Stumbur, Stephanie et al. (2021) International Worm Meeting "C. elegans regulates its behavior via serotonergic signaling to find food and hydrogen peroxide protection."

McGowan, Natalie, Serkin, William, Sevedolmohadesin, Maedeh, Venkatachalam, Vivek, Stumbur, Stephanie, Apfeld, Javier, Schiffer, Jodie

[International Worm Meeting, 2021]

Hydrogen peroxide is a pervasive chemical weapon used by many species to damage their prey or to protect themselves from their pathogens. Cells rely on conserved defense mechanisms, including catalases, to avoid the damage that hydrogen peroxide inflicts on their macromolecules We recently discovered that C. elegans represses those defenses in response to sensory perception of E. coli, the nematode's food source in the lab, because E. coli can deplete hydrogen peroxide from the local environment and thereby protect the nematodes1. Here, we investigated the extent to which C. elegans can tell the difference between bacteria that provide hydrogen peroxide protection and bacteria that do not. To address that question, we determined the extent to which hydrogen peroxide modulates the behavior of C. elegans in food lawns of wildtype E. coli and mutant E. coli unable to degrade hydrogen peroxide. In the absence of hydrogen peroxide in the environment, C. elegans did not exhibit a preference between these two bacteria stains. However, in the presence of hydrogen peroxide, C. elegans remained on the wildtype bacteria lawn but was much more likely to leave the lawn of the bacteria that do not degrade hydrogen peroxide. This nematode behavior was conserved when we used catalase-positive and catalase-negative bacterial strains from the natural C. elegans microbiome, suggesting that this decision-making occurs in the natural environment. To determine how C. elegans senses and responds to bacteria and environmental hydrogen peroxide, we measured calcium dynamics in ciliated sensory neurons. We identified several neuronal classes that respond to hydrogen peroxide in a bacteria-dependent manner. In addition, genetic analysis showed that serotonin signaling regulates this nematode behavior. Our findings demonstrate that the cross-kingdom interactions between C. elegans and bacteria in their microbiome determine the nematode food-leaving behavior via serotonergic signaling, enabling nematode populations to find safety from hydrogen peroxide. 1. Schiffer JA et al. (2020). Caenorhabditis elegans processes sensory information to choose between freeloading and self-defense strategies. eLife.

Schiffer, Jodie et al. (2017) International Worm Meeting "How do worms determine their readiness to cope with environmental stress?"

Stumbur, Stephanie, Heath, William, Tam, Hannah, McGowan, Natalie, Vogelaar, Abigail, Schiffer, Jodie, Apfeld, Javier, Stanley, Julian

[International Worm Meeting, 2017]

At any time in their life, worms may encounter harmful environmental conditions such as high concentrations of oxidants or high temperature. We are interested in understanding how worms prepare for these potentially lethal events. We want to know: what establishes how prepared they are? To determine the mechanisms that control survival under harmful conditions, we are examining the effects of strong loss-of-function and null mutants in a collection of intercellular signaling receptors and transcription factors regulated by these receptors. We measure survival under oxidative stress and at high temperature using a "Lifespan Machine" cluster of flat-bed scanners [1]. This automated technology presents substantial advancements in throughput and sensitivity compared to manual methods. Using this approach, we have identified several signaling receptors and transcription factors that regulate survival under oxidative conditions. Interestingly, we identified both receptors and transcription factors that function to either confer or limit oxidative-stress resistance. This indicates that the combined level of activity of these genes sets the worm's readiness to cope with oxidative stress. We are currently investigating whether these genes act together or independently to determine the worm's normal level of resistance to various stressors, and the mechanisms that establish the normal activity levels of each of these genetic determinants of stress resistance. Reference: 1. Stroustrup N, Ulmschneider BE, Nash ZM, Lopez-Moyado IF, Apfeld J, Fontana W. The Caenorhabditis elegans Lifespan Machine. Nature Methods. 2013;10(7):665-70.

Schiffer, J. et al. (2019) International Worm Meeting "Sensory neurons specifically regulate hydrogen-peroxide protection by target tissues via a multistep hormonal relay."

