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WB Derry et al. (1999) International C. elegans Meeting "Analysis of the C. elegans homolog of the FHIT tumor suppressor gene"

[

International C. elegans Meeting,

1999]

Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.

Wolf FW et al. (1998) West Coast Worm Meeting "Yes! you can suppress vab-8"

Wolf FW, Garriga G

[

West Coast Worm Meeting,

1998]

vab-8 acts in posteriorly directed cell and axon growth cone migrations in a cell autonomous manner. vab-8 functions in posterior ALM cell migration but not in later anterior ALM axon migration, and continuous vab-8 expression in the ALM can reorient anterior ALM axon outgrowth posteriorly. This finding suggests that the direction of migration can be regulated at the level of vab-8 activity. vab-8 encodes two isoforms of a novel intracellular protein, and the opal null allele e1017 is predicted to remove both activities. Our analysis of vab-8 has led us to propose that there exists at least one mechanism for the global guidance of posterior migrations. To identify other genes that function in posterior guidance, we suppressed the highly penetrant CAN cell migration defect of vab-8(e1017) mutants. Extragenic suppressors could identify genes that act downstream of vab-8 as well as genes that act in parallel pathways. From screening over 20,000 genomes we isolated four partially penetrant van (variably abnormal normal) suppressors, each defining a distinct gene. van-1(gm128) is a semi-dominant suppressor of vab-8 cell and axon outgrowth defects that is allele non-specific but at least so far is gene specific. Alas, van-1 has no phenotype on its own. In dosage studies van-1(gm128) appears to alter gene function. We have mapped van-1 to LGI between stu-4 and unc-11 and transformation rescue experiments are underway.

van Schendel, R. et al. (2017) International Worm Meeting "Polymerase theta is a key driver of genome evolution, of CRISPR/Cas9-mediated mutagenesis and of mutagen-induced deletion allele formation."

Tijsterman, M., van Schendel, R.

[

International Worm Meeting,

2017]

For more than half a century, genotoxic agents have been used to induce mutations in the genome of model organisms to establish genotype-phenotype relationships. We have recently demonstrated for two of the most widely used mutagens in C. elegans, i.e. ethyl methanesulfonate (EMS) and photo-activated trimethylpsoralen (UV/TMP), that deletion mutagenesis is the result of polymerase Theta (POLQ)-mediated end joining (TMEJ) of double strand breaks (DSBs) 1, 2. This discovery allowed us to survey many thousands of available deletion alleles, which reveals a step-wise and versatile model for the in vivo mechanism of TMEJ, explaining the molecular nature of mutagen-induced deletion alleles 3. We also found that CRISPR/Cas9-induced genomic changes are exclusively generated through POLQ action, refuting a previously assumed requirement for NHEJ in their formation; Unlike somatic cells, which use NHEJ to repair DNA breaks, germ cells use an alternative route 4. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification. New insights into the mechanism of genome engineering, genome maintenance and evolution will be discussed. 1. Koole,W. et al. A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites. Nat. Commun. 5, 3216 (2014). 2. Roerink,S.F., van Schendel,R., & Tijsterman,M. Polymerase theta-mediated end joining of replication-associated DNA breaks in C. elegans. Genome Res. 24, 954-962 (2014). 3. van Schendel R., van Heteren J., Welten,R., & Tijsterman,M. Genomic Scars Generated by Polymerase Theta Reveal the Versatile Mechanism of Alternative End-Joining. PLoS. Genet. 12, e1006368 (2016). 4. van Schendel,R., Roerink,S.F., Portegijs,V., van den Heuvel,S., & Tijsterman,M. Polymerase Theta is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis. Nat. Commun. 6, 7394 (2015).

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[

International Worm Meeting,

2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

van Luenen HGAM et al. (1995) International C. elegans Meeting "Mechanism of Tc3 transposition"

van Luenen HGAM, Plasterk RHA

[

International C. elegans Meeting,

1995]

The strain Bristol N2 has no endogenous Tc3 transposition activity, but forced expression of Tc3 transposase results in somatic transposition of endogenous Tc3 elements as well as Tc3 elements located on a transgene (Van Luenen et al. 1993 EMBO J. 12:2513-2520). This provides us with an assay for the requirements of Tc3 transposition in vivo.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[

International Worm Meeting,

2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Vangheel, M. et al. (2015) International Worm Meeting "GLB-33: when a globin hooks up with a G-protein coupled receptor."

Tilleman, L., Hoogewijs, D., De Henau, S., Moens, L., Van Doorslaer, S., Schoofs, L., Helbo, S., Berghmans, H., Fago, A., Beets, I., Dewilde, S., Germani, F., Braeckman, B. P., Desmet, F., Vangheel, M.

[

International Worm Meeting,

2015]

