11 Papers found (0.009 seconds)

Akira Nukazuka et al. (2007) International Worm Meeting "Semaphorin - Plexin signal controls epidermal ray morphogenesis by stimulating mRNA translation via eIF2a in C. elegans."

Hajime Fujisawa, Mie Chikuma, Shin Takagi, Yoichi Oda, Toshifumi Inada, Akira Nukazuka

[

International Worm Meeting,

2007]

Semaphorins comprising a protein family in animals regulate diverse developmental events, and plexins serve as semaphorin receptors to transduce the signals into cytoskeletal reorganization. Genetic analysis has revealed that the semaphorin/plexin system regulates epidermal morphogenesis in C. elegans: in semaphorin (smp) and plexin (plx-1) mutants, epidermal cells in the larval male tails are aberrantly arranged, which results in an ectopic positioning of sensory ray 1 in the adult stage. We here report on in vivo evidence that mRNA translation is stimulated by SMP - PLX-1 signal via reduced phosphorylation of a translational regulator, eukaryotic initiation factor 2a (eIF2a). Higher phosphorylation of eIF2a, which was observed in smp / plx-1 mutants, reduced to the normal level by restoring the SMP - PLX-1 signal, implying that this signal is necessary to lower the eIF2a phosphorylation. Overexpression of a phosphomimetic eIF2a, as well as inactivation of translation machineries (eIF2a, eIF2b, eIF4G), produced the ray phenotype comparable to smp / plx-1 mutants, indicating that downregulated protein synthesis is attributed to the ray defect in mutants. On the other hand, lowered eIF2a phosphorylation caused by the loss-of-function mutations in GCN1 and PEK/PERK, both of which normally inhibit mRNA translation by participating in the eIF2a phosphorylation, suppressed the ray phenotype in smp / plx-1 mutants, suggesting that enhanced protein synthesis is sufficient for SMP - PLX-1 signaling. Thus, these data demonstrate an essential role for translation stimulation mainly via reduced eIF2a phosphorylation in SMP-induced proper epidermal ray morphogenesis. In our current research, we aim to identify target protein(s) that is synthesized in response to the SMP - PLX-1 signal. In this meeting, we will also make a progress report on this issue and further discuss the mechanism of cytoskeletal reorganization during the semaphorin/plexin-mediated cellular morphogenesis.

Akira Nukazuka et al. (2006) East Asia C. elegans Meeting "Semaphorin - Plexin signal controls epidermal ray morphogenesis by stimulating mRNA translation via eIF2α in C. elegans"

Toshifumi Inada, Shin Takagi, Yoichi Oda, Mie Chikuma, Akira Nukazuka, Hajime Fujisawa

[

East Asia C. elegans Meeting,

2006]

Semaphorins, which comprise a protein family in animal kingdom, regulate diverse developmental events including axonal guidance. Transmembrane protein plexins serve as receptors for semaphorins and transduce their signals into cytoskeletal reorganization. Genetic analysis has revealed that the semaphorin/plexin system regulates epidermal morphogenesis in C. elegans: in semaphorin (SMPs) and plexin (PLX-1) mutants, epidermal cells in the larval male tails are aberrantly arranged, which results in an ectopic positioning of sensory ray 1 in the adult stage. We here report on necessity and sufficiency of translation stimulation for SMPs - PLX-1 signaling unexpectedly by reduced phosphorylation of a master translational regulator, eukaryotic initiation factor 2α (eIF2α). In SMPs / PLX-1 mutants, higher phosphorylation of eIF2α was observed, implying downregulated global protein synthesis. Overexpression of a phosphomimetic form of eIF2α phenocopied SMPs / PLX-1 mutants, indicating that the elevated eIF2α phosphorylation is attributed to the ray defect in mutants. On the other hand, lowered eIF2α phosphorylation caused by the loss-of-function mutations in GCN1 and PEK/PERK, both of which normally inhibit mRNA translation by participating in the eIF2α phosphorylation, suppressed the ray phenotype in SMPs / PLX-1 mutants, raising a possibility that enhanced protein synthesis is sufficient for SMPs - PLX-1 signaling. Thus, these data indicate an essential role for translation stimulation via reduced eIF2α phosphorylation in SMPs-induced proper epidermal ray morphogenesis. In our current research, we aim to identify target protein(s) that is synthesized in response to the SMPs - PLX-1 signal. In this meeting, we will also make a progress report on this issue and further discuss the mechanism of cytoskeletal reorganization during the semaphorin/plexin-mediated cellular morphogenesis.

Birgit Gerisch et al. (2007) International Worm Meeting "RNAi screen for genes that regulate stress resistance and life span."