Stumbur, Stephanie, Schiffer, J., Johnsen, Sean, Apfeld, Javier, Stanley, Julian, Servello, Francesco, Vogelaar, Abigail, Heath, William, McGowan, Natalie

[International Worm Meeting, 2019]

Hydrogen peroxide is a common weapon that species use for offensive purposes. Cells use conserved defense mechanisms to prevent and repair peroxide damage, but whether these responses are coordinated across cells in multicellular organisms is poorly understood. We determined whether sensory neurons control hydrogen-peroxide protective responses in C. elegans</em>. We measured the peroxide tolerance of a collection of sensory-neuron genetic-ablation strains using a Lifespan Machine assay and identified ten types of taste, smell, oxygen- and temperature- sensing neurons with significant positive or negative effects on peroxide tolerance. How do specific sensory neurons control whether target tissues protect the animal from hydrogen peroxide? Using genetic and site-of-action studies, we found that ASI sensory neurons secrete DAF-7, a TGF? hormone, to specifically lower hydrogen peroxide tolerance. The DAF-7 receptor, DAF-1, acts redundantly in at least three different sets of interneurons to lower peroxide tolerance. DAF-1 inhibits transcription by the DAF-3 coSMAD. We find this inversion of the ASI signal ensures that peroxide tolerance stays low until a decrease in DAF-7 causes multiple sets of interneurons to activate DAF-3. DAF-3 does not regulate peroxide tolerance via dauer, germline, or fat pathways. Instead, we find that by lowering the expression of DAF-2 insulin/IGF1 receptor ligands (Narasimhan et al., 2011; Shaw et al., 2007) DAF-3 triggers a peroxide-protective response via de-repression of DAF-16 FOXO transcription in neurons and intestine. Together, TGF? and insulin/IGF1 pathways form a hormonal relay that may enable ASI to sensitively induce a peroxide-protective response in target tissues. Both ingestion and perception of E. coli appear to regulate DAF-7 signaling. E. coli ingestion increases peroxide tolerance but inhibits DAF-3. As a result, DAF-3 alleviates the decrease in peroxide tolerance that occurs when E. coli ingestion is reduced. Perception of E. coli likely lowers the worm's peroxide tolerance via DAF-7, because E. coli promotes daf-7 expression in ASI (Chang et al., 2006; Entchev et al., 2015) . Why does E. coli perception by ASI lower the worm's peroxide tolerance? We find that hydrogen peroxide degrading enzymes from E. coli protect C. elegans from hydrogen peroxide killing. We propose that C. elegans uses sensory information to decide whether it needs to induce its own hydrogen-peroxide protection or bacteria will protect them instead.

Schiffer, Jodie et al. (2021) International Worm Meeting "Caenorhabditis elegans processes sensory information to choose between freeloading and self-defense strategies"

McGowan, Natalie, Tam, Hannah, Stroustrup, Nicholas, Amrit, Francis Raj Ghandi, Heath, William, Stanley, Julian, Vogelaar, Abigail, Schiffer, Jodie, Servello, Francesco, Martin, Olivier, Xu, Yuyan, Stumbur, Stephanie, Apfeld, Javier, Eder, Matthias, Johnsen, Sean, Serkin, William, Brennan, Sarah, Ghazi, Arjumand

[International Worm Meeting, 2021]

Hydrogen peroxide is the preeminent chemical weapon that organisms use for combat. Individual cells rely on conserved defenses to prevent and repair peroxide-induced damage, but whether similar defenses might be coordinated across cells and tissues in animals remains poorly understood. Here, we screen a collection of sensory neuron genetic ablations in the nematode C. elegans to determine their effects on resistance to peroxide. We identify a neuronal circuit that processes information perceived by two of those sensory neurons to control the induction of hydrogen peroxide defenses in the organism. In the presence of E. coli, C. elegans'food source, the animal's neurons signal via TGFb-insulin/IGF1 relay to target tissues to repress expression of catalases and other hydrogen peroxide defenses. We found that catalases produced by E. coli can compensate for the animal's downregulation of catalases by depleting hydrogen peroxide from the local environment and thereby protecting C. elegans. This adaptive strategy is the first example of a multicellular organism modulating its defenses when it expects to freeload from the protection provided by molecularly orthologous defenses from another species.

Broitman-Maduro, Gina et al. (2013) International Worm Meeting "Enhanced RNA virus susceptibility results from defects in lipid metabolism."

Ding, Shou-wei, Coffman, Stephanie, Guo, YuanYuan, Carranza, Francisco, Maduro, Morris, Pompura, Saige, Broitman-Maduro, Gina

[International Worm Meeting, 2013]

