Yates JA et al. (1994) J Parasitol "Infectivity and normal development of third stage Brugia malayi maintained in vitro."

Yates JA, Nelson FK, Rajan TV, Schmitz KA

[J Parasitol, 1994]

Shipment of infective-stage filarial larvae (L3s) usually has been accomplished by transporting living infected vectors or L3s cryopreserved in liquid nitrogen. Our objective was to find culture conditions for transporting L3s that would promote survival of Brugia malayi larvae without altering their capacity to infect susceptible animals. In preliminary studies we observed that Ham's nutrient mixture F-12, with antibiotics and 1% fetal calf serum, could support L3s without apparent development for at least 10 days. In order to evaluate the effect of culture temperatures on infectivity, fresh L3s were divided into groups that were either immediately injected into jirds (infectivity control) or incubated for 24, 48, or 120 hr in tightly sealed tubes maintained horizontally at either 0 C, 20 C, or 37 C, before they were injected into jirds. Necropsies were performed on the jirds 120-130 days after injection to recover and count adult worms. Levels of microfilaremia were also determined. We found that L3s held overnight at 0 C, although apparently viable, were unable to survive in jirds. However, larvae kept at 20 C and 37 C produced patent infections with adult worms in normal locations even after 120 hr of in vitro cultivation. There was no statistical difference in mean worm recovery or size of worms from jirds infected with freshly harvested L3s and jirds injected with larvae that were maintained overnight at 20 C or 37 C. When cultured L3s were shipped from Michigan to Connecticut by overnight air courier, along with infected living mosquitos, the L3s appeared to be 99% viable upon arrival. L3s shipped in F-12 produced patent infections in C.B.-17 scid/scid mice with worm recoveries comparable to those observed in mice injected with L3s freshly obtained from shipped mosquitos.

Solorzano E et al. (2011) BMC Evol Biol "Shifting patterns of natural variation in the nuclear genome of caenorhabditis elegans."

Bergeron RD, Sung W, Okamoto K, Thomas WK, Solorzano E, Datla P

[BMC Evol Biol, 2011]

BACKGROUND: Genome wide analysis of variation within a species can reveal the evolution of fundamental biological processes such as mutation, recombination, and natural selection. We compare genome wide sequence differences between two independent isolates of the nematode Caenorhabditis elegans (CB4856 and CB4858) and the reference genome (N2). RESULTS: The base substitution pattern when comparing N2 against CB4858 reveals a transition over transversion bias (1.32:1) that is not present in CB4856. In CB4856, there is a significant bias in the direction of base substitution. The frequency of A or T bases in N2 that are G or C bases in CB4856 outnumber the opposite frequencies for transitions as well as transversions. These differences were not observed in the N2/CB4858 comparison. Similarly, we observed a strong bias for deletions over insertions in CB4856 (1.44: 1) that is not present in CB4858. In both CB4856 and CB4858, there is a significant correlation between SNP rate and recombination rate on the autosomes but not on the X chromosome. Furthermore, we identified numerous significant hotspots of variation in the CB4856-N2 comparison.In both CB4856 and CB4858, based on a measure of the strength of selection (ka/ks), all the chromosomes are under negative selection and in CB4856, there is no difference in the strength of natural selection in either the autosomes versus X or between any of the chromosomes. By contrast, in CB4858, ka/ks values are smaller in the autosomes than in the X chromosome. In addition, in CB4858, ka/ks values differ between chromosomes. CONCLUSIONS: The clear bias of deletions over insertions in CB4856 suggests that either the CB4856 genome is becoming smaller or the N2 genome is getting larger. We hypothesize the hotspots found represent alleles that are shared between CB4856 and CB4858 but not N2. Because the ka/ks ratio in the X chromosome is higher than the autosomes on average in CB4858, purifying selection is reduced on the X chromosome.

Ju J et al. (2023) Nucleic Acids Res "Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis."

Tomita K, Ju J, Kuroyanagi H, Aoyama T, Yashiro Y, Yamashita S

[Nucleic Acids Res, 2023]

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

Julia Grabitzki et al. (2006) European Worm Meeting "Identification of O-linked N-acetylglucosamine Modified Proteins in Caenorhabditis elegans"

Gunter Lochnit, Rudolf Geyer, Michael Ahrend, Brigitte Schmitz, Julia Grabitzki

[European Worm Meeting, 2006]

