da Silva AF et al. (2022) Neurotoxicology "JM-20 affects GABA neurotransmission in Caenorhabditis elegans."

Arantes LP, Soares MV, Nunez-Figueredo Y, Cordeiro LM, da Silveira TL, Soares FAA, Baptista FBO, Machado ML, Zamberlan DC, da Silva AF, Rodriguez EO

[Neurotoxicology, 2022]

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodiazepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a promising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the development of worms after 48h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM-20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, computational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model.

Killeen M et al. (2002) Dev Biol "UNC-5 function requires phosphorylation of cytoplasmic tyrosine 482, but its UNC-40-independent functions also require a region between the ZU-5 and death domains."

Culotti J, Pawson T, Tong J, Steven R, Killeen M, Krizus A, Scott I

[Dev Biol, 2002]

Members of the UNC-5 protein family are transmembrane receptors for UNC-6/netrin guidance cues. To analyze the functional roles of different UNC-5 domains, we sequenced mutations in seven severe and three weak alleles of unc-5 in Caenorhabditis elegans. Four severe alleles contain nonsense mutations. Two weak alleles are truncations of the cytodomain, but one is a missense mutation in an extracellular immunoglobulin domain. To survey the function of different regions of UNC-5, wild-type and mutant unc-5::HA transgenes were tested for their ability to rescue the unc-5(e53) null mutant. Our data reveal partial functional requirements for the extracellular domains and identify a portion of the cytoplasmic juxtamembrane (JM) region as essential for rescue of migrations. When nine cytodomain tyrosines, including seven in the JM region, are mutated to phenylalanine, UNC-5 function and tyrosine phosphorylation are largely compromised. When F482 in the JM region of the mutant protein is reverted to tyrosine, UNC-5 tyrosine phosphorylation and in vivo function are largely recovered, suggesting that Y482 phosphorylation is critical to UNC-5 function in vivo. Our data also show that part of the ZU-5 motif is required for UNC-40-independent signaling of UNC-5.

Hartman PS et al. (1995) International C. elegans Meeting "UV RADIATION INHIBITS CELL DIVISION IN THE P1 LINEAGE MORE THAN IN THE AB LINEAGE"

Buckles D, Hartman PS

[International C. elegans Meeting, 1995]

Two-cell embryos were irradiated with germicidal (254nm) radiation and subsequent cell divisions were determined using Nomarski DIC microscopy. A dose of 30 Jm-2 (which gave ca. 10% survival) delayed P1 and P2 divisions by 27%, versus only 8% for the equivalent AB divisions. Similarly, although exposure to 150 Jm-2 abolished cytokinesis in some AB descendants (resulting in occasional binucleated cells), the next two cell cycles were only 22% longer than in unirradiated controls. Conversely, the P1 and P2 cell cycles averaged 95% longer. The delays in the EMSt, E, and MSt cell cycles were similar to those in the AB lineage, suggesting the longer delays were limited to the P4 progenitors. "Cell-cycle checkpoints" have been documented in other organisms. These afford additional time for restitution of DNA damage. Such checkpoints are apparently largely inoperative in the AB lineage (which generates soma exclusively) but at least partially functional in the cells which give rise to the germ line.

A Smardon et al. (1999) International C. elegans Meeting "EGO-1, required for germline development, is related to plant rna-directed rna polymerases"

EM Maine, JM Spoerke, N Mackin, A Smardon, SC Steven

[International C. elegans Meeting, 1999]

ego-1 has been described as a gene required for various aspects of germline development, including efficient DTC-to-germ line signaling (Qiao et al. '95). More recently, we reported the cloning of what appeared to be a distinct, although very tightly linked gene, ego-6 (Smardon et al. '98 WBG). Based on molecular studies, we can now say that ego-1 and " ego-6 " are in fact the same gene. The loss of ego-1 gene function is associated with premature meiosis, occasional abnormal nuclei in the distal (mitotic) region, an expanded mitosis-to-pachytene transition zone, defects in gametogenesis, and a slight reduction in germ cell numbers. Identified by their ability to enhance a mild glp-1(ts) phenotype in the germ line, ego-1 alleles also enhance a mild lag-1(ts) phenotype in the germ line. ego-1 was placed on the physical map by identification of a deletion associated with ego-1(om84) . This deletion disrupts a gene who structure subsequently was determined by recovery and sequencing of cDNAs and RT-PCR products. The gene includes 15 exons and is trans-spliced to SL1. RNAi and mutation analyses confirmed the identity of this gene as ego-1 . It is predicted to encode a 1632 amino acid protein with limited homology to the products of several other eukaryotic genes (see below). Two strong mutant alleles, om84 and om97 , contain extremely premature stop codons and thus are likely to be null. The weaker om71 allele contains a Pro-Ser substitution at position 930; this residue is conserved among all known EGO-1 family members. A single ego-1 transcript is detected in L4 and adult animals. It is present predominantly, if not entirely, in the germ line. Anti-EGO-1 antibodies are being generated to examine the protein expression pattern. EGO-1-related proteins include a set of RNA-directed RNA polymerases recently identified in tomato and four other plant species (Schiebel et al. '98). Their limited homology to EGO-1 may indicate a common domain, such as one that binds RNA, rather than conserved overall function. Functions of the three ego-1 paralogs are being examined using RNAi. Qiao et al. (1995) Genetics 141, 551-569; Smardon et al. (1998) WBG 15, 31; Schiebel et al. (1998) Plant Cell 10, 2087-2102.

