Blum, Elyse et al. (2015) International Worm Meeting "Mechanism of peel-1 toxicity."

Blum, Elyse, Cattin, Jerome, Ailion, Michael

[International Worm Meeting, 2015]

In C. elegans, hybrid incompatibility between the Bristol N2 and Hawaiian CB4856 strains is mediated by the peel-1/zeel-1 genetic element. peel-1 encodes a sperm-expressed toxin and zeel-1 encodes its zygotically-expressed antidote. We are interested in determining the cellular mechanism of PEEL-1 toxicity. To identify factors required for PEEL-1 toxicity, we conducted a screen for suppressors of the lethal phenotype caused by ectopic PEEL-1 expression driven by a heat-shock promoter (hs::peel-1). Thus far, we have isolated five suppressors. All five suppressors are healthy and have no obvious visible phenotypes. They still express a heat-shock driven GFP transgene at normal levels, indicating that they do not suppress hs::peel-1 due to failed induction of the heat-shock response. The two strongest suppressors fully suppress hs::peel-1 and are allelic. The other three suppressors are partial suppressors belonging to two complementation groups and preferentially suppress hs::peel-1 in younger larvae. Thus, it seems likely that only a small number of genes can be mutated to suppress hs::peel-1. We have mapped these suppressors to single chromosomes and performed whole genome sequencing to clone the genes. We also tagged heat-shock driven PEEL-1 with GFP on either its N or C terminal. In both cases, induction of heat-shock leads to death of the worms, indicating that the tagged PEEL-1 protein is still functional. GFP is localized to a perinuclear compartment that may be the endoplasmic reticulum. Previously, we showed that PEEL-1 kills either muscle or neurons cell autonomously when specifically expressed in these cell types. We now show that PEEL-1 is also toxic when expressed in the intestine. Endogenous PEEL-1 is transmitted paternally to kill zeel-1(-) embryos. We wondered whether maternal expression would also kill zeel-1(-) embryos. We expressed peel-1 in the maternal germline using the pie-1 promoter and found that it did not lead to death of zeel-1(-) progeny. Thus, it appears that paternal expression and transmission is critical for the embryonic lethal phenotype.

Posch, G et al. (2019) International Worm Meeting "A small conserved membrane protein is required for PEEL-1 toxicity."

Posch, G, Cattin, J, Ailion, M

[International Worm Meeting, 2019]

PEEL-1 and ZEEL-1 are a toxin and antidote pair that mediate an intraspecific hybrid incompatibility in C. elegans. We are interested in determining the mechanism of PEEL-1 toxicity and ZEEL-1 anti-toxicity. PEEL-1 is expressed paternally and delivered to embryos via sperm. In the absence of embryonically expressed ZEEL-1, PEEL-1 leads to embryonic arrest at a the two-fold stage. Ectopically expressed PEEL-1 causes a cell-autonomous necrotic cell death that does not depend on the apoptotic caspase CED-3. To identify factors required for PEEL-1 toxicity, we performed a forward genetic screen for suppressors of lethality caused by heat-shock induced expression of PEEL-1. We isolated six independent suppressors belonging to four different complementation groups. Extent of suppression ranges from partial suppression of lethality primarily in larval stages to full suppression of lethality at all stages. We molecularly identified two genes required for PEEL-1 toxicity. Two partial hs::peel-1 suppressors carry missense mutations in the gene nxf-1, encoding a nuclear-export factor required for general export of mRNAs out of the nucleus. Though null mutants in nxf-1 are lethal, our nxf-1 missense mutants are indistinguishable from wild-type, suggesting that the peel-1 mRNA may be particularly dependent on the function of this RNA export factor. Two strong PEEL-1 suppressors carry mutations (missense and deletion) in the gene F47B7.1, encoding a 59 amino acid transmembrane protein that belongs to a family of similar small transmembrane proteins previously uncharacterized in C. elegans. Transgenic expression of F47B7.1 rescues the peel-1 suppression phenotype of the deletion mutation yak103, confirming the gene identification. We also show that yak103 suppresses the embryonic lethal phenotype caused by endogenous PEEL-1 protein supplied from sperm, demonstrating that F47B7.1 is required for the toxic effects of both ectopically and endogenously expressed PEEL-1. We are using GFP constructs to determine the expression pattern and cellular localization of F47B7.1. We are also investigating whether mutations in F47B7.1 affect localization of ectopically expressed GFP-tagged PEEL-1.