Julia Grabitzki1, Michael Ahrend2, Brigitte Schmitz2, Rudolf Geyer1 and Gnter Lochnit1. The posttranslational modification N-acetylglucosamine O-glycosidically linked (O-GlcNAc) to serine and threonine residues of proteins has been shown to be ubiquitous amongst eukaryotic proteins of the nucleus, cytoskeleton, cytoplasm, and has also been detected on cytosolic tails of membrane proteins [1]. O-GlcNAcylated proteins can form reversible multimeric complexes with other polypeptides or structures. The modification is often accompanied by phosphorylation/ dephosphorylation. O-GlcNAc can act either simultaneously or in a reciprocal fashion with phosphorylation. According to the Yin-Yang hypothesis, the phosphorylation/ dephosphorylation regulates O-GlcNAc-modified protein function (z.B. signal transduction and protein-protein interaction) in concert with phosphorylation [2-4]. The addition of O-GlcNAc to and the removal from the protein backbone is dynamic with rapid cycling in response to cellular signals or cellular stages.. Despite the fact, that Caenorhabiditis elegans is the best studied model organism, there have been no studies on O-GlcNAcylation in this organism so far. Therefore, to elucidate the role of O-GlcNAcylation, we investigated the proteome of a C. elegans mixed-stage population by two-dimensional gelelectrophoresis and subsequent western-blotting with the O-GlcNAc-specific antibody CTD 110.6 for the occurrence of this modification and identified the modified proteins by mass-spectrometry. We detected and identify several O-GlcNAc-modified proteins in C. elegans. Most of the identified proteins are involved in metabolic pathways. The prediction of the cellular localisation of the identified proteins revealed a predominant cytosolic occurrence of the O-GlcNAc modification.. References:. [1]. Rex-Mathes, M., J. Koch, Werner, S., Griffith, L. S and B. Schmitz. 2002. Methods Mol Biol 194: 73-87.. [2] Zachara, N.E. and G.W. Hart, Chem Rev, 2002. 102(2): p.431-8.. [3]. Griffith, L. S. and B. Schmitz. 1999. Eur J Biochem 262(3): 824-31.. [4] Wells, L. and G. W. Hart. 2003. FEBS Lett 546(1): 154-8.

Rose JK et al. (2022) Front Behav Neurosci "Neuroligin Plays a Role in Ethanol-Induced Disruption of Memory and Corresponding Modulation of Glutamate Receptor Expression."

Liang J, Lin CHS, Rose JK, Parvand M, Butterfield M, Rankin CH

[Front Behav Neurosci, 2022]

Exposure to alcohol causes deficits in long-term memory formation across species. Using a long-term habituation memory assay in Caenorhabditis elegans, the effects of ethanol on long-term memory (> 24 h) for habituation were investigated. An impairment in long-term memory was observed when animals were trained in the presence of ethanol. Cues of internal state or training context during testing did not restore memory. Ethanol exposure during training also interfered with the downregulation of AMPA/KA-type glutamate receptor subunit (GLR-1) punctal expression previously associated with long-term memory for habituation in C. elegans. Interestingly, ethanol exposure alone had the opposite effect, increasing GLR-1::GFP punctal expression. Worms with a mutation in the C. elegans ortholog of vertebrate neuroligins (nlg-1) were resistant to the effects of ethanol on memory, as they displayed both GLR-1::GFP downregulation and long-term memory for habituation after training in the presence of ethanol. These findings provide insights into the molecular mechanisms through which alcohol consumption impacts memory.

Russell GJ et al. (1989) Biochim Biophys Acta "Colchicine binding in the free-living nematode Caenorhabditis elegans."

Lacey E, Russell GJ

[Biochim Biophys Acta, 1989]

The [3H]colchicine-binding activity of a crude supernatant of the free-living nematode Caenorhabditis elegans was resolved into a non-saturable component and a tubulin-specific component after partial purification of tubulin by polylysine affinity chromatography. The two fractions displayed opposing thermal dependencies of [3H]colchicine binding, with non-saturable binding increasing, and tubulin binding decreasing, at 4 degrees C. Binding of [3H]colchicine to C.elegans tubulin at 37 degrees C is a pseudo-first-order rate process with a long equilibration time. The affinity of C. elegans tubulin for [3H]colchicine is relatively low (Ka = 1.7 x 10(5) M(-1)) and is characteristic of the colchicine binding affinities observed for tubulins derived from parasitic nematodes. [3H]Colchicine binding to C. elegans tubulin was inhibited by unlabelled colchicine, podophyllotoxin and mebendazole, and was enhanced by vinblastine. The inhibition of [3H]colchicine binding by mebendazole was 10-fold greater for C. elegans tubulin than for ovine brain tubulin. The inhibition of [3H]colchicine binding to C. elegans tubulin by mebendazole is consistent with the recognised anthelmintic action of the benzimidazole carbamates. These data indicate that C. elegans is a useful model for examining the interactions between microtubule inhibitors and the colchicine binding site of nematode tubulin.

Miyazaki H et al. (2012) Biophys J "CLC anion channel regulatory phosphorylation and conserved signal transduction domains."

Parton A, Kim S, Yamada T, Miyazaki H, Beth AH, Morrison R, Strange K

[Biophys J, 2012]

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a 176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first -helix (1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 1.

