Akwasi Anyanful et al. (2005) International Worm Meeting "Paralysis and killing of C. elegans by enteropathogenic E. coli requires the bacterial tryptophanase gene"

Daniel Kalman, Jennifer Dolan-Livengood, Lisa Kalman, Melanie Sherman, Mark DeZalia, Taiesha Lewis, Guy Benian, Akwasi Anyanful, Seema Sheth

[International Worm Meeting, 2005]

Enteropathogenic E. coli (EPEC), a major cause of food and water-borne disease causes high morbidity and mortality in developing countries. We have developed a model of EPEC virulence based on our observation that these bacteria paralyze and kill C. elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting mediation by a secreted toxin. Virulence however did require tryptophan and bacterial tryptophanase because lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyze or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a co-cultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal nonpathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyze and kill the nematodes. Furthermore, tryptophanase activity was required for full activation of LEE promoters, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including daf-2, age-1, hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes, including daf-16, mev-1, mek-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Hwang, W. et al. (2017) International Worm Meeting "Glutamine 5'-tRNA-drived small RNAs promote longevity via increasing mitochondrial activity through AMPK."

Shin, G.W., Nam, H.G., Seo, M., Lee, S.J.V., Kim, Y.E., Jung, G.Y., Hwang, W., Lee, Y., Ahn, G.O., Ha, C.M.

[International Worm Meeting, 2017]

Transfer RNA-derived small RNAs (tsRNAs) are abundant small non-coding RNAs discovered in various organisms, including C. elegans. Studies have begun to unravel the biological functions of tsRNAs, but their roles in aging processes remain elusive. Here we demonstrate the functional importance of specific tsRNAs for C. elegans longevity. We first determined age-dependent changes in tsRNA levels by analyzing available small RNA sequencing data (1) and confirmed the results by using Northern blot analysis. We found that the level of various tsRNAs gradually increased during aging. We overexpressed one of such tsRNAs, glutamine (Gln) 5'-tsRNA, by using an RNA polymerase III-responsive U6 (K09B11.12) promoter. Interestingly, we found that overexpression of the Gln 5'-tsRNA significantly extended lifespan. Our results suggest that the level of life-extending Gln 5'-tsRNA is increased during normal aging as a compensatory response. We then asked which longevity factors were required for the life extension. We showed that mutations in aak-2/the catalytic alpha subunit of AMP-dependent protein kinase (AMPK) fully suppressed the Gln 5'-tsRNA-mediated longevity. Through performing RNA seq. analysis, surprisingly, we found that the expression of all 12 mitochondrial DNA-encoded genes was highly increased by Gln 5'-tsRNA overexpression. We further showed that Gln 5'-tsRNA overexpression increased mitochondrial DNA contents in an AMPK-dependent manner and enhanced muscle-specific mitochondrial RFP intensity. Together, our data raised the possibility that age-dependent increases in Gln 5'-tsRNA levels delay aging and promote longevity by enhancing mitochondrial functions through AMPK. Currently we are testing whether our findings regarding the role of tsRNAs in mitochondrial function in C. elegans are conserved in mammals by using mice. Our study provides key information regarding anti-aging and mitochondria-enhancing functions of tsRNAs, which are universal small non-coding RNAs. (1) Kato et al., 2011, RNA

Reis-Rodrigues, Pedro et al. (2015) International Worm Meeting "N-acylethanolamines modulate C. elegans longevity at warm temperatures."

S. Gill, Matthew, Harrison, Neale, Reis-Rodrigues, Pedro

[International Worm Meeting, 2015]

