lin-32, suggesting that LIN-32 acts as a primary determinant of V ray fate, while MAB-3 potentiates LIN-32 activity.

Baird, Scott E. et al. (2011) International Worm Meeting "Ray Pattern Variation in C. elegans: Mapping a Major-Effect QTL on LGV."

Baird, Scott E., Bailey, Daniel

[International Worm Meeting, 2011]

Cryptic variation of ray pattern has been observed in recombinant-inbred lines (RIL) derived from crosses of N2 and CB4856 strains of C. elegans (Guess et al., 2007). The variant pattern consists of the anterior displacement of ray 3 into a position immediately adjacent to ray 2. QTL analyses of these RIL identified a major-effect QTL on the left arm of chromosome V (QTL-V). One RIL, QX34, possessed a recombinant chromosome V that contained CB4856 alleles from -12.72 to -7.93 cM, a region that precisely corresponded to QTL-V. When this chromosome (QX34-V) was crossed into an otherwise N2 background (strain PB2034), the variant ray pattern was observed at a frequency of 0.50. Based on genotypes of other RIL with recombination breakpoints in this region, it appeared that QTL-V resulted from allelic variation at two or more loci. To confirm this result, we are mapping the gene(s) in this region responsible for the observed variation in ray pattern. Initial experiments demonstrated that the CB4856 alleles responsible for QTL-V were recessive to the corresponding N2 alleles. From deletion heterozygotes, it was determined that at least one gene responsible for QTL-V lies to the right of -9.00 cM. However, deletion of a region from -12.06 to -8.15 did not uncover the ray pattern variant. Therefore, either allelic variation at two or more genes is required for QTL-V or the gene responsible for QTL-V resides between -8.15 and -7.93 cM. Attempts at recombination mapping of QTL-V are ongoing but have been hindered by an apparent suppression of recombination in PB2034/N2 heterozygotes.

Millard, J. et al. (2017) International Worm Meeting "Exploring the effects of the V-ATPase inhibitor luteolin on development, germline apoptosis, and FB-MO acidification in C. elegans."

Henderson, M., Millard, J., Dexter, K.

[International Worm Meeting, 2017]

The Vacuolar-type Adenosine Triphosphatase (V-ATPase) is a large multi-subunit enzyme complex found within intracellular and plasma membranes of animals, fungi, and plants. Contemporary research efforts have elucidated the role of V-ATPases in neoplastic maladies. Increased V-ATPase expression has been shown to support neoplastic metabolism and migration. The extensive involvement of V-ATPases in pathological conditions qualifies it as a suitable target for pharmaceutical intervention. Luteolin is a flavone found in a variety of plants, and is frequently consumed as a dietary supplement. Luteolin has been demonstrated to inhibit V-ATPase activity in mammalian cell culture. During spermatogenesis in C. elegans, the fibrous body-membranous organelles (FB-MOs) act as secretory vesicles responsible for the delivery of proteins to the surface of the sperm prior to fertilization. V-ATPases function to acidify the FB-MOs, contributing to their maturation to spermatozoa. Successful spermatogenesis is contingent on the fidelity of the V-ATPase in this acidification process. This change in pH can be detected by the use of LysoSensor Blue (Molecular Probes) staining. Disruption or inhibition of the V-ATPase complex is reliably reported by the LysoSensor staining assay. This allows for screening of potential V-ATPase inhibiting compounds such as luteolin. The toxicity of luteolin to C. elegans was demonstrated by growth and development assays. Examination of the effects of luteolin on germline apoptosis were performed using the ced-1::gfp reporter strain and acridine orange staining. Successful inhibition of FB-MO acidification supports the role of the V-ATPase in spermiogenesis as well as the potential therapeutic role of luteolin. These methods also provide an efficient medium for investigation of other V-ATPase inhibitory compounds.

Hediye Nese Cinar et al. (2007) International Worm Meeting "C. elegans: A model host for Vibrio cholerae and other Vibrio species."

Mahendra Kothary, Kivanc Bilecen, Robert Sprando, Ben D Tall, Fitnat Yildiz, Hediye Nese Cinar, Barbara McCardell

[International Worm Meeting, 2007]

Vibrio species are responsible for foodborne illnesses around the world, particularly in underdeveloped countries. Recent studies have shown that the human pathogens Vibrio cholerae (1) and Vibrio vulnificus (2) cause lethal infections in C. elegans. Using V. cholerae wild type strain A1552, we found that, in addition to the induction of the slow killing in adult worms, A1552 also killed larvae and caused escape behavior when killing assays were performed on brain heart infusion (BHI) agar plates. V. cholerae: OP50 mixed inoculation (1:10000) was as effective as the V. cholerae single inoculation in killing adults and larvae, indicating that the infectious dose of V. cholerae is very low. Furthermore, L4 stage worms which were exposed to a GFP-tagged V. cholerae strain for one hour and subsequently fed with OP50, exhibited gut colonization of V. cholerae which resulted in worm death similar to that observed in the continuous feeding experiments. To determine whether C. elegans prefers OP50 over V. choleare, 20 <font face=symbol>m</font>l of each bacterial culture were seeded on opposite sides of BHI plates and worms were placed at the midline. N2 adults were equally distributed to the OP50 and V. cholerae sides of the plate which suggests that they do not show a preference for OP50. However, worms on the OP50 side of the plate were located on the bacterial lawn while worms on the V. cholerae side tended to wander about and did not show a preference for the bacterial lawn. Once the OP50 lawn became contaminated with V. cholerae, which was carried through worms, all the worms on the plate adopted &#145;wandering about behavior. These observations suggest that C. elegans may sense V. cholerae as a harmful agent and possibly avoids it upon exposure. Alternatively, V. cholerae may have destroyed sensory processes in worms resulting in loss of their ability to locate the food. Other clinical isolates of V. cholerae are being tested in killing and avoidance assays. A V. cholerae transposon insertion library will also be screened to identify genes required for the observed phenotypes. These studies will be extended to other pathogenic Vibrio species. (1) Vaitkevicius K. et al. PNAS, 103 (2006) 9280-9285 (2) Dhakal BK et al. Biochemical and Biophysical Research Communications, 346 (2006) 751-757.

Glen Ernstrom et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Defining the Role of the V-ATPase in Neurotransmission"

Erik Jorgensen, Glen Ernstrom, Mark Palfreyman, Shigeki Watanabe

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

Neurotransmitter release depends on the fusion of neurotransmitter-loaded vesicles with the plasma membrane. Recent studies in yeast and Drosophila have implicated subunits of the Vo sector of the V-ATPase as acting at a late step of neurotransmitter release. The Vo sector is one half of a multi-subunit complex that functions as a proton pump when assembled with an ATP-hydrolyzing V1 sector. A proton gradient established by the V-ATPase is necessary for vesicular neurotransmitter loading, but the yeast and Drosophila studies implicate Vo subunits as having an additional role in vesicle fusion. We are investigating the role of V-ATPase by characterizing neuromuscular transmission in two C. elegans partial loss-of-function V-ATPase mutants: vha-12(n2915) disrupts the "B" subunit of ATP-hydrolyzing V1 sector of the V-ATPase, and unc-32(e189) eliminates a neural-specific isoform of the Vo "a" subunit. Using a vesicle-targeted pH-sensitive GFP, we find that both n2915 and e189 mutants have acidification defects. Interestingly, we find no reduction in the amplitudes of the miniature postsynaptic current events. We hypothesize acidification is inefficient in the V-ATPase mutants, but the mutant V-ATPase can eventually provide a gradient that allows neurotransmitter loading to proceed. Analysis of the frequency of postsynaptic events in the V-ATPase mutants reveals a reduction in the rate of fusion events in both V1 and Vo mutants. We are now testing a model in which partially acidified vesicles are not competent to fuse -- a checkpoint mechanism that is enhanced by hypomorphic mutations of the V-ATPase.

Yonju Ji et al. (2003) International Worm Meeting "The E subunit of vacuolar-type H+-ATPase (vha-8) is essential for embryogenesis and yolk transfer in C. elegans"

Yonju Ji, Joohong Ahnn, Kyu Yeong Choi

[International Worm Meeting, 2003]

Vacuolar H+-ATPases (V-ATPases) are ATP-dependent proton pumps composed of peripheral V1 sector and membrane-bound V0 sector. It is localized at membranes of intracellular acidic organelles and plasma membranes of various cell types. By virtue of its regulation in acidification, V-ATPase is required for many intracellular processes such as receptor-mediated endocytosis and protein sorting. We have characterized the E subunit of V-ATPase in C. elegans. This subunit is one of the most well conserved subunits sharing approximately 57% identity with the human homologue, ATP6E. Green fluorescent protein (GFP) and whole-mount immunostaining analyses showed that V-ATPase E subunit (vha-8) abundantly expresses in the H-shaped excretory cell, consistent with the expression patterns observed for other V-ATPase subunits. RNA mediated interference targeted to vha-8 resulted in embryonic and larval lethality indicating that vha-8 is essential during early developmental processes. Recently, we have isolated a deletion mutant of vha-8, which shows embryonic lethality. We are currently characterizing this mutant in order to elucidate the functional roles of V-ATPase during early embryogenesis as well as in receptor-mediated endocytosis.