Seidel, Hannah et al. (2009) International Worm Meeting "Balancing selection maintains paternal-effect-by-zygotic incompatibility among C. elegans wild isolates."

Rockman, Matthew, Seidel, Hannah, Kruglyak, Leonid, Ailion, Michael

[International Worm Meeting, 2009]

C. elegans strains Bristol (N2) and Hawaii (CB4856) exhibit a paternal-effect-by-zygotic incompatibility involving two genes - peel-1 and zeel-1. peel-1 and zeel-1 interact like a toxin and an antidote: peel-1 is a paternal-effect gene that induces developmental arrest in embryos that do not inherit the zygotically expressed gene zeel-1. peel-1 and zeel-1 are located adjacent to one another in the Bristol genome, and Hawaii carries a deletion spanning both genes. We have cloned peel-1 and zeel-1 by transgenic complementation. peel-1 encodes a predicted transmembrane protein with no homology to any gene in any species. peel-1::gfp transcriptional fusions are expressed in spermatocytes, and immunostaining localizes PEEL-1 to the cell membrane of mature sperm. Embryos affected by the paternal-effect lethality arrest paralyzed at the two-fold stage, suggesting that PEEL-1 causes a defect in muscle development or function. zeel-1 encodes a protein homologous to the substrate recognition subunit of an E3 ubiquitin ligase. Unlike most members of its family, ZEEL-1 contains a predicted transmembrane domain, and this transmembrane domain is required for the ability of zeel-1 to rescue the paternal-effect lethality. zeel-1::gfp translational fusions are expressed ubiquitously in the embryo, beginning approximately 4 hours post-fertilization and ending before hatching. The observation that zeel-1 expression does not begin until mid-embryogenesis raises the possibility that sperm-supplied PEEL-1 persists for several hours after fertilization. The zeel-1/peel-1 incompatibility is not specific to a cross between Bristol and Hawaii. We have genotyped a large panel of wild strains at the zeel-1/peel-1 locus and found that approximately two thirds of strains carry zeel-1 and peel-1 (like Bristol), whereas the remaining strains carry a deletion of the genes (like Hawaii). The Bristol- and Hawaii-like haplotypes exhibit elevated sequence divergence, and the allele frequency spectrum at the locus is indicative of balancing selection. We propose that balancing selection acting on zeel-1, peel-1, or a tightly linked locus preserves both haplotypes in the population despite the lethality caused by their interaction.

Caro, Lews et al. (2021) International Worm Meeting "Dissecting the Molecular Mechanism of the peel-1/zeel-1 Selfish Genetic Element"

Caro, Lews, Posch, Galen, Ailion, Michael

[International Worm Meeting, 2021]

Selfish genetic elements persist in species despite their negative impacts on host fitness. Toxin-antidote selfish elements kill progeny that do not inherit the genetic element thus increasing their representation in the next generation. The peel-1/zeel-1 gene pair is a toxin-antidote system responsible for a hybrid incompatibility between C. elegans Bristol N2 and Hawaiian CB4856 strains. PEEL-1 is a sperm-delivered toxin that arrests embryonic development. However, embryos inheriting the zeel-1 antidote gene can escape PEEL-1 toxicity. We are investigating the molecular mechanism of PEEL-1 toxicity and ZEEL-1 anti-toxicity. We used a forward genetic screen to discover that the small, conserved protein, F47B7.1 is necessary for PEEL-1 toxicity. Expression of PEEL-1 and F47B7.1 together induces cell swelling and cell death in HEK293T mammalian cells. ZEEL-1 expression suppresses this toxicity. Therefore, I have reconstituted the toxin and antidote activity in a heterologous system, suggesting that we have identified the minimal set of proteins involved in toxicity and anti-toxicity. My continued work uses C. elegans, HEK293T cells, and in vitro biochemistry to dissect the molecular mechanism of toxicity and anti-toxicity.

Lamelza, Piero et al. (2013) International Worm Meeting "If we have children together, will they be less fit? Hybrid incompatibilities in Caenorhabditis species."

