Cattin, Jerome et al. (2015) International Worm Meeting "Molecules involved in the biogenesis of dense-core vesicles."

Ailion, Michael, Cattin, Jerome, Topalidou, Irini

[International Worm Meeting, 2015]

The regulated release of neuromodulators is mediated by a specialized type of cellular vesicle, the dense-core vesicle (DCV). The biogenesis, maturation, and trafficking of DCVs are not well characterized, mainly because few proteins specific to DCV function are known. In a genetic screen for suppressors of constitutively active Galphaq we identified mutations in the small GTPase RAB-2, the RAB-2 effectors RUND-1 (a RUN domain protein) and CCCP-1 (a coiled-coil protein), and an uncharacterized WD40 domain-containing protein (Y87G2A.11). Here we investigate the specific function of these proteins both in C. elegans and in neuroendocrine cell lines. We show that mutants of these genes have the characteristic "unmotivated" phenotype characterized by little spontaneous movement on food but capable of coordinated locomotion once stimulated. All proteins function in the same genetic pathway, in a cell-autonomous way, and are required for trafficking of DCV but not SV (synaptic vesicle) cargos. We show that all these proteins are expressed in neuroendocrine cell lines. With the exception of Y87G2A.11, which localizes to the cytoplasm, the three other players co-localize with each other in a perinuclear region both in C. elegans and in neuroendocrine cells. Co-localization experiments in insulinoma cells (INS-1) show that these proteins partially co-localize with the trans Golgi and with proinsulin, suggesting a role in the biogenesis or early transport of secretory vesicles. We have successfully knocked out the Y87G2A.11 ortholog TSSC1 in INS-1 cells using the CRISPR technology and are now performing insulin secretion assays to investigate its role in DCV secretion. CRISPR-induced knock out of CCCP-1 and RUND-1 orthologs is underway. We are also performing in vitro binding assays of these proteins to characterize their physical interactions and yeast two hybrid and mass spec experiments to identify new interactors. Finally, we have characterized biophysical properties of CCCP-1. CCCP-1 is predicted to have long coiled-coil domains like golgins. We show that it forms oligomers and has an elongated shape, raising the possibility that it may act as a tether that links together yet unknown membranes. We are assaying its ability to bind lipids and tether membranes.

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.

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.