Prior work has shown that C. elegans can support replication of (+) strand RNA viruses, including the Flock House Virus (FHV), and the Orsay virus. We have previously reported that transgene expression of FHV RNA1, which encodes the viral RdRP and a viral suppressor of RNAi (B2), can direct its own replication (Lu et al., 2005). By replacing the coding region of B2 with that of GFP (FR1gfp) we can visualize viral replication in backgrounds that are virus-susceptible. In an RNAi screen for factors that influence replication of FR1gfp (see abstract by Stephanie Coffman), we identified genes that are known to affect storage or metabolism of lipids, such as nhr-68 (Ashrafi et al., 2003). To evaluate the relationship between lipid storage and RNA virus replication, we examined fat stores using Oil Red O in chromosomal mutants for other genes identified by the screen, and directly assessed viral replication in strains that are known to have fat storage defects. Our results confirm that conditions known to cause increases in fat stores, such as the germlineless glp-4(bn2) background, result in a significant increase in viral replication. As lipid storage defects have been recently shown to be correlated with autophagy (Hansen et al., 2013), we have tested the role of autophagy in lipid storage-mediated viral susceptibility. Overexpression of the lipase lipl-4 appears to be sufficient to increase viral susceptibility, in a manner that is partially dependent on the autophagy gene atg-1. The connection between lipid storage and susceptibility to RNA virus replication is not immediately obvious, and our data suggest that the interactions are complex, though one possibility is that autophagy regulates some components of the RNAi machinery (Gibbings et al., 2012). Results of characterization of viral replication (both FR1gfp and Orsay virus) in various mutant backgrounds will be presented.

Harris TW et al. (1997) International C. elegans Meeting "unc-26 ENCODES A PHOSPHOTIDYL INOSITOL-5-PHOSPHATASE"

Jorgensen EM, Harris TW

[International C. elegans Meeting, 1997]

Synaptojanin is a phosphotidyl inositol-5-phosphatase, which is highly enriched at nerve terminals and binds proteins implicated in synaptic vesicle endocytosis. Four overlapping ESTs isolated by Yuji Kohara are highly homologous to rat synaptojanin. These ESTs hybridize to YAC Y67H2 which spans the cosmid gap between cosmids JC8 and W02A2 on LG IV. Using the ESTs as a launch pad into the gap, we have been able to reconstruct the entire synaptojanin genomic region by screening phage libraries and by PCR crawling into regions not represented in any library. The genomic region comprising synaptojanin extends greater than 25kb. cDNA sequence reveals a protein conserved for each of the four synaptojanin domains, that is, the Sac1, the OCRL, the phosphatase, and the proline-rich domains. We have also identified cDNAs lacking both the phosphatase and proline-rich domains, and splice variants that add or remove a single 20bp exon in the OCRL domain. Within the JC8/W02A2 cosmid gap lies unc-26. unc-26 mutants are resistant to acetylcholinesterase inhibitors, have behavioral phenotypes similar to the endocytic mutants dyn-1, snt!1, unc-11, and unc-41, and are severely depleted for synaptic vesicles. These data suggest that UNC-26 may be required for synaptic vesicle endocytosis and thus is a likely candidate for the gene encoding synaptojanin. We have now demonstrated that unc-26 encodes synaptojanin by sequencing mutant alleles: s1710 (kindly provided by D. Baillie) is a frameshift in the fifth exon, and n1307 and n702 contain 600bp insertions in the first intron. Strikingly, the sequence of these insertions do not resemble Tc1-Tc5, and may represent a novel class of transposons active in the wild-type background. Given the large size of the locus and our current lack of upstream regulatory regions we have been unable to rescue unc-26. However, Stephanie Chissoe has isolated fosmids of this region using 5' regions of the gene as a probe, and we are currently determining the ability of these fosmids to rescue unc-26.

Grant BD et al. (1998) East Coast Worm Meeting "Endocytosis Mutants and The Yolk Receptor in C. elegans"

Zhang YH, Pedraza LG, Wallenstein E, Hirsh DI, Grant BD

[East Coast Worm Meeting, 1998]