Lamelza, Piero, Cattin, Jerome, Ailion, Michael, Wilson, Vanessa, Topalidou, Irini

[International Worm Meeting, 2013]

Speciation often occurs by the accumulation of genetic incompatibilities between populations. We are studying the genetic basis of hybrid incompatibility in several Caenorhabditis species. In C. elegans, hybrid incompatibility between the Bristol and Hawaiian strains is mediated by the peel-1/zeel-1 genetic element. peel-1 encodes a sperm-expressed toxin and zeel-1 encodes its zygotically-expressed antidote. We are interested in determining the cellular mechanism of PEEL-1 toxicity. To identify other factors required for PEEL-1 toxicity, we conducted a screen for suppressors of ectopic PEEL-1 driven by a heat-shock promoter. Thus far, we have isolated five suppressors. The strongest suppressor fully suppresses PEEL-1 toxicity and is not allelic to the heat-shock transcription factor gene hsf-1. We have also expressed GFP-tagged PEEL-1 under a galactose-inducible promoter in yeast. Preliminary data suggest that PEEL-1 strongly inhibits growth of the yeast when induced. We have also begun a search for hybrid incompatibility genes in other Caenorhabditis species. We performed crosses between two recently isolated Caenorhabditis species, C. sp. 17 and C. sp. 29. When C. sp. 17 males were crossed to C. sp. 29 females, all F1 progeny arrested as embryos. However, when C. sp. 29 males were crossed to C. sp. 17 females, a very small percentage of F1 progeny did not arrest as embryos, but instead grew to the adult stage. Thus, as with peel-1, a parental effect contributes to the hybrid incompatibility. Almost all of the viable hybrids were female, indicating that the cross obeys Haldane's rule. The few F1 hybrid males are sterile. F1 hybrid females did not give viable progeny when crossed to C. sp. 29 males, but gave large numbers of viable F2 progeny and some arrested embryos when crossed to C. sp. 17 males. Among the surviving F2, there are more females than males, indicating that this cross also obeys Haldane's rule. There is significant variability in the relative proportion of males, females, and arrested embryos in the F2 generation from cross to cross, suggesting that particular combinations of genes in the rare surviving F1 females determine the proportions seen in the F2 crosses.

John, S. et al. (2017) International Worm Meeting "Hyperglutamylation of microtubules causes a reduction in brood size."

Dominguez, J, Peel, N, Hepurker, A, John, S.

[International Worm Meeting, 2017]

Microtubule glutamylation is a reversible covalent modification of the microtubules, which can regulate microtubule function. The addition of glutamate to the polymerized microtubule is catalyzed by the tubulin tyrosine ligase like (TTLL) enzymes and its removal is carried out by the cytosolic carboxypeptidase (CCPP) family of enzymes. A mutation in C. elegans ccpp-1 results in tubulin hyperglutamylation and ciliary fragmentation, suggesting an essential function for ccpp-1 in maintenance of microtubule stability in the cilia (O'Hagan et al, 2011). This neuronal phenotype parallels defects observed in mice when the murine homolog, CCP1, is lost. Interestingly these murine CCP1 mutants also show a reduction in female fertility, leading us to investigate the function of CCPP-1 in the C. elegans germline. We have found that mutations in ccpp-1 lead to a temperature sensitive reduction in brood size, although embryonic viability is unperturbed. Breeding mutant hermaphrodites with wild type males did not restore the normal brood size and ccpp-1 males were able to sire a normal brood when mated with wild-type hermaphrodites. Thus the reduced fertility can be attributed to a defect in oogenesis. To further investigate the underlying cause of this fertility defect, we first examined the gross morphology of the ccpp-1 mutant germline, but found no overt problems with germline size or organization. We are now undertaking an analysis of whether increased apoptosis leads to a reduction in gamete production. This work will ultimately allow us to better understand the function of tubulin glutamylation in the worm.

Hannah Seidel et al. (2008) Development & Evolution Meeting "Zygotic-by-paternal-effect incompatibility maintained by balancing selection"

Leonid Kruglyak, Hannah Seidel, Matt Rockman

[Development & Evolution Meeting, 2008]

Natural selection is expected to eliminate genetic incompatibilities from interbreeding populations. We have discovered a globally distributed incompatibility in Caenorhabditis elegans that is maintained despite its negative consequences for fitness. The incompatibility causes embryonic lethality through a paternal-by-zygotic interaction whereby embryos homozygous for a naturally occurring deletion of the zygotically acting gene, zeel-1, arrest if their sperm parent carries an incompatible allele of a second, paternal effect locus, peel-1. The two interacting loci are tightly linked, with incompatible alleles occurring in linkage disequilibrium in two common haplotypes. These haplotypes exhibit elevated sequence divergence and an allele frequency spectrum indicative of balancing selection acting to preserve both in the population. Our data suggest that long-term maintenance of a balanced polymorphism has permitted the incompatibility to persist despite gene flow across the rest of the genome. We have cloned zeel-1 by transgenic complementation, and it belongs to a previously uncharacterized family of C. elegans-specific genes with homology to a recognition subunit of an ubitquitin ligase complex. A zeel-1::GFP fusion shows expression during mid to late embryogenesis, and embryos affected by the incompatibility arrest paralyzed at the two-fold stage, suggesting they may have a defect in elongation or muscle morphogenesis. Naturally occurring mutations disrupting peel-1 function map to the intergenic region downstream of zeel-1, implying that peel-1 may be a cryptic or non-coding gene or a regulatory element of zeel-1 itself.