Svendsen BA et al. (1988) Worm Breeder's Gazette "effects of UV radiation on embryonic DNA synthesis and cell division (subtitle: tough guy polymerases from the worm?)."

Reddy J, Hartman PS, Svendsen BA

[Worm Breeder's Gazette, 1988]

Synchronous populations of young embryos were exposed to various fluences of 254 radiation and incubated in the presence of tritiated thymidine. At selected times, samples were processed to determine the effects of irradiation on DNA synthesis (measured as the incorporation of tritium into TCA precipitable material) and cell division [measured by counting nuclei a la Gossett and Hecht (1980. J. Histochem. Cytochem. 28, 507-510)]. Despite the fact that over 50% of embryos failed to develop into adults after fluences of 25 Jm-2 or greater, they displayed control (unirradiated) levels of both cell division and replication at fluences of up to 250 Jm-2. This resistance was not due to UV light attenuation, since both parameters were strongly inhibited in two UV radiation hypersensitive mutants (rad-1 and rad-3) by fluences of 10 Jm-2 and greater. Other systems (e.g., bacteria, mammalian cells, yeast) are much more sensitive to the effects of UV radiation on DNA replication. Analysis of the sizes of newly synthesized DNA in irradiated and unirradiated embryos may provide an explanation for this extreme radiation resistance. Synchronous embryos were irradiated, pulse labeled with tritiated thymidine, and chased for various time intervals. After lysis with proteinase K and SDS, samples were subjected to centrifugation through alkaline sucrose gradients. Inclusion of [14C]-labeled T4 and T7 DNA's allowed determination of molecular weights. Fluences of up to 270 Jm-2 failed to reduce the molecular weight of newly synthesized DNA relative to unirradiated controls. This was unexpected, since damage to the parental strand in other organisms suppresses the size of nascent molecules by preventing elongation of DNA polymerase. It can be calculated, based upon the induction frequency of cyclobutane dimers as well as the size of the newly synthesized DNA, that greater than ten cyclobutane dimers were induced in the template opposite each nascent fragment. This leads to the provocative possibility that a DNA polymerase from C. elegans is capable of trans-lesion synthesis through radiation-induced DNA damage. These data are in keeping with the observation, described in the preceding paragraph, that DNA synthesis and cell division during embryogenesis are refractory to super-lethal doses of UV radiation.

PW Faber et al. (1999) International C. elegans Meeting "Analysis of polyglutamine-mediated cellular dysfunction in C. elegans."

B Westlund, ME MacDonald, JM Spoerke, AC Hart, JR Alter, PW Faber

[International C. elegans Meeting, 1999]

Huntington's Disease (HD) is one of 8 dominant human neurodegenerative disorders caused by expansion of an unstable CAG-trinucleotide repeat encoding polyglutamine (polyQ). How expanded polyQ stretches cause disease is unknown. We generated a C. elegans model for polyQ-mediated cellular dysfunction by expressing fragments of huntingtin, the protein product of the HD locus, containing polyQ stretches of 2, 23 (control) or 95 and 150 (toxic) residues in the ASH sensory neuron. Expression of Htn-Q150, but not Htn-Q2, Htn-Q23 or Htn-Q95 causes an ASH dye-filling defect, but no ASH cell death, in 8-day-old animals. Despite the absence of cell death this dye-filling defect is dependent on ced-3 function, suggesting that Htn-Q150 activates the apoptotic cell death pathway. Htn-Q150 coexpression with subthreshold levels of a second toxic (but non-apoptotic!) transgene, an OSM-10::GFP fusion protein, causes enhanced dye-filling defects and cell death in 3- and 8-day-old animals. This cell death depends on ced-3 function, supporting the notion that Htn-Q150 activates the apoptotic cell death pathway. Htn-Q150 and OSM-10::GFP coexpression (but neither alone) leads to a strong defect in nose touch response (Faber et al., 1999 PNAS 96, 179-184). Our current efforts include 1) Targeting Htn-Q150 expression to other cell types to create phenotypes more amenable to high throughput screens. 2) Screening (10,000 animals to date) for recessive enhancers of the Htn-Q150 mediated ASH dye-filling defect in 3d animals (unenhanced rt13 ) and one integrated array ( rtIs12 ) that maintain their enhancing potential in 4x backcrossed (4x) animals. Another putative enhancer has just been isolated. rt13 enhancement is array dependent; 79% (n = 160) and 1% (n = 150) of rt13 (4x) ASH neurons are dye-filling defective at 3d in the presence or absence of an Htn-Q150 array, respectively. Importantly, two new Htn-Q150 arrays generated in rt13 (4x) animals by microinjection reproduce the ASH dye-filling defect of 73% (n = 162). Further analysis of rt13 is underway.