We are using C. elegans genetics to look for new genes involved in receptor-mediated endocytosis. We have isolated 23 mutants, in 11 complementation groups, defective in the endocytosis of YP170-GFP by oocytes. Some of these mutants are temperature sensitive lethals. We anticipate that our mutants will identify key components of the general endocytic machinery as well as gene products that specifically mediate yolk uptake. The most severe YP170-GFP uptake defective mutants identify a gene encoding a putative receptor for vitellogenins (yolk proteins) in the C. elegans oocyte. During the normal maturation of oocytes within the C. elegans germline yolk proteins (YPs), encoded by the vit genes and secreted by the intestine, are actively endocytosed from the psuedocoelomic space and stored within membrane bound compartments sometimes called yolk platelets. A vit-2::gfp transgene, encoding YP170-GFP, has allowed us to visualize this process in vivo and study its properties by fluorescence microscopy. RNAi analysis in this genetic background allowed us to test the function of potential components of the C. elegans endocytic machinery identified by the genome project. This analysis indicates that YP170 endocytosis requires clathrin coated pit components (clathrin heavy chain, adaptins, dynamin) and regulators of endosomal function (rab5, rab7, rab11), as expected from studies of lipoprotein uptake in other organisms. &#9;One complementation group, comprised of three alleles, appears to mutate T11F8.c, a partially sequenced member of the LRP family of lipoprotein and proteinase receptors. We are currently confirming this inference by transformation rescue and sequencing of mutant alleles. RNAi data by Bossinger et al. (1997 IWM Abstract 61) had previously indicated a possible role for T11F8.c in yolk uptake. Our results show that T11F8.c RNAi blocks YP170-GFP uptake. We are tentatively naming this gene lrp-2, in anticipation of the complete genomic sequence which is in progress at the sequencing consortium (Many thanks to Bob Waterston and Stephanie Chissoe). Immunofluorescence experiments, using polyclonal antisera we produced against intracellular or extracellular LRP-2 fusion proteins, indicate that LRP-2 is an abundant plasma membrane protein expressed specifically in oocytes. Our putative lrp-2 mutants all show an absence or mislocalization of LRP-2 in oocytes. &#9;We are currently engaged in a more detailed phenotypic analysis of endocytic defects in the other ten complementation groups identified in our genetic screen utilizing anti-LRP-2 antibodies and other specific antisera we have developed against general endocytic pathway components identified in our RNAi screen (see abstract by Y. Zhang et al.).

Kuwabara PE et al. (1998) European Worm Meeting "VAB-17, A NOVEL WD-40 PROTEIN INVOLVED IN MORPHOGENESIS"

Kuwabara PE, Hodgkin JA

[European Worm Meeting, 1998]

cr7 was identified as a viable mutant defective in head morphogenesis, somewhat similar in phenotype to vab-1,-2, and -3 mutants. cr7 mutants are characterised by having a highly penetrant abnormal dorsal head bulge and a less penetrant tail bulge. The cr7 allele is unlikely to be null, because animals carrying cr7 in trans to a deficiency arrest as embryonic lethals. Based on these initial phenotypic observations, the gene identified by cr7 has been named vab-17. As a first step toward understanding the role played by vab-17 in C. elegans morphogenesis, we used positional cloning to identify the gene. vab-17 was genetically mapped to LGV; its physical location was further refined using PCR to characterise deficiency breakpoints. Cosmids spanning the region were tested for their ability to rescue vab-17(cr7) mutants, however, none of these cosmids appeared to rescue. Consequently, fosmid clones that partially cover a cosmid gap in the region were tested and some were shown to rescue vab-17 (fosmid information kindly provided by Stephanie Chissoe). Further analysis indicated that at least one of these fosmids also rescues the lethality associated with vab-17/Df. A 10 kb subclone (pPK333) was subsequently identified that also rescued the Vab phenotype; the sequence of this subclone led to the identification of several overlapping Kohara cDNAs. These cDNA clones (kindly provided by Y. Kohara) were found to carry alternatively spliced variants of a single gene. Surprisingly, the sequences of some of these cDNAs extended beyond the 5' end of the rescuing 10 kb subclone. RT-PCR was used to obtain additional cDNAs extending to the 5' end of the vab-17 transcript; vab-17 appears not only to have alternatively spliced mRNA variants, but also to have to have multiple 5' ends, each of which is trans-spliced to SL1. Based on our analyses of cDNA sequences, the vab-17 gene covers a region of 16.5 kb. The largest predicted protein encoded by the vab-17 locus has a molecular mass of 180 kDa. The sequence of this protein is mostly novel, but it contains a series of potential WD-40 repeats, a motif that has been shown to mediate the assembly of macromolecular complexes. Although pPK333 does not carry the entire vab-17 coding region, it is capable of rescuing vab-17(cr7). Therefore, in order to obtain a preliminary picture of VAB-17 expression, pPK333 was used to create an in-frame GFP fusion. The fusion protein appears to localise to the cytoplasmic face of adherens junctions, but unlike the adherens junction staining displayed by MH27, the VAB-17 fusion is not associated with embryonic hypodermal adherens junctions. Further characterisation of VAB-17 expression is in progress. In the course of genetically mapping vab-17, several marker genes were found to exhibit synthetic lethality with vab-17(cr7). Understanding the basis of this lethality may provide clues to the role vab-17 plays in morphogenesis. We have taken advantage of this synthetic lethality to identify a series of vab-17 intragenic revertants, which are weakly Vab, yet suppress the synthetic lethality. We are in the process of identifying the molecular lesions of these vab-17 mutants.