Dominguez, J. et al. (2019) International Worm Meeting "Characterization of microtubule deglutamylating enzyme function in the C. elegans germline."

Dominguez, J., John, S., Peel, N., Hepurker, A., Oliver, T.

[International Worm Meeting, 2019]

Microtubule glutamylation is a reversible process that regulates microtubule function by modifying them with the addition of glutamate residues. This post translational modification of the polymerized microtubule is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes, while the removal of glutamate is carried out by a family of cytosolic carboxypeptidase (CCP) enzymes. C. elegans has two deglutamylating enzymes, CCPP-1 and CCPP-6, homologs of mammalian CCP1 and CCP5 respectively. Loss of mouse CCP1 function leads to neurodegeneration and reduced female fertility, whereas loss of CCP5 causes defects of the sperm flagella. C. elegans ccpp-1 mutants show hyperglutamylation and ciliary fragmentation, suggesting that CCPP-1 functions in maintaining ciliary microtubule stability (O'Hagan, 2011). Although ccpp-1(ok1821) mutants show normal brood size at 20 deg C we have identified a heat-induced reduction in brood size. This fertility defect originates in oogenesis and does not seem to result from increased germline apoptosis. In contrast, mutation of ccpp-6, does not affect brood size and the brood size defect is not worse in a ccpp-1(ok1821); ccpp-6(ok382) double mutant, suggesting that redundancy is not present in germline. In addition, we have found that ccpp-1 mutants show increased sensitivity to the microtubule destabilizing drug colchicine and to the cold. We are currently investigating the underlying cause of the ccpp-1 reduced fertility.

Shah, B. et al. (2019) International Worm Meeting "Microtubule glutamylation aids cold and colchicine tolerance in C. elegans."

Lee, J., Suri, P., Peel, N., Shah, B., Chawla, D.

[International Worm Meeting, 2019]

Glutamylation, the covalent attachment of glutamic acid to tubulin in the polymerized microtubule, is enriched on long-lived microtubules. It is thought to contribute to centriole stability, cilia motility and axon function. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase like (TTLL) enzymes. Comprehensive in vivo analyses of the function of tubulin glutamylation have proved challenging because of the existence of a large family of TTLL enzymes in many species. To investigate the function of tubulin glutamylation we have generated a ttll-4(tm3310); ttll-11(tm4059); ttll-15(tm3871) ttll-5(tm3360) ttll-9(tm3889) 'quint' mutant that lacks all five C. elegans glutamylating TTLL enzymes. The quint mutant shows normal embryonic viability and brood size and the centrosomes organize a spindle that is competent for cell division. Our data therefore suggest that, contrary to expectations, microtubule glutamylation is not essential for C. elegans viability. Glutamylation is enriched on stable microtubules, but whether it contributes to this stability remains unclear. We find that our quint mutants show enhanced colchicine and cold-sensitivity, suggesting that microtubule stability is altered by the loss of glutamylation. In human cells glutamylation modulates microtubule severing by spastin and katanin. To investigating whether glutamylation regulates microtubule stability via this mechanism we combined our quint mutant with mei-1(GOF) and mei-1(LOF) alleles of katanin, but did not observe suppression of the mei-1-associated lethality. We are currently undertaking a detailed analysis of microtubule dynamics to determine the underlying cause of altered microtubule properties in the quint mutant.

Martin Gutternigg et al. (2006) European Worm Meeting "Glycosidases of Caenorhabditis elegans involved in N-glycan processing"

Martin Gutternigg, Matthias Hackl, Ute Stemmer, Iain B. H. Wilson, Petra Geier, Gunter Lochnit, Ramona Ranftl, Katharina Paschinger, Verena Jantsch, Dorothea Lubich

[European Worm Meeting, 2006]

Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.