Albert C Lee et al. (2005) International Worm Meeting "Probing the Role of APH-1 in Vulval Development and Function"

Caroline Goutte, Valerie A Hale, Albert C Lee, Julia M Claggett

[International Worm Meeting, 2005]

APH-1 is one of four components of the secretase activity responsible for the final intramembrane cleavage of Notch Receptor, -amyloid precursor protein, and a growing list of other substrates. In C. elegans, the aph-1 gene was identified based on its Notch-like mutant phenotype that leads to maternal-effect embryonic lethality. Reduction of aph-1 activity can also cause post-embryonic Notch-type phenotypes such as defects in germline proliferation and defects in vulval development. For example, the recessive aph-1(or28) mutation causes a fully penetrant egg laying defect which we have characterized as a failure in uterine pie cell induction, similar to that shown for sel-12 mutants, aph-2 mutants, and partial loss-of-function lin-12 mutants. The miss-specification of pie cells leads to a physical block between the uterine and vulval tissues in aph-1(or28) animals. In contrast, these animals are not defective in the AC-VU decision that is also mediated by the Notch receptor LIN-12. In order to better follow the role of APH-1 during vulval development we have characterized the expression pattern of an aph-1::gfp fusion construct, and found expression in neurons near the vulval region. The expression pattern does not correlate with a role for APH-1 in the well characterized events of AC-VU interaction or pie cell induction, but rather suggests yet another situation in which aph-1 activity may be involved. Thus far we have been unable to detect defects in neurons or neuronal components in aph-1 mutant worms, so we have turned to analyzing neuronal function by assaying the ability of aph-1 mutants to lay eggs in response to drug stimulation (for these studies we have used aph-1 mutants that do not cause physical blocks to egg laying, as well as aph-1 RNAi). Our progress on these analyses will be presented.

Hunter-Ensor M et al. (1998) East Coast Worm Meeting "DAUER-SPECIFIC BEHAVIOR"

Horvitz HR, Hunter-Ensor M

[East Coast Worm Meeting, 1998]

In response to environmental cues, C. elegans undergoes a switch in its development. Rather than committing to reproduction, the L1 larva responds to increased temperature, pheromone and decreased food supply by altering its physiology and committing to the formation of a third-stage dauer larvae. The dauer larvae is behaviorally distinct. Dauers do not feed, and they spend much of their time lying motionless on the plate. They are not dormant, however. They respond to touch and are capable of rapid locomotion when disturbed. In addition, dauers display a behavior not displayed by other larva or adult worms. Dauers will climb objects that project from the surface of a petri plate. For example, on moldy food-exhausted plates, many dauers can be seen climbing up mold stalks, holding on with their tails, and waving their heads in the air. This distinctive dauer-specific behavior, presumably for dispersal, is known as nictation. We would like to understand whether neuronal changes underlie dauer-specific behaviors. Previous work by Albert and Riddle1 indicates that sensory neuron morphology is altered in dauers and that these alterations are reversible, as they are not observed in post-dauer L4 and adult worms. We are looking for changes in neuronal morphology, synaptic structure and transmitter expression. We are also screening for dauer-specific behavioral mutants. 1 Albert, P.S. and Riddle, D.L., 1983, J. Comp. Neurol. 219: 461-481.

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.

Hsu, Jiun-Min et al. (2013) International Worm Meeting "A Neomorphic Mutation of mec-12/alpha-Tubulin Redirects Synaptic Vesicle Transport in C. elegans by Enhancing Dynein Activity."

Hsu, Jiun-Min, Pan, Chun-Liang, Garriga, Gian, Chen, Chun-Hao, Gurling, Mark, McDonald, Kent, Chen, Yen-Chih, Lee, Albert

[International Worm Meeting, 2013]

Polarized cargo transport into axons or dendrites is mediated by specific molecular motors. Synaptic vesicles (SV) are targeted to the axon by the plus end-oriented motor UNC-104/KIF1A. Since they are also associated with the minus end-oriented motor dynein, it is unclear why they are prevented from entering the dendrite, which contains abundant minus end-out microtubules. The C. elegans gene mec-12 encodes an a-tubulin enriched in the touch neurons that is required for SV transport. We identified a missense mutation of mec-12, gm379, which not only prevented SV from reaching synaptic regions, but also mistargeted them to the non-axon compartment of the PLM neuron. gm379 altered a conserved C-terminus glycine residue and behaved as a neomorphic mutation, as SV mistargeting was not seen in the mec-12 null and could be completely abolished by mec-12 RNAi. Interestingly, reducing UNC-104/KIF1A function worsened vesicle mistargeting, and excess UNC-104 partially rescued it. By contrast, elimination of the dynein heavy chain DHC-1 suppressed SV mistargeting and significantly rescued vesicle transport defects in gm379, mimicking the effects of UNC-104 overexpression. Amino acid substitution at G416 revealed a charge-based principle governing the tubulin selectivity for KIF1A or dynein, with excess negative charge enhanced microtubules-dynein interaction and reciprocally reduced microtubules-KIF1A interaction. Our work extends previous in vitro structural studies and reveals mechanistic insights on the electrostatic interactions between the molecular motors and the C-terminus of alpha tubulin in the regulation of polarized SV transport. This work is supported by a National Health Research Institute Career Development Grant (NHRI-EX101-10119NC), the National Science Council, Taiwan (NSC100-2320-B-002-095-MY3), and NTU (NTU-CDP-102R7810).

H Hsin et al. (1999) International C. elegans Meeting "Signals from the Reproductive System that Regulate the Lifespan of C. elegans"

CJ Kenyon, H Hsin

[International C. elegans Meeting, 1999]

Understanding how the aging process can be regulated is a fascinating and fundamental problem in biology. We have found that signals from the reproductive system influence the lifespan of the nematode C. elegans . If the cells that give rise to the germline are killed with a laser microbeam, the lifespan of the animal is extended (Hsin and Kenyon, Nature , in press). Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1-like pathway known to regulate the aging of this organism. Mutants with reduced activity of the insulin/IGF-1 receptor homolog DAF-2 have been shown to live twice as long as normal (1,2,3), and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators (1,2,4,5). We find that in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (6,7). In addition, our findings suggest that signals from the somatic gonad have an opposite effect on lifespan, and that this effect appears to require DAF-2 activity. Together our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the rate of aging of the animal. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity. 1. C. Kenyon, et al., Nature 366 , 461-4 (1993). 2. P. Larsen, P. Albert, D. Riddle, Genetics 139 , 1567-83 (1995). 3. K. Kimura, H. Tissenbaum, Y. Liu, G. Ruvkun, Science 277 , 942-6 (1997). 4. K. Lin, J. Dorman, A. Rodan, C. Kenyon, Science 278 , 1319-22 (1997). 5. S. Ogg, et al., Nature 389 , 994-9 (1997). 6. W-H. Yeh. Ph.D. Thesis, University of Missouri, Columbia (1991). 7. D. Riddle, P. Albert, in C. elegans II D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Eds. (Cold Spring Harbor Laboratory Press, 1997) pp. 739-68.

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.