Hartman PS (1984) Worm Breeder's Gazette "effects of age and liquid holding on the UV radiation sensitivities of N2 and radiation-sensitive (rad) dauer larvae."

Hartman PS

[Worm Breeder's Gazette, 1984]

Wild-type (N2) and four radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, were not hypersensitive. Irradiation also resulted in a significant delay in development. For example, over 95% of the unirradiated N2 dauer larvae completed development within three days of food presentation; however, fluences of 78 Jm-2 and 156 Jm-2 resulted in average delays of approximately 40 and 50 hours, respectively. In fact, higher fluences resulted in developmental periods as long as 7.5 days. Brood sizes decreased in a fluence-dependent fashion; however, most N2 survivors which were delayed in development for up to four days still produced greater than 10 offspring per animal. The fact that dauer larvae do not resume development until placed in the presence of food was exploited to test two aspects of their radiation response. First, the effect of dauer age on radiation sensitivity was examined by irradiating N2 dauer larvae at 0, 25 or 50 days after harvesting. Animals were collected and either irradiated immediately or incubated in PBS for 25 or 50 days before irradiation. There were no significant differences in radiation sensitivities. Second, the effect of post irradiation holding on survival was tested on N2 dauer larvae. Animals were irradiated with either 116 or 194 Jm- 2 and incubated in a non-nutritive medium (PBS) for various intervals before exposure to food. Liquid holding recovery (LHR) refers to the increased survival observed in many instances when irradiated organisms are held in a nonnutritive medium before plating. Genetic and biochemical evidence in both bacteria and yeast indicate that increased survival can be attributed to DNA repair that occurs during holding. No liquid holding recovery (LHR) occurred at either fluence; in fact, survival decreased after approximately five days of post- irradiation holding. In addition, no differences were observed in the amount of developmental delay imposed by radiation. These results imply that this developmental state is not a period of active DNA repair.

Suzuki K (1980) Worm Breeder's Gazette "radiobiological properties of a strain of free-living nematode, R Tokai."

Suzuki K

[Worm Breeder's Gazette, 1980]

For the last one year, we have investigated radiobiological properties of the strain of nematode reported in J. Exp. Geront. 13: 323 (1978). When eggs were irradiated with ultraviolet light (UV), hatchability was reduced to about 10 per cent of that of unirradiated control eggs at 100 Jm(-2). It was also found that during the process to hatching there is a stage exhibiting the maximum UV-sensitivity. In order to analyze this stage, we have visualized the whole process of development by 16 mm cine-film. The work is now in progress. Larvae and mature nematodes were much more resistant to UV than eggs. For example, the average life span of 10 day-old virgin females was reduced to 32 per cent of that of control nematodes at 400 Jm(-2). The nematode was also quite resistant to X-rays. Fluence of 4 krads did not shorten the life span significantly. Even at 60 krads, the life span was shortened by about 10 days as compared to that of unirradiated control animals (60 days at 30 C). Different from the case of UV-irradiation, however, it was observed that various kinds of abnormally shaped adults were produced among X-irradiated populations when worms were irradiated in a larval stage. The results led us to an expect that the nematode could be used as an experimental animal for screening teratorogenic agents including radiations. It was also found that strand breaks in DNA produced by X-irradiation are efficiently repaired as evidenced by the sedimentation profiles in sucrose density gradients.

Klosterman S M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Regulation of tomosyn (TOM-1) trafficking in C. elegans"

Klosterman S M, Richmond J E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Regulated exocytosis of secretory vesicles is critically dependent on the assembly of SNARE complexes between the plasma membrane SNAREs, synatxin and SNAP-25 and vesicle-associated synaptobrevin, which render vesicles fusion competent. Tomosyn, a syntaxin-binding protein, forms a complex with syntaxin and SNAP-25 in competition with synaptobrevin predicted to limit fusogenic SNARE complex formation (Fujita et al, 1998). Consistent with these data, the highly conserved C. elegans tomosyn homolog, TOM-1, has been shown to negatively regulate both synaptic and dense-core vesicle release (Gracheva et al., 2006; Gracheva et al.,2007). TOM-1 appears to be associated with both synaptic vesicles (Takamori et al., 2006) and dense core vesicles (Gracheva et al., 2007) via an unknown mechanism. Consequently, the synaptic localization of TOM-1 is dependent on the anterograde vesicle kinesin motor (UNC-104) (McEwen et al., 2006). Given the importance of TOM-1 in the regulation of synaptic strength, we are interested in identifying the mechanism responsible for the synaptic localization of this protein. Neither syntaxin nor SNAP-25, the two established tomosyn-interacting proteins appear to be involved in TOM-1 transport to synapses (McEwen et al., 2006). In order to screen for TOM-1 trafficking defective mutants, we have generated an integrated transgene expressing TOM-1::GFP. We have verified that this protein is expressed at synapses and localizes in an UNC-104-dependent manner. We are currently screening known vesicle-associated candidate proteins by RNAi or by crossing the TOM-1::GFP transgene into reference alleles to examine their potential role in the regulation of tomosyn trafficking. Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, YokoyamaS, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH, Takai Y (1998) Tomosyn: asyntaxin-1-binding protein that forms a novel complex in the neurotransmitterrelease process. Neuron 20:905-915. Gracheva EO, Burdina AO, Touroutine D, Berthelot-Grosjean M, Parekh H,Richmond JE (2007) Tomosyn negatively regulates CAPS-dependent peptide releaseat Caenorhabditis elegans synapses. J Neurosci 27:10176-10184. Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD,Hadwiger G, Nonet ML, Weimer RM, Richmond JE (2006) Tomosyn inhibits synapticvesicle priming in Caenorhabditis elegans. PLoS Biol 4:e261.McEwen JM, Madison JM, Dybbs M, Kaplan JM (2006) Antagonistic regulationof synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51:303-315. Takamori S et al. (2006) Molecular anatomy of a trafficking organelle.Cell 127:831-846.

Chuang, Chien-Hui et al. (2017) International Worm Meeting "ZYG-9 and TAC-1 are involved in early meiotic spindle assembly in C. elegans oocytes."

Bowerman, Bruce, Chuang, Chien-Hui

[International Worm Meeting, 2017]

Oocytes of many animals form bipolar spindles in the absence of centrosomes, and how these acentrosomal spindles establish bipolarity remains unclear. In C. elegans oocyte meiosis I, spindle microtubules first form a cage-like structure peripheral to the chromosomes, with multiple small and peripheral spindle poles appearing as assembly progresses. These small poles later merge together and ultimately form two mature poles on the opposite sites of the aligned chromosomes (Wolff et al., 2016). Previous studies have documented defects in oocyte meiotic spindle structure in ZYG-9 or TAC-1 depleted oocytes (Matthews LR et al., 1998; Bellanger JM and Gonczy P, 2003; Yang et al., 2003), but when these assembly defects first appear is not known. Using live imaging, we are investigating the temporal requirements for ZYG-9 and TAC-1 during meiotic spindle assembly. Here we show that ZYG-9 and TAC-1 are required very early in spindle formation, with defects that then further interfere with the establishment of spindle bipolarity. Using GFP and mCherry fusions to mark microtubules and chromosomes, respectively, in zyg-9 (-) oocytes, we have detected defects very early in meiotic spindle assembly. Rather than forming a cage-like structure with all microtubules restricted to the periphery, we instead observed a cage-like structure with some microtubules extending across the interior, projecting through the volume occupied by chromosomes. This abnormal microtubule scaffold subsequently assembled into a multi-polar spindle network, with individual bivalents sometimes surrounded by discrete small bipolar spindles. Ultimately chromosomes often segregated toward more than two poles. Similarly, using GFP fused to ASPM-1 to mark spindle poles, we found that wild-type oocytes had two stable ASPM-1 foci established by metaphase, while zyg-9 (-) oocytes had multiple ASPM-1 foci that dynamically coalesced and dispersed throughout most of meiosis I. ZYG-9 and TAC-1 form a complex that promotes microtubule assembly (Bellanger JM and Gonczy P, 2003; Le Bot N et al., 2003; Srayko M et al., 2003), and TAC-1 depleted oocytes showed similar phenotypes to the zyg-9 (-) oocytes. We are currently investigating the localization of the ZYG-9/TAC-1 complex during early meiotic spindle assembly to further advance our understanding of how these proteins contribute to the acentrosomal assembly of bipolar spindles during oocyte meiotic cell division. References: Wolff et al., Mol Biol Cell, 2016; Matthews LR et al., J Cell Biol., 1998; Bellanger JM and Gonczy P, J Cell Biol., 2003; Yang et al., Dev Biol., 2003; Le Bot N et al., Curr Biol., 2003; Srayko M et al., Curr Biol., 2003.