Jentsch TJ et al. (2018) Physiol Rev "CLC Chloride Channels and Transporters: Structure, Function, Physiology, and Disease."

Jentsch TJ, Pusch M

[Physiol Rev, 2018]

CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory -subunits for function. CLC proteins come in two flavors: anion channels and anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analysis identified residues involved in ion permeation, anion-proton coupling and gating and led to attractive biophysical models. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl^- channels, whereas ClC-3 through ClC-7 are 2Cl^-/H⁺-exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl^- channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. In this review, emphasis is laid on biophysical structure-function analysis and on the cell biological and organismal roles of mammalian CLCs and their role in disease.

Yusuke Hokii et al. (2006) East Asia C. elegans Meeting "Spatio temporal distribution patterns of C.elegans small ncRNAs"

Yusuke Hokii, Yuko Endo, Yuko Mizuki, Chisato Ushida, Yuki Sugawara, Fumitoshi Harada, Hyouta Himeno, Takayuki Konno, Akira Muto

[East Asia C. elegans Meeting, 2006]

CeR 3, 4, 6, 7, 8, 9, 19 RNAs that were encoded in the regions corresponding to the introns of protein coding genes were isolated from Caenorhabditis elegans. These seven RNAs showed typical snoRNA secondary structures. CeR 19 RNA is included in C/D snoRNA and the other RNAs were classified in H/ACA snoRNAs. Four of the seven isolated snoRNAs (CeR 6, 7, 9, 19) could form base pairings with parts of rRNAs, suggesting that they have a potential to guide pseudouridylation and methylation of rRNAs. CeR 6 RNA may guide modification of U1254 of 18S rRNA. The predicted targets of CeR 7 RNAs are U778 and U827 of 18S rRNA, U866, U1058, U2400 and U2546 of 26S rRNA and U89 of 5.8S rRNA. CeR 9 RNA guides probably pseudouridylation of U513 of 18S rRNA. CeR 19 RNA has two base paring sites that ribose 2 O methylation of U308 of 26S rRNA and C642 of 18S rRNA. In the recent study of Schmitz et al. the targets of CeR 3, 4 and 8 RNA were predicted. These RNAs perhaps guide modification U3035 (CeR 3), U2558 (CeR 4), U2098 and U3035 (CeR 8) of rRNAs, respectively. Here we examined whole mount in situ hybridization to know the localization of these RNAs in cells and their spatiotemporal distribution patterns in worms. As expected, all these RNAs are localized in the nucleolus of most cells of the worm. Their expressions were observed from early embryonic stage to adult. Chemical modification experiments of rRNAs indicated that a predicted target of CeR 9 RNA, U513 of 18S rRNA, was pseudouridine. Further analysis will reveal the whole positions of C.elegans rRNA pseudouridine and demonstrate whether the potential target sites of CeR RNAs are indeed modified or not.

Nathalie Strutz et al. (2001) International C. elegans Meeting "Identification of functional ion channel domains of C. elegans glutamate receptor subunits"

Nathalie Strutz, David Madsen, Michael Hollmann, Andres V Maricq

[International C. elegans Meeting, 2001]

Glutamate receptors are not only abundant in the nervous system of vertebrates but also of invertebrates. In Caenorhabditis elegans , 10 putative ionotropic glutamate receptor subunits have been identified so far (Brockie et al. 2001, J. Neurosci. 21, 1510). Two of these, GLR-1 and GLR-2, have been expressed in Xenopus oocytes and failed to show ligand-activated currents. Therefore, we set out to investigate if the pore-forming domains of these two subunits are intrinsically functional ion conducting domains. We have previously shown that domain transplantation between different subtypes of glutamate receptors can generate functional chimeras that allow the characterization of those domains. To test for pore function, we transplanted the pore-forming region of GLR-1 or GLR-2 into either rat GluR1, as a representative of the AMPA receptor family, or rat GluR6, as a representative of the KA receptor family. The resulting chimeras GluR1-PGLR1, GluR1-PGLR2, GluR6-PGLR1, and GluR6-PGLR2, as well as the respective wildtypes GluR1, GluR6, GLR-1, and GLR-2 were expressed and characterized in Xenopus oocytes. Surprisingly, GluR1-PGLR1, GluR1-PGLR2, and GluR6-PGLR2 produced ligand-activated currents, despite the GLR-1 and GLR-2 wildtype receptor's apparent lack of ion channel function. The maximal current amplitudes of GluR6-PGLR2 were comparable to wildtype GluR6. GluR1-PGLR1 and GluR1-PGLR2 showed reduced current amplitudes in comparison to wildtype GluR1. The ion channel properties of the chimeras classify GLR-1 and GLR-2 into the group of non-NMDA receptor subunits, consistent with conclusions derived from sequence homology data.