Adam Antebi, Birgit Gerisch, Hans Lehrach

[

International Worm Meeting,

2007]

Management of stress appears to be intimately related to the regulation of animal life span. Notably, most known long-lived mutants tested so far are resistant to various forms of environmental stress. In particular, long-lived mutants of the daf-2/insulin-like receptor (insulin/IGF-1 signaling) show increased resistance to heat and oxidative stress. We are using the correlation between longevity and stress resistance to identify genes that are involved in the process of life span regulation. We screened over 900 RNAis corresponding to phosphatases and kinases, as well as some kinase associated or regulated genes with wild type for enhanced survival under stress. We obtained 40 RNAis that result in strong resistance to oxidative stress, 37 with resistance to heat stress, and 6 with resistance to both. Some of the identified genes were previously shown to play a role in stress resistance and longevity, such as the guanylyl cyclase gcy-23 (Inada et al., 2006 and Hamilton et al., 2006). Selected candidate RNAis are currently being tested in aging experiments. In a similar approach we also screened the kinase RNAis for suppression of daf-2. Using a long-lived and stress resistant daf-2 mutant, we screened for reduced survival under heat and/or oxidative stress. Twelve candidates were identified and analyzed in aging experiments. Further characterization and assignment of the identified genes to pathways should yield insights into cellular processes involved in stress resistance and longevity.

Karla Opperman et al. (2007) International Worm Meeting "A structure-function analysis of the C. elegans L1CAMs."

Lihsia Chen, Xuelin Wang, Karla Opperman, Shan Zhou

[

International Worm Meeting,

2007]

Cell adhesion molecules play important roles during development and in maintaining tissue integrity. L1CAMs are a family of immunoglobulin (Ig)-like cell adhesion molecules that are important in vertebrate nervous system development. L1CAMs are conserved in C. elegans, and are encoded by the lad-1/sax-7 and lad-2 genes. SAX-7 and LAD-2 have distinct functions in the nervous system despite overlapping neuronal expression. SAX-7 is required to maintain neuronal positioning while LAD-2 participates in axon pathfinding. We are performing a structure-function analysis to determine how both proteins mediate their functions. Because the cytoplasmic tails between both proteins are distinct, we are focusing our analysis on how the cytoplasmic tail regulates L1CAM functions. Indeed, while the extracellular domains of SAX-7 and LAD-2 are conserved, the LAD-2 cytoplasmic tail is completely divergent and does not contain any of motifs conserved in SAX-7 and vertebrate L1CAMs. These motifs include the ankyrin-binding motif, which links L1CAMs to the spectrin-actin cytoskeleton, the PDZ-binding motif, and conserved tyrosine residues that are phosphorylated. Our preliminary results reveal that both conserved motifs, as well as phosphorylation one of the tyrosine residues, which regulates ankyrin binding, all contribute to the function of SAX-7 in neuronal position maintenance. (1) Chen L, Ong B, Bennett V. J Cell Biol. 2001 Aug 20;154(4):841-55. (2) Wang X, Kweon J, Larson S, Chen L. Dev Biol. 2005 Aug 15;284(2):273-91. (3) Sasakura H, Inada H, Kuhara A, Fusaoka E, Takemoto D, Takeuchi K, Mori I. EMBO J. 2005 Apr 6;24(7):1477-88. Epub 2005 Mar 17.

Kagoshima, Hiroshi et al. (2009) International Worm Meeting "Systematic analyses of AFD-specific enhancers in C. elegans."

Kajiwara, Junko, Kagoshima, Hiroshi, Kohara, Yuji

[

International Worm Meeting,

2009]

How can transcription factors control gene expression in the right place and at the right time? To investigate the mechanism of the cell-specific transcriptional regulation, we chose AFD thermosensory neuron for our target cell. We performed systematic deletion analyses of nine AFD-specific genes using promoter::GFP reporters, and narrowed down the DNA sequences required for AFD expression to relatively small regions (from 50 bp to 600 bp). Among these AFD-specific promoters, gcy-8 and gcy-18 promoters are particularly interesting, because these genes may have arisen from gene duplication and most probably they were controlled by the same regulatory mechanism. This notion is supported by following data: their expression were similar in spatial pattern (exclusively expressed in AFD) [Inada et al. 2006] and in temporal control (initiated at pre-comma stage), and were downregulated by either of the mutants, ttx-1 or ceh-14 (both of which encode homeobox transcription factors expressed in AFD) [Satterlee et al. 2001 and this work]. Moreover, the expression of gcy-8 and gcy-18 are completely abolished in ttx-1; ceh-14 double mutant background. We also showed that forced expression of both transcription factors in AWB chemosensory neuron could induce ectopic expression of gcy-8 and gcy-18 in AWB. These results suggest that ttx-1 and ceh-14 genes play pivotal roles in gcy-8 and gcy-18 expression. We found these promoters shared common 7-8 bp DNA motifs, which are conserved in five Caenorhabditis species (C. elegans, C. briggsae, C. brenneri, C. remanei and C. japonica). We are currently analyzing the promoters of gcy-8 and gcy-18 orthologs of the Caenorhabditis species in C. elegans. Further analysis will be reported at the meeting.