Baer, Charles F. et al. (2013) International Worm Meeting "Revisiting the effects of spontaneous mutations on the (micro)environmental variance in Caenorhabditis."

Farhadifar, Reza, Baer, Charles F., Andersen, Erik C., Needleman, Daniel

[International Worm Meeting, 2013]

An obvious feature of living organisms is that their development is robust to variation in the environmental circumstances in which the organism finds itself - within limits. That is, the phenotype is more or less "canalized" (canalized "robust"). The "more or less" is of considerable interest to evolutionary biologists, for a variety of reasons, i.e., under what environmental or genetic circumstances does development become more or less canalized? Environmental canalization can be quantified as the phenotypic variation among genetically identical individuals raised in a uniform environment - the "(micro)environmental variance" in the lingo of quantitative genetics, V(E). Here we provide quantitative estimates of the effects of spontaneous mutations on V(E) for a variety of phenotypic traits subject to different selective regimes. Three general quantitative trends emerge. First, in almost all cases, mutation accumulation tends to de-canalize the phenotype (i.e., V(E) increases), typically at a rate similar to the rate of change of the trait itself. Second, there is a strong positive association between the rate of increase of V(E) for a trait and the mutational variance, V(M) for the trait itself. Third, and most intriguingly, mutations affecting V(E) for a trait appear to be usually under stronger selection than mutations affecting the trait itself.

Mallapur S et al. (1996) East Coast Worm Meeting "MAPPING THE BIOLOGICAL ACTIVITIES OF NETRIN UNC-6 TO SPECIFIC PROTEIN DOMAINS"

Wadsworth WG, Mallapur S

[East Coast Worm Meeting, 1996]

UNC-6 is a laminin related protein required for circumferential migrations of pioneering axons and mesodermal cells in C. elegans. Vertebrate homologs, the netrins, have been proposed to act as chemoattractants and chemorepellants for migrating axons in the embryonic nervous system. UNC-6 is predicted to be a secreted protein with five structural domains. The N-terminal domains VI and V (V-1,V-2,V-3) are homologous to laminin-g except small region of domain VI and domain V-2 that are homologous to laminin-b. The C-terminal, C domain, is unique. The homology of domain VI to laminin suggests that UNC-6 interacts with the basement membrane through this domain. Molecular analysis of unc-6 alleles suggests that specific functions are coded by different domains. For example, alleles that delete specifically domain V-2 affect only dorsal migrations whereas an allele carrying a mutation in domain VI selectively disrupts ventral migrations. Another allele that has a different mutation in domain VI disrupts mesodermal but not axonal migrations. We have suggested that the V-2 domain mediates binding to the UNC-5 receptor for dorsal migrations and that mutations in domain VI affects the display of UNC-6 on the face of the basement membrane. Our goal is to study structure-function relationships of UNC-6. Since several EGF-like repeats of laminin have been proposed to form binding sites for cellular receptors, the unc-6 domains V-1, V-2, V-3 might be the ligands for receptors on growth cones and migrating cells. Therefore, we have constructed synthetic alleles of unc-6 containing deletions of EGF-like repeats. We have also constructed an allele that has the V-2 domain replaced by domain V-3 to test whether domain V-3 can functionally substitute for domainV-2. If so, it may indicate that the spacing between EGF-like repeats is critical and not the sequence. We have constructed an allele with a deletion of C domain to examine its effect on axonal and mesodermal migrations. The C domain is rich in basic amino acids and may tether netrins to other molecules for display on the cell surfaces or it may bind to unique matrix sites. We will also test whether a distinct function is associated with the unique and highly conserved laminin-b like region of domain VI. For these experiments, we have designed an assay system that provides a strong selection for loss of function alleles. Our screen first measures the ability to rescue a select phenotype, either movement (dorsal guidance function) or egg laying (ventral guidance function). Further analysis details if other functions of unc-6 are affected. This study may discover new functions associated with specific domains of unc-6. Since netrins are highly conserved from nematodes to chordates and contain structures found in other extracellular matrix proteins, this analysis will provide insights into how such protein domains interact with their cellular receptors and the extracellular matrix.