Kim, E.J. et al. (2019) International Worm Meeting "CU-Eudragit-PVA nanoparticles for antiaging of dopamine neurons in Caenorhabditis elegans ."

Kim, E.J., Cho, J.H., Lee, J.E., Choi, S.S., de Guzman, A.C.V.

[International Worm Meeting, 2019]

Curcumin (CU) is a natural polyphenol found in Curcuma longa which has been used for foods and traditional medicines. CU exerts a wide range of health promotional benefits including antioxidant, antibacterial, anti-inflammatory, antitumor, anticancer and neuroprotective activities. The poor solubility of CU in aqueous solutions, however, has been a challenge for its clinical applications with bioavailability. In this study, we encapsulated CU into Eudragit S100-Polyvinyl alcohol (PVA) nanoparticles to increase its aqueous solubility and fed these nanoparticles into C. elegans to determine their neuroprotective efficacy. CU-Eudragit-PVA nanoparticles efficiently suppressed the aging or degeneration of dopaminergic neurons through the activation of anti-aging and dopamine metabolism genetic regulatory pathways. These results demonstrate that CU-Eudragit-PV nanoparticles have a potential for a therapeutic agent to inhibit neuronal dysfunctions in Parkinson's disease.

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.

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.

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.

Musset-Bilal F et al. (1996) East Coast Worm Meeting "Structural and functional similarities between mammalian and C. elegans SPARC proteins"

Musset-Bilal F, Schwarzbauer JE, Fitzgerald MC

[East Coast Worm Meeting, 1996]

The basement membranes of nematodes and vertebrates are composed of similar protein components including laminin, perlecan, type IV collagen, and SPARC. The sequence conservation of these proteins suggests that basement membrane structure and function are conserved. To determine the extent of the structural similarities between C. elegans and vertebrate SPARC proteins, we have raised polyclonal antibodies against a bacterial fusion protein containing one of the calcium binding domains of nematode SPARC. Immunoblots of whole nematode lysates and of NP40 soluble proteins showed that nematode SPARC is a 27 kD protein (1). When electrophoresed under nonreducing conditions, SPARC shows increased mobility demonstrating that it has compact, disulfidebonded structure. Similarly, treatment with N-glycosidase F, which removes complex carbohydrates, resulted in increased mobility. Therefore, SPARC is glycosylated, probably on the conserved asparagine residue in the cysteine-rich domain. Together these biochemical analyses show that nematode SPARC is structurally very similar to its vertebrate counterparts. In mammalian cell culture, excess SPARC protein has been shown to reverse cell adhesion and cause cell rounding. Overexpression of wild type SPARC in nematodes causes body deformities and paralysis which could result from defects in muscle cell adhesion (2). To determine the effects of SPARC on muscle cell structure, a transgenic line was generated that carries extra copies of the SPARC gene (ost-l) along with the rol-6(d) gene N2;Ex[ost-1;rol-6(d)]). L4 larvae from this line develop an Unc phenotype with a protruding vulva, paralysis, and sterility. These animals were stained with fluorescently labeled phalloidin to visualize actin filament organization. While wild type animals form distinct sex muscle cell structures that attach at the vulva, the N2;Ex[ost-l;rol-6(d)] animals show no actin organization in their sex muscle cells. We conclude that the vulval protrusion observed in the presence of excess SPARC results from improper cytoskeletal organization of the sex muscle cells. As actin filament structure is dependent on cell adhesion, these results support the idea that in these transgenic animals SPARC is disrupting muscle cell adhesion. 1. Schwarzbauer, J.E., F. MussetBilal, and C.S. Ryan (1994) The extracellular matrix associated protein SPARC/osteonectin in Caenorhabditis elegans. Meth. Enzymol. 245, 257-270. 2. Schwarzbauer, J.E. and C.S. Spencer (1993) The Caenorhabditis elegans homolog of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol. Biol. Cell 4, 941- 952.

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).

Morck, Catarina et al. (2011) International Worm Meeting "Soulless nematodes: KIN-29 mediates the essential function of the CAN neurons."

Garriga, Gian, Morck, Catarina

[International Worm Meeting, 2011]

The canal-associated neurons (CANL/R) are two bilaterally symmetric neurons that are born in the head and migrate to the middle of the embryo. Each neuron extends two processes: one process grows to the head and one to the tail (1). The CANs are essential for viability. Sulston and Hodgkin originally discovered that killing the CANs with laser microsurgery resulted in a starved appearance and larval arrest, leading them to propose that the CANs were the nematode soul (2). Similarly, ceh-10(gm58) mutants lack CANs and die as larvae (3). In vab-8(e1017) mutants, the CAN neurons fail to migrate, and their posterior processes only extend a short distance. As a consequence, the part of the body that lacks CAN processes becomes much thinner and the worms exhibit a withered tail phenotype (4). We identified a mutation that suppresses the withered tail phenotype of vab-8 mutants and the larval lethality of ceh-10 mutants. Surprisingly, the double mutants still retain defective CANs, which indicates that the suppressor mutation bypasses the requirement for CAN function. The suppressor encodes kin-29, a serine threonine kinase most homologous to members of the ELKL motif kinase (EMK) family and salt-induced kinase family (5). KIN-29 and its homologs phosphorylate and inhibit class II histone deacetylases. In C. elegans, KIN-29 inhibits HDA-4, which acts with the MADS domain transcription factor MEF-2 to regulate chemoreceptor gene expression (6). Loss of HDA-4 or MEF-2 is not lethal, indicating that KIN-29 must have other targets that mediate CAN function. The pseudocoelom fills with fluid in animals with compromised CAN function. This phenotype and the association of the CAN processes with the excretory canals led us to test the model where the CANs essential function is to regulate the activity of the excretory canals. The analysis of CAN and excretory canal mutants suggest that the CANs regulate other cells, and we are currently attempting to identify CAN target cells by addressing where KIN-29 functions. REFERENCES: 1.Sulston, J.E., E.Schierenberg, J.G. White and Thomson, J.N. (1983) Dev. Biol. 100: 64-119 2.Sulston, J.E. and Hodgkin, J.A. Worm Breeder's Gazette 5(1): 19 3.Forrester, W.C., Garriga, G. (1997) Development 124(9): 1831-43. 4.Manser, J. Wood, W.B. (1990) Dev Gen 11:49-64 5.Lanjuin, A., Sengupta, P. (2002) Neuron 33(3): 369-81 6.van der Linden, A.M., Nolan, K., Sengupta, P. (2007) EMBO. 26(2): 358-70.