The relationship between environment and longevity is perhaps best illustrated by the effect of temperature on lifespan across multiple species. Contrary to previous thought, it has been shown that temperature mediates its effects on lifespan in part through highly coordinated genetic pathways (Lee et al. 2009, Xiao et al. 2013). We have recently reported that the N-acylethanolamine (NAE) system in worms reduces C. elegans lifespan at 25oC but not at 15oC (Harrison et al. 2014), phenocopying the effect of ablation of the AFD thermosensory neuron (Lee et al. 2009). Here we show that deletion of faah-1, an NAE hydrolyzing enzyme, recapitulates the effect of over-expression of the biosynthetic enzyme nape-1. We also find that the effect of faah-1 deletion on lifespan is dependent on daf-9 and daf-12, indicating that the NAE signal acts via the same mechanism as that described for thermosensory mutants (Lee et al. 2009). In other systems, NAEs act as short-range signaling molecules and thus we hypothesized that NAEs directly influence neuronal activity to control lifespan cell non-autonomously. In accordance with this, we observed that genetic ablation of the ASJ neuron by cell-specific caspase expression rescues the effect of faah-1 on lifespan at 25oC. We are currently evaluating the requirement for thermosensory neurons in mediating the effects of faah-1 deletion.We are also interested in determining which specific NAEs are responsible for signaling this effect. NAEs are synthesized from phospholipid precursors and differ in their fatty acid moiety. We have previously reported that eicosapentaenoyl ethanolamine (EPEA), a C20:5n3-containing NAE, influences dauer entry and suppresses DR-induced longevity in worms. To test if this molecule could be responsible for the temperature-dependent effects of NAEs, we crossed nape-1 over-expressers into a fat-1 mutant, which lacks the fatty acid precursor needed for EPEA production. In this background we found that fat-1 is not required for the effects of nape-1 over-expression on lifespan, indicating that the effect of the NAE system on temperature-dependent longevity is independent of EPEA. We are currently trying to identify the specific NAE(s) responsible for this effect, as well as further defining the mechanism by which the NAE system affects lifespan in a temperature dependent manner.

Son, H.G. et al. (2017) International Worm Meeting "Prefoldin 6 promotes longevity in daf-2/insulin/IGF-1 receptor mutants via acting together with heat shock factor 1 (HSF-1)."

Baek, H., Lee, S.J.V., Jang, S.K., Choi, E., Nam, H.G., Park, S., Kim, E., Son, H.G., Seo, M., Ahn, G.O., Seo, K., Hsu, A.L.

[International Worm Meeting, 2017]

Heat shock factor 1 (HSF-1) is a master transcription factor that regulates cellular responses to proteostatic stresses. In C. elegans, HSF-1 promotes long lifespan by acting as a downstream factor of insulin/IGF-1 signaling (IIS) pathway. Here we aimed at identification of novel factors that regulate lifespan acting together with HSF-1. We first carried out a genome-wide modifier RNAi screen and identified 17 RNAi clones that enhanced the sterile phenotype of reduction-of-function hsf-1(sy441) mutants. Among them we found that pfd-6/prefoldin 6 was required for the longevity of daf-2/insulin/IGF-1 receptor mutants by using RNAi and pfd-6(gk493446) mutations. We found that HSP-70, one of direct target chaperones of HSF-1, physically interacted with PFD-6, by employing a split GFP system. Therefore, HSF-1 appears to increase the levels of HSP-70 that binds PFD-6, which contributes to the longevity of daf-2 mutants. We then showed that pfd-6p::pfd-6::GFP was expressed in the hypodermis and the intestine, where tissue-specific RNAi knockdown of pfd-6 significantly suppressed the longevity of daf-2(e1370) mutants. Next, we asked how PFD-6 contributed to longevity. PFD-6 is a component of prefoldin complex as well as R2TP (Rvb1, Rvb2, Tah1/TPR-containing protein, Pih1/Protein interacting with Hsp90)/prefoldin-like complex, which have multiple functions including those as chaperones. We found that many components of R2TP/prefoldin-like complex were partially but specifically required for the longevity of daf-2 mutants, whereas the majority of prefoldin complex components were not. Thus, PFD-6 appears to act as a component of R2TP/prefoldin-like complex to mediate longevity. In conclusion, our work suggests a novel mechanism by which PFD-6, a component of R2TP/prefoldin-like complex, regulates longevity acting together with HSF-1 in IIS pathway.

Lim, Daisy S. et al. (2019) International Worm Meeting "daf-42 is an Essential Gene for Dauer Development of Caenorhabditis elegans."

Kim, Wonjoo, Lee, Junho, Kim, Jun, Kim, Nari, Lim, Daisy S.

[International Worm Meeting, 2019]

Dauer is an important survival strategy for nematodes across free-living and parasitic nematodes. In C. elegans, L1 and L2d larvae are able to integrate surrounding environmental condition and choose to develop into dauer stage in harsh condition, surviving up to several months until they encounter favorable environment. While studies on dauer formation in C. elegans have provided insights in the decision processes prior to dauer commitment, the downstream factors that directly participate directly in dauer development have remained elusive. This is in part due to difficulty in generating dauer stage-specific lethal mutants by random mutagenesis. We have discovered a novel mutant strain that exhibits developmental defect specifically during dauer development and identified its causative nonsense mutation in the daf-42 gene. daf-42 mutants develop normally into reproducing adult animals under normal laboratory conditions, but become trapped in their own cuticle and fail to molt into dauer larvae in harsh conditions. daf-2; daf-42 double mutant animals started to develop into dead dauer corpses when dauers started to form in daf-2 mutants, and had the same dauer-commitment timepoint as that of daf-2 mutant. Analysis of RNA-seq data of larvae in dauer development (Lee et al, 2017) showed that daf-42 expresses briefly during dauer entry, and we will show its temporal and spatial expression. As daf-42 is already known to have homologs in other Caenorhabditis species, we seek to elucidate both molecular mechanism of daf-42 in dauer development and its evolutionary conservation among nematode species. Reference: Lee, J. S., Shih, P. Y., Schaedel, O. N., Quintero-Cadena, P., Rogers, A. K., & Sternberg, P. W. (2017). PNAS, 114(50), E10726-E10735.

Nayoung Suh et al. (2003) International Worm Meeting "Identifying GLD-2 target mRNAs in C. elegans"

Nayoung Suh, Judith Kimble

[International Worm Meeting, 2003]

We are interested in understanding the molecular controls of how germ cells are regulated to leave the mitotic cell cycle and differentiate. The gld-2 gene is a central player in this control in the nematode Caenorhabditis elegans (Kadyk and Kimble, 1998). The C. elegans GLD-2 protein belongs to the DNA nucleotidyltransferase superfamily; its closest homologs are S. pombe Cid1 and Cid 13, S. cerevisiae TRF4/TRF5, and classical eukaryotic poly(A) polymerase (PAP). Unlike conventional PAP, GLD-2 does not contain any apparent RNA binding motif. In the simplest model, GLD-2 acts as a heterodimer with the RNA binding partner (Wang et al., 2002). By interacting with different RNA binding proteins, GLD-2 PAP activity might target diverse specific mRNAs. Therefore, identifying target mRNAs of GLD-2 is necessary to understand how an essential regulator of the germ line governs its development and early embryogenesis. To identify target mRNAs of GLD-2, we will employ two different approaches. For the biochemical approach, we will co-immunoprecipitate GLD-2 associated mRNAs. This strategy has been used successfully to identify GLD-1 targets (Lee and Schedl, 2001). To date, we have made the DNA construct for an epitope-tagged GLD-2. We will establish the transgenic animal expressing this construct and test if it is functional. The other approach is to measure the poly(A) tail length of candidate target mRNAs to test for GLD-2-dependent polyadenylation. We will use a "Poly(A) test (PAT)" assay, a PCR-based technique that amplifies poly(A) tails. We are currently establishing the experimental techniques for examining the length of specific mRNA tails. Kadyk and Kimble (1998) Development 125, 1803-13; Lee and Schedl (2001) Genes Dev. 15, 2408-20; Wang et al. (2002) Nature 419, 312-16.

Lee, Myon-Hee et al. (2009) International Worm Meeting "FOG-3 phosphorylation by MAPK controls the C. elegans sperm fate."

Lee, Myon-Hee, Nykamp, Keith, Kimble, Judith

[International Worm Meeting, 2009]

FOG-3 TOB/BTG family protein is essential for sperm fate specification in C. elegans (Chen et al. 2000) and the primary C. elegans ERK/MAPK, known as MPK-1, has also been implicated in sperm fate specification (Lee et al. 2007). We have obtained several lines of genetic evidence that suggest a key role for MPK-1 in controlling the sperm fate. To learn how MPK-1 might function in this important cell fate decision, we examined the FOG-3 amino acid sequence and found a potential MAPK-docking site and four predicted MAPK phosphorylation sites (i.e S/TP). We then test the idea that MPK-1 might control the sperm fate by FOG-3 phosphorylation. In vitro, we have found that FOG-3 can be directly phosphorylated by murine ERK/MAP kinase; in vivo, we have generated a battery of FOG-3 transgenes and found that FOG-3 phosphorylation is critical for sperm fate specification. Importantly mammalian Tob is also a substrate of MAPK (Maekawa et al. 2002; Suzuki et al. 2002). We suggest therefore that the C. elegans germline may use an ancient regulatory cassette for control of the sperm/oocyte decision. References: Lee et al. (2007) Multiple functions and dynamic activation of MPK-1 extracellular signal-regulated kinase signaling in Caenorhabditis elegans germline development. Genetics 177, 2039-62. Maekawa et al. (2002) Identification of the anti-proliferative protein Tob as a MAPK substrate. J Biol Chem 277, 37783-7. Suzuki et al. (2002) Phosphorylation of three regulatory serines of Tob by Erk1 and Erk2 is required for Ras-mediated cell proliferation and transformation. Genes Dev 16, 1356-70. Chen et al. (2000) A novel member of the Tob family of proteins controls sexual fate in Caenorhabditis elegans germ cells. Dev Biol 217, 77-90.

Nicole F Liachko et al. (2005) International Worm Meeting "Characterization of putative DAF-16 target genes"

Siu Sylvia Lee, Nicole F Liachko

[International Worm Meeting, 2005]

The forkhead transcription factor DAF-16 is a key mediator of longevity, metabolism, and development. An informatic search using the known DAF-16 DNA recognition sequence to predict putative DAF-16 target genes has been performed (1). To further characterize the predicted DAF-16 target genes, we are optimizing a chromatin immunoprecipitation (ChIP) procedure using larvae and adult worms to demonstrate the presence of DAF-16 at the promoter of the target genes. In addition, we are using a promoter GFP fusion strategy to examine the expression of a subset of the DAF-16 target genes in worms. This approach allows study of when and where the target genes are expressed, as well as study of the dynamic regulation of the expression of the target genes in reponse to environmental stresses. Preliminary results show cell-type and developmental stage specific expression of the genes of interest. (1) S.S. Lee, S. Kennedy, A.C. Tolonen, G. Ruvkin, Science 300, 644 (2003).

Minna Roh et al. (2007) International Worm Meeting "Investigating the role of the Arp2/3 complex during gastrulation."

Minna Roh, Daniel J. Marston, Bob Goldstein

[International Worm Meeting, 2007]

Gastrulation is a crucial process during embryonic morphogenesis that shapes the body plan of organisms. Regulated cell movements are critical for gastrulation, and these movements require intricate coordination of cytoskeletal components. Although gastrulation is imperative for development, the molecular mechanisms underlying gastrulation cell movements in Caenorhabditis elegans are still largely unknown. Cellular actin architecture is remodeled based on signalling events and upstream actin regulators in diverse cell types. One such regulator is the Arp2/3 complex. This complex acts to nucleate new actin filaments off existing actin filaments and, thus, affects overall actin organization. In C. elegans, gastrulation is initiated by the ingression of two endodermal precursor cells, Ea and Ep. The current model for how this ingression event occurs is through constriction of the apical region of the E cells, thereby pulling in the neighbouring cells such that they fill in the gap left by the E cells as they ingress (Lee and Goldstein, 2003). Myosin is activated in the apical region of the E cells, as indicated by an accumulation of phosphorylated myosin light chain (Lee et al., 2006). Severson et al., in 2002, discovered that depleting C. elegans Arp-related proteins, ARX-2 and ARX-1, results in gastrulation defects. In these embryos, gastrulation is not initiated and the E cells divide on the surface of the embryo. We would like to understand the effects of Arp2/3 components during gastrulation and on actin regulation in the early embryo. We have found that in Arp2/3 depleted embryos, E cells are polarized normally. In addition, myosin is activated in the apical region of the E cells, but the E-cells still fail to ingress. Surprisingly, using live imaging techniques to visualize actin, we found that specific neighbouring cells extend short Arp2/3-dependent actin-rich structures near their apical borders with the E cells. These dynamic extensions occur where cell flattening has previously been reported by SEM (Nance and Priess, 2002). Thus, these results suggest a new layer to our existing model of C. elegans gastrulation, in that in addition to apical constriction, E cell ingression may also involve Arp2/3-dependent, F-actin-rich extensions from specific neighbouring cells.