Kotaro Kimura et al. (2001) International C. elegans Meeting "Reverse genetic approach to reveal sensory signal transduction pathway involving TTX-4 nPKC-epsilon"

Kotaro Kimura, Yoshifumi Okochi, Makoto Suzaki, Kunihiro Matsumoto, Naoki Hisamoto, Ikue Mori

[

International C. elegans Meeting,

2001]

We found that TTX-4, a homolog of nPKC ("novel" PKC)-epsilon, is essential for sensation of environmental signals such as temperature, volatile and soluble compounds, and osmolarity (Okochi et al., this meeting). Biochemical analyses mostly based on mammalian cell cultures have revealed that nPKC species are activated only by diacylglycerol (DAG) or other lipids such as phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) or phosphatidylserine without requirement of calcium ion which is necessary for cPKC ("conventional" PKC) activation. Little evidence has been reported, though, to prove in vivo activation of nPKC by any of those lipids. Moreover, in vivo substrates of nPKC, whose activities are regulated by nPKC-dependent phosphorylation and required for transducing specific signal from nPKC, are poorly understood. To address these questions, we are undertaking reverse and molecular genetic approaches to identify upstream and downstream molecules of the TTX-4 nPKC-epsilon. We identified C. elegans homologs of candidate genes for nPKC pathway from the genome database, and have been making GFP reporter constructs of those genes. If the candidates are expressed in the same cells as those expressing ttx-4 , we will screen TMP/UV library for knockout mutants (Inada et al., this meeting). (I) Upstream candidates: DAG is a strong candidate molecule as an activator of TTX-4. DAG can be generated through hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC) or through dephosphorylation of phosphatidic acid (PA) by PA phosphatase (PAP). Thus, those enzymes are candidates for upstream gene products of TTX-4. We are currently analyzing expression pattern of these genes. (II) Downstream candidates and binding proteins: Several proteins have been reported to be phosphorylated by nPKCs in mammals. Biological significance of those phosphorylations however remain questionable. We are currently analyzing a couple of homologs for these proteins, beta'-COP (F38E11.5) and adducin (F57F5.4). Interestingly, the adducin (F57F5.4) is located adjacent to ttx-4 (F57F5.5) on the physical map. We are also screening yeast two hybrid library using wild type or kinase negative TTX-4 catalytic domain as a bait.

Hiroyuki Sasakura et al. (2010) East Asia Worm Meeting "Regulation of thermosentation by SRTX-1(GPCR) in AFD thermosensory neurons"

Keita Suzuki, Ikue Mori, Hiroko Itoh, Hiroyuki Sasakura

[

East Asia Worm Meeting,

2010]

C. elegans senses the environmental temperature by AFD and AWC sensory neurons. Genetic analysis suggested that signal transduction of thermosensory system in AFD and AWC is similar to that of olfactory and visual system in C. elegans and other animals (Mori et al., 2007; Kuhara et al., 2008). Based on the analogy to olfactory and visual system, it is plausible to hypothesize that G protein-coupled receptors (GPCRs) sense the environmental temperature. Previous reports showed that srtx-1 encoding GPCR is specifically expressed in sensory ending of both AFD and AWC neurons (Colosimo et al., 2004; Biron et al., 2008). We evaluated the expression of srtx-1 in detail by comparing fluorescence intensities of srtx-1promoter::GFP in AFD and AWC. Our results suggest that srtx-1 is expressed strongly in AFD, very weakly in AWC OFF neuron, and not expressed in AWC ON neuron. We generated two deletion mutants, srtx-1(nj62) and srtx-1(nj63), and found that both mutants exhibited impaired thermotaxis. Wild type animals migrated to the previous cultivation temperature after cultivation at 17, 20 or 23 degree on a temperature gradient. The srtx-1 mutants however migrated to colder region than the cultivation temperature after cultivated at 23 degree, and slightly but significantly migrated to colder region than the cultivation temperature after cultivation at 20 degree. By contrast, srtx-1 mutants migrated to higher region than the cultivation temperature after cultivation at 17 degree. The expression of strx-1cDNA only in AFD restored the abnormalities of thermotaxis. When strx-1cDNA was overexpressed in AFD of wild type animals, the distribution on a temperature gradient of animals cultivated at 17 and 20 degree was shifted to higher region. According to all these results, we propose that STRX-1(GPCR) is a key component for thermosensation in AFD, somewhat regulates temperature sense range.We thank D. Garbers forgcy-8p promoter, H. Oliver forceh-36 partial promoter, H. Inada for TMV-UV library, T. Kohashi for advice to assay systems.

Hiroko Ito et al. (2004) East Asia Worm Meeting "Genetic analysis of guanylyl cyclases and a cyclic nucleotide-gated cation channel involved in thermosensation"

John Satterlee, Piali Sengputa, Hitoshi Inada, Ikue Mori, Hiroko Ito

[

East Asia Worm Meeting,

2004]

C. elegans senses temperature mainly by a pair of sensory neurons (AFD) and shows a characteristic behavior towards the temperature called thermotaxis. Several mutants that are defective in thermotaxis have been isolated, and these mutants can be divided into three classes: cryophilic (cold-seeking), thermophilic (heat-seeking), and athermotactic (non-temperature responsive) phenotype. Analyses of these mutants identified several genes involved in thermotaxis. tax-4 encoding a cyclic nucleotide-gated cation channel (CNG channel) is essential for thermosensation in AFD. tax-4 is also required for olfaction by sensory neuron AWC, implicating similar components in thermosensory and olfactory signal transduction. In AFD, three guanylyl cyclase genes, gcy-8, gcy-18 and gcy-23 , play an essential role for thermosensation (see abstract by Inada et al .). Analysis of eat-16 mutant, which is defective in a regulator of G protein signaling (RGS), revealed that AWC olfactory neuron functions as a thermosensory neuron and that odr-1 guanylyl cyclase is involved in thermosensory signal transduction in AWC (Okumura et al ., submitted). To investigate genetic interaction of these four guanylyl cyclases and TAX-4 CNG channel, we constructed tax-4;gcy-23 gcy-8 gcy-18 and gcy-23 gcy-8 gcy-18;odr-1 quadruple mutants, and examined their thermotaxis phenotype. While gcy-23 gcy-8 gcy-18 mutants showed cryophilic and athermotactic phenotype, tax-4;gcy-23 gcy-8 gcy-18 mutants showed athermotactic phenotype like tax-4 mutants, suggesting that tax-4 is epistatic to gcy-8, gcy-18 and gcy-23 . gcy-23 gcy-8 gcy-18;odr-1 mutants showed athermotactic phenotype, although odr-1 single mutants showed almost normal thermotactic phenotype. This result suggests that ODR-1, which is an essential component in olfactory signal transduction, functions in thermosensory signal transduction in AWC. Our results are consistent with a molecular model in which three guanylyl cyclases (GCY-8, GCY-18 and GCY-23) and ODR-1 function upstream of TAX-4 CNG channel for thermosensation in AFD and AWC, respectively.

Hiroyuki Sasakura et al. (2007) International Worm Meeting "Analysis and screening of genes involved in thermosensory signaling."

Keita Suzuki, Hiroko Ito, Hiroyuki Sasakura, Ikue Mori, Takuma Sugi

[

International Worm Meeting,

2007]

C. elegans senses the environmental temperature by AFD and AWC sensory neurons. AFD neurons are specialized sensory neurons for temperature reception. AWC, known as olfactory neurons, also senses the temperature (Kuhara et al., this meeting). Genetic analysis suggested that signal transduction of temperature is similar to that of olfactory and visual system in C. elegans and other animals. Namely, cGMP produced by guanylate cyclases is used as second messenger and cGMP dependent cation channel TAX-2 and TAX-4 are crucial for activation of AFD and AWC. Although thermoreceptor itself is not identified, it is plausible that GPCRs sense the environmental temperature based on analogy to olfactory and visual system. Consistent with this possibility, G alpla ODR-3 and negative regulator of G protein EAT-16 are important for thermosensation in AWC sensory neurons (Kuhara et al., this meeting). GPCRs expressed in AFD and/or AWC become good candiates for thermoreceptors. It has been reported that B0496.5 encodes GPCR and expressed in sensory ending of both AFD and AWC (Colosimo et al., 2004). To examine B0496.5 gene function, we isolated two deletion mutants, nj62 and nj63. From deletion sites, both alleles are likely to be null. When nj62 and nj63 mutants are cultivated in 20 degree, they exhibited significantly smaller isothermal tracking than wild type. We also tested these mutants on linear thermal gradient. When nj62 and nj63 mutants are cultivated in 20 or 23 degree, they migrated to the lower temperature than wild type animals. From behavioral tests, B0496.5 gene likely has an important role on thermosensation. Overexpression experiments and physiological analