Tristan Kooistra et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Tissue-specific TIR-1/NSY-1/SEK-1-dependent MAPK Activation Confers Pathogen Resistance Through Neuroendocrine Signaling in C. elegans"

Tristan Kooistra, Robert Shivers, Dennis Kim

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

A conserved TIR-1/NSY-1/SEK-1/PMK-1 MAPK pathway is required for immunity in C. elegans. We investigated the tissue-specific functions of TIR-1/NSY-1/SEK-1-dependent MAPK signaling in response to pathogen infection. We determined that TIR-1/NSY-1/SEK-1-dependent MAPK signaling in the intestine provides a major contribution to pathogen resistance, but does not completely restore pathogen resistance. Surprisingly, we defined an additional tissue-specific role for TIR-1/NSY-1/SEK-1-dependent MAPK signaling in chemosensory neurons in resistance to pathogen killing. We are currently defining the neuroendocrine signaling circuitry that functions downstream of TIR-1/NSY-1/SEK-1-dependent MAPK signaling to confer pathogen resistance in C. elegans.

Bryan T. Phillips et al. (2007) International Worm Meeting "Reciprocal asymmetry of SYS-1/b-catenin and POP-1/TCF controls asymmetric divisions in C. elegans."

Jeff Hardin, Ryan King, Judith Kimble, Ambrose R. Kidd III, Bryan T. Phillips

[International Worm Meeting, 2007]

<font face=symbol>b</font>-catenins are conserved regulators of metazoan development that function with TCF DNA-binding proteins to activate transcription. SYS-1/<font face=symbol>b</font>-catenin and POP-1/TCF regulate several asymmetric divisions, including that of the somatic gonadal precursor cell (SGP). In the distal but not the proximal SGP daughter, SYS-1/<font face=symbol>b</font>-catenin and POP-1/TCF transcriptionally activate ceh-22 to specify the distal fate. Using a rescuing transgene, VNS::SYS-1, which fuses VENUS fluorescent protein to SYS-1, we find more VNS::SYS-1 in distal than proximal SGP daughters, a phenomenon we call "SYS-1 asymmetry." In addition, SYS-1 asymmetry is seen in many other tissues, consistent with the idea that SYS-1 regulates asymmetric divisions broadly during C. elegans development. In particular, SYS-1 is more abundant in E than MS, and SYS-1 is critical for the endodermal fate. In all cases, SYS-1 is reciprocal to POP-1 asymmetry: cells with higher SYS-1 have lower POP-1, and vice versa. SYS-1 asymmetry is controlled posttranslationally and relies on frizzled and dishevelled homologs, which also control POP-1 asymmetry. Therefore, upstream regulators modulate the SYS-1 to POP-1 ratio by increasing SYS-1 and decreasing POP-1 within the same cell. By contrast, SYS-1 asymmetry does not rely on WRM-1, which appears specialized for POP-1 asymmetry. We suggest a two-pronged pathway for control of SYS-1:POP-1, which can robustly accomplish differential gene expression in daughters of an asymmetric cell division. Our data suggest SYS-1 asymmetry is regulated at the level of protein stability, with increased SYS-1 degradation in those daughter cells with high nuclear POP-1. Studies testing this hypothesis are currently ongoing and will be presented at the meeting.

Tomoaki Mizuno et al. (2007) International Worm Meeting "The C. elegans adaptor protein SHC-1 regulates KGB-1 MAPK pathway in stress response."

Kunihiro Matsumoto, Naoki Hisamoto, Tomoaki Mizuno, Aya Sasakawa

[International Worm Meeting, 2007]

The JNK MAP kinase (MAPK) pathway plays a pivotal role in the various stress responses of evolutionarily diverse species. In C. elegans, a JNK-like MAPK signaling pathway composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 JNK-like MAPK controls stress response. The KGB-1 cascade is inactivated by VHP-1 MAPK phosphatase. The vhp-1 loss-of-function mutation or expression of constitutively activated MEK-1(SSEE) causes hyperactivation of the KGB-1 pathway, resulting in lethality. However, components mediating this pathway remain unknown. Recently, mapping of the C. elegans interactome network has revealed that MEK-1 physically interacts with SHC-1, an adaptor protein highly homologous to mammalian Shc. We have conducted the genetic and biochemical analysis to elucidate the function of SHC-1 in the KGB-1 cascade. We found that the shc-1 deletion mutation is defective in activation of KGB-1 and results in hypersensitivity to heavy metals, whose phenotypes are also observed in mlk-1 and mek-1 mutants. The shc-1 mutation can suppress the lethality caused by the vhp-1 mutation but not that by expression of MEK-1(SSEE). This suggests that SHC-1 acts at the level of MEK-1. Consistent with this possibility, SHC-1 is necessary for activation of MEK-1. Thus, the C. elegans Shc protein plays a critical role in stress response by mediating activation of MEK-1 in the KGB-1 MAPK signaling pathway.

Lin, Rueyling et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "WRM-1 functions as a scaffold for phosphorylation of POP- 1 by LIT-1 kinase"

Huang, Shuyi, Robertson, Scott, Lin, Rueyling

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

During C. elegans embryogenesis, Wnt and MAP kinase signaling pathways specify the endoderm precursor E. We showed previously that whereas Wnt increases the level of ?-catenin/SYS-1, MAP kinase reduces the level of TCF/POP-1, resulting in a robust increase in the SYS-1-to-POP ratio and transcriptional activation of endoderm genes in E. The ?-catenin SYS-1 was shown to bind to the conserved N-terminus of POP-1, functioning as an essential coactivator for POP-1 to mediate Wnt signaling. A second ?-catenin protein, WRM-1, is also required for E specification. WRM-1 does not, however, bind to the N-terminal domain of POP-1, nor does it function as a coactivator of POP-1. Instead, WRM-1 has been shown to be an obligate component of the LIT-1 kinase complex, together phosphorylating POP-1 which promo tes its nuclear export, resulting in lowered nuclear POP-1 levels in signal-receiving cells. While the requirement of WRM-1 for LIT-1 activity has been clearly established, the molecular mechanism by which WRM-1 regulates LIT-1 kinase activity remains unclear. We show here that WRM-1 functions as a scaffold protein that brings POP-1 and LIT-1 into close proximity. Unlike SYS-1 binding to the N-terminal domain, WRM-1 binds to the C-terminal domain of POP-1. POP-1 deleted of the C-terminal domain does not bind to WRM-1, nor does it exhibit LIT-1-mediated phosphorylation in tissue culture cells. In vivo, POP-1 delta C is symmetric between A-P nuclei, and has a reduced activity, both in repression and activation. We show that binding between WRM-1 and POP-1 is regulated: binding decreased when LIT-1 kinase was coexpressed in mammalian tissue culture cells, with the decrease preferentially in hyperphosphorylated POP-1. An epitope at the C-terminal domain is differentially p resented in different populations of POP-1, suggesting a possible mode of regulation in vivo.

Huaqi Jiang et al. (2002) Mid-west Worm Meeting "An essential function for AHA-1: Evidence that AHA-1 dimerizes with CKY-1 in the pharynx"

Shu Wu, Huaqi Jiang, Jo Anne Powell-Coffman

[Mid-west Worm Meeting, 2002]

The basic-helix-loop-helix-PAS (bHLH-PAS) proteins are a family of transcription factors that mediate diverse processes, including cellular adaptations to environmental signals and developmental cell fate decisions. The C.elegans genome encodes 5 bHLH-PAS proteins. These include AHR-1, HIF-1, and AHA-1. The AHA-1 protein dimerizes with multiple bHLH-PAS partners and is expressed in most, if not all, somatic cells. Although ahr-1, hif-1 double mutants are viable, animals homozygous for null mutations in aha-1arrest development as young larvae. To understand the essential functions of aha-1, we examined the expression of the two remaining bHLH-PAS genes, T01D3.2 and cky-1/C15C8.2. A T01D3.2:GFP reporter is expressed in two neurons. CKY-1:GFP is expressed in most non-neuronal pharyngeal cells. These data suggested two non-exclusive models to explain the essential developmental function of AHA-1: i) AHA-1 might form a complex with CKY-1 in pharyngeal cells; or ii) AHA-1 might function as a homodimer or independently of other bHLH-PAS proteins. In support of the first model, AHA-1 and CKY-1 form a DNA-binding complex in vitro, and AHA-1 is localized to the nucleus of cells that express CKY-1:GFP. To determine whether expression of AHA-1 in the pharynx could rescue aha-1-deficient animals, we constructed a cky-1:aha-1 chimeric gene, in which cky-1 5' regulatory sequences directed the expression of the aha-1 transcript. We injected cky-1:aha-1 into the aha-1mutant background, thereby creating animals mosaic for AHA-1 expression. The transgenic animals were viable and fertile. We conclude that AHA-1 function is essential, and we propose that AHA-1 and CKY-1 have a role in pharyngeal function.

Clari Valansi et al. (2001) International C. elegans Meeting "GENETIC INTERACTIONS BETWEEN TWO CELL FUSION MUTANTS"

Benjamin Podbilewicz, Clari Valansi, Tamar Gattegno

[International C. elegans Meeting, 2001]

Hypodermal cells undergo programmed cell fusion to generate multi-nucleated cells during embryonic development in C.elegans . Analysis of mutants that have epithelial cells that fail to fuse provides an opportunity to clone the genes involved. duf-1 ( zu316cs ) (Dorsal UnFused) is a zygotic lethal mutant that arrests at 1.5 to 2- fold stage of elongation with fewer dorsal hypodermal cell fusions (1). Strains expressing jam-1 ::GFP fusion protein that is localized to the adherens junctions of epithelial cells were used for fusion analysis on living animals that were also analyzed by Nomarski optics. eff-1 (hy21) is a recessive viable mutant with lumpy, Dpy, Pvl, and Egl phenotype that is defective in all the epithelial cell fusion processes (e.g. vulva, embryonic hypodermis, Pn.p cells, seam cells.) (2) To investigate the possible genetic interactions between eff-1 and duf-1 we constructed and analyzed an eff-1 ( hy21ts ) homozygous duf-1 ( zu316cs ) heterozygous strain. We found that duf-1 /+; eff-1 segregates 25% duf-1;eff-1 embryos that arrest with all hypodermal cells unfused and 75% eff-1 viable unfused animals. Since the duf-1 ; eff-1 arrested embryos had a 100% unfused hypodermis (both dorsal and ventral) and eff-1 ; duf-1 /+ were viable unfused identical to eff-1 , thus, eff-1 is epistatic to duf-1 concerning hypodermal dorsal fusion while duf-1 is also independently required for embryonic viability. In summary, duf-1 embryos arrested with partially unfused dorsal hypodermis while the duf-1; eff-1 embryos arrested with completely unfused hypodermis. Both eff-1; duf-1 /+ and eff-1 worms were viable with the Eff (Epithelial Fusions Failed) phenotype. We propose that duf-1 controls dorsal epidermal fusions by regulating the activity of eff-1 that may be a general Effector of Fusion active in all epithelial cells. 1. Gattegno T. and Podbilewicz B. IWM (1999) Abstract, p. 328. 2. Shemer et al. Mohler et al. (2001) 13 th International C. elegans Meeting. duf-1 ------> eff-1 ------> dorsal hypodermal fusion

Ken-ichi Ogura et al. (2001) International C. elegans Meeting "Translational control of maternal glp-1 mRNA by POS-1 and its interacting protein PIP-1"

Yuji KOHARA, Ken-ichi Ogura, Keiko Gengyo-Ando, Shohei MItani

[International C. elegans Meeting, 2001]

A maternal factor POS-1 regulates cell fate determination of early embryos (Tabara et al., Development, 126, 1-11, 1999). POS-1 has two TIS11 type (CCCH) zinc finger motifs, and is present in cytoplasm of posterior blastomeres, being a temporary component of P granules. At the previous meeting (12th IWM, abstract 83, 1999), we reported that POS-1 binds to PIP-1 that has an RNP type RNA binding motif. PIP-1 is present in cytoplasm of oocytes and early embryos, also being a temporary component of P granules. RNAi analysis showed that PIP-1 is also required for cell fate determination of early embryos. This time, we have isolated a deletion null mutant pip-1(tm291) and found that the phenotypes are identical to those of RNAi, and that POS-1 and PIP-1 regulate maternal glp-1 mRNA translation. Translation of the maternal glp-1 mRNA is temporally and spatially regulated by the 3' UTR (Evans et al., Cell, 77, 183-194, 1994). In wild type embryos, GLP-1 begins to be detected in anterior AB blastomeres at two-cell stage. At four-cell stage, GLP-1 is only detected in anterior blastomeres ABa and ABp, but not in posterior EMS and P2. We found that GLP-1 was not detected in pip-1 embryos, and that GLP-1 was ectopically detected in the posterior blastomeres in pos-1 embryos. At four-cell stage embryos of pos-1 , GLP-1 was detected in posterior EMS and P2 in addition to anterior ABa and ABp. GLP-1 was not detected in pos-1 oocytes and one-cell stage embryos, suggesting that the temporal regulation is normal. We examined POS-1 localization in pip-1 , and PIP-1 localization in pos-1 , and found that their localizations were unchanged. These results suggest that PIP-1 is a positive regulator and POS-1 is a negative regulator of the translation of maternal glp-1 mRNA. In pos-1; pip-1 double RNAi embryos, the GLP-1 localization was identical to that of pos-1 embryos, suggesting that pos-1 is genetically epistatic. Finally, by using the three hybrid system, we found that POS-1 specifically interacted with spatial control region (SCR) identified in the 3' UTR of glp-1 mRNA (Rudel and Kimble, Genetics, 157, 639-654, 2001), but PIP-1 did not. We will discuss possible mechanisms of the translational regulation of maternal glp-1 mRNA.