Globin-folded proteins are present in a wide range of life forms and crucial in a variety of physiological processes. Here, we characterize globin 33 (GLB-33) of Caenorhabditis elegans, a soil dwelling nematode. The glb-33 gene encodes a globin domain which is preceded by a G-protein coupled receptor domain (GPCR), making it the longest of all the C. elegans globins [1]. GLB-33 is expressed in sensory, inter- and motor neurons possibly linking sensory input with the animal's locomotion [1]. GLB-33's presence in the ALN and PLN neuron pairs hints at oxygen sensing. This hypothesis is strengthened by the biochemistry of the globin domain, which appears to have features of a typical O2 sensor with high O2 affinity, next to a fast nitrite reductase activity [2]. At present, the neuropeptide ligand of the GPCR still needs to be identified, but its similarity with the FMRFamide receptor of another nematode (Ascaris suum) is apparent [2]. Both ligands are likely influencing the conformation of the entire molecule, making it difficult to predict any biological effect purely based on biochemical information. Therefore the study of the protein's function needs an integrated approach, ranging from biochemical to physiological techniques. Preliminary experiments have indicated that GLB-33 is not required for survival and fecundity under normal or induced oxidative stress conditions. However animals lacking GLB-33 have a slower recovery rate after a 24-h oxygen deprivation period.[1] Hoogewijs D., Geuens E., Dewilde S., Vierstraete A., Moens L., Vinogradov S. & Vanfleteren J.R. (2007) BMC Genomics, 8:356[2] Tilleman L., Germani F., De Henau S., Helbo S., Desmet F., Berghmans H., Van Doorslaer S., Hoogewijs D., Schoofs L., Braeckman B.P., Moens L., Fago A. & Dewilde S. (2015) The Journal of Biological Chemistry.

Catherine M Dempsey et al. (2005) International Worm Meeting "A genetic screen for the targets of dopamine in the egg-laying paradigm."

Scott M Mackenzie, Catherine M Dempsey, Ji Y Sze

[

International Worm Meeting,

2005]

Serotonin and dopamine act antagonistically to modulate egg-laying behaviour in C. elegans. Serotonin signals via the G-coupled serotonin receptor SER-1 in the vulval muscle to stimulate egg-laying, but the mode of the inhibitory action of dopamine remains largely unknown. To date three dopamine receptors have been identified in C. elegans, one D1 like (dop-1) and two D2 like (dop-2, dop-3). No dopamine receptor has yet been implied in the egg-laying paradigm (1,2). MOD-1 was originally characterised as an ionotropic serotonin receptor controlling the rate of locomotion of C. elegans in response to food. Surprisingly, MOD-1 appears to be essential for the inhibitory action of dopamine on egg-laying. mod-1 (ok103) deletion mutants continue to lay eggs in the presence of dopamine and following prolonged starvation (3). Inhibitory dopaminergic signaling via MOD-1 is restricted to the egg-laying paradigm as mod-1 (ok103) mutants are still susceptible to paralysis induced by dopamine. In order to determine the role and pathway of MOD-1 in this dopamine signalling pathway we are conducting a genetic screen for mutants resistant to dopamine inhibition of egg-laying. Our screen will use the following approach: F2 progeny of singled ethylmethane sulfonate mutagenized animals are exposed to 5 mg/ml serotonin and 3 mg/ml dopamine. mod-1 (ok103) mutants continue to lay eggs under such treatment whereas egg-laying in wild-type animals is inhibited. We have recovered several mutants that show similar dopamine resistance as mod-1 (ok103) mutants. 1. Sanyal, S., Wintle, R. F., Kindt, K. S., Nuttley, W. M., Arvan, R., Fitzmaurice, P., Bigras, E., Merz, D. C., Hebert, T. E., van der Kooy, D., Schafer, W. R., Culotti, J. G. & Van Tol, H. H. M. (2004) Embo Journal 23, 473-482. 2. Chase, D. L., Pepper, J. S. & Koelle, M. R. (2004) Nature Neuroscience 7, 1096-1103. 3. Dempsey, C. M. Mackenzie S. M., Gargus A., Blanco G. & Sze J. Y. (2005).. Genetics (In press).

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[

International Worm Meeting,

2007]

Hydrogen sulfide (H2S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H2S to begin to elucidate the molecular mechanisms of H2S action. We show that nematodes exposed to H2S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H2S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H2S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H2S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H2S-induced increased thermotolerance. These data suggest that H2S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H2S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H2S.

T.J. van Ham et al. (2007) International Worm Meeting "A C. elegans model links novel modifiers of a-synuclein inclusion formation to aging."

K.L. Thijssen, E.A.A. Nollen, T.J. van Ham, R.H.A. Plasterk

[

International Worm Meeting,

2007]

Accumulation and aggregation of a-synuclein in the brain is characteristic of, and considered to be causatively related to, several age-related neurodegenerative diseases such as Parkinsons disease (PD). To study early stages of aS accumulation and to identify modifier genes, we created transgenic C. elegans expressing the human aS gene fused to YFP in the body wall muscle. These transgenic animals show localization of aS-YFP to discrete foci during aging, which is not seen for YFP alone, indicating specificity to aS. These foci are present as early as day two after hatching, and become more abundant during aging. By fluorescence recovery after photobleaching (FRAP) we determined that the foci contain mobile material until day 11. However, at old age also immobilized aggregated protein is present. The amount of aS-foci increases to day 11 and decreases thereafter. Interestingly, this decrease in foci formation is associated with a relative increase in the amount of immobilized aggregates. We performed a genome-wide RNAi screen for modifiers of foci formation, and found 108 genes that repeatedly showed increased foci formation when silenced. Remarkably, none of these genes overlaps with genes found in a screen for polyglutamine aggregation we performed in C. elegans1. This might indicate that different mechanisms are involved in these diseases. To further characterize genes found in the screen, deletion strains of a selection of the genes representing different functional classes were crossed into the aS-YFP strain. Deletion of a gene - involved in aging and encoding one of the most potent suppressors of foci-formation - resulted in a twofold increase in the amount of foci on day thirteen. We are currently further characterizing the deletion strains. In conclusion, we developed a new animal model that captures features of age-related PD. We used the model to dentify novel modifiers of aS-YFP foci formation, of which we are further characterizing the effect of deletion of these genes on aS-YFP foci formation. These results can provide insight into the early cellular mechanisms involved in aS accumulation, and could thereby yield molecular targets for therapy. 1 Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H.A. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.

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