Ayuna Hattori et al. (2010) East Asia Worm Meeting "The C. elegans JNK signaling pathway regulates FOS-1 transcription factor in stress response"

Tomoaki Mizuno, Ayuna Hattori, Naoki Hisamoto, Kunihiro Matsumoto

[East Asia Worm Meeting, 2010]

The JNK MAP kinase (MAPK) pathway plays a pivotal role in the various stress responses of evolutionarily diverse species. In C. elegans, a JNK-like MAPK pathway composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK controls stress response and larval development. The KGB-1 pathway is negatively regulated by VHP-1 MAPK phosphatase. The vhp-1 loss-of-function mutation causes hyperactivation of the KGB-1 pathway, resulting in larval arrest phenotype. To identify the novel downstream factors regulated by KGB-1, we performed yeast two-hybrid screening using KGB-1 as a bait. As a result, we isolated FOS-1, a bZIP transcription factor homologous to human Fos, as a KGB-1 binding protein. Further biochemical analysis showed that FOS-1 is phosphorylated by KGB-1 at Thr-304. In addition, the knock down of FOS-1 by RNAi suppressed the stress sensitivity of kgb-1 mutants. Overexpression of the phospho-defective form of FOS-1 caused heavy metal sensitivity and suppressed the vhp-1 mutant phenotype, while the phospho-mimetic of FOS-1 causes larval arrest. These results suggest that KGB-1 negatively regulates FOS-1 through phosphorylation of FOS-1 at Thr-304.

Chiang, Wei-Chung et al. (2009) International Worm Meeting "Regulation of longevity by two novel heat-shock factor-1 regulators, ddl-1 and ddl-2."

Hsu, Ao-lin, Lee, Hee-chul, Chiang, Wei-Chung, Ching, Tsui-ting

[International Worm Meeting, 2009]

Heat-shock transcription factor-1 (HSF-1) acts as a cellular survival factor against various stresses, including but not limited to the heat stress. The activation of HSF-1 increases thermotolerance and longevity in C. elegans. However, how HSF-1 response to stress and influence longevity at molecular level remains largely unclear. Recently, two genes, ddl-1 and ddl-2 (ddl, daf-16 dependent longevity), were shown to influence lifespan and thermotolerance in C. elegans when inactivated by RNAi. To confirm this observation, we analyzed the lifespan of animals overexpressing ddl-1 and ddl-2. Consistent with previous findings, overexpression of ddl-1 or ddl-2 shortens lifespan. It has been previously suggested by yeast-two hybrid results that DDL-1 may physically interacts with DDL-2 and heat-shock factor binding protein-1 (HSB-1), a known negative regulator of HSF-1. Therefore we asked whether ddl-1 and ddl-2 regulate longevity by modulating hsf-1 activity. Our result showed that ddl-1 and ddl-2 RNAi fails to increase lifespan in hsf-1 mutant (sy441), suggesting that hsf-1 functions downstream of ddl-1 and ddl-2. Our biochemical evidences also showed that knock-down of ddl-1 and ddl-2 by RNAi increases HSF-1 activity. Taken together, our data suggest that DDL-1 and DDL-2 might influence longevity by regulating HSF-1 activity. We hypothesize that DDL-1and DDL-2 regulate HSF-1 activity by forming a complex with HSF-1, and this complex is important for keeping HSF-1 in an inactive form. Our preliminary results have confirmed the interaction between DDL-1 and HSF-1; DDL-1 and HSB-1; DDL-1 and DDL-2 by co-immunoprecipitation. By understanding the role of DDL-1 and DDL-2 in regulating HSF-1, we will be able to reveal the underlying mechanism by which HSF-1 regulates heat-shock response and promotes longevity in C. elegans.

Kiyokazu Morita et al. (2002) West Coast Worm Meeting "Identification of the downstream genes of lon-1"

Kiyokazu Morita, Min Han

[West Coast Worm Meeting, 2002]

DBL-1 regulates the body length of C. elegans dose dependent manner 1) . DBL-1 negatively regulates the expression level of LON-1 2) . Overexpression of DBL-1 causes Lon phenotype like lon-1 , and overexpression of LON-1 causes Sma phenotype like dbl-1 . LON-1 has evolutionary conserved protein domain from yeast, plant to human, but its biological function is not clear. To understand the mechanism of LON-1, we try to get the genes downstream of lon-1. lon-1 overexpressed allele nkIs10 shows Sma phenotype like dbl-1. We screened the suppressor of Sma phenotype by EMS mutagenesis. Over the 50, 000 genomes, we identified 5 Lon and 10 wild type body length suppressores. We are currently mapping them. 1)K. Morita, et al (1999) Development 2)K. Morita, et al (2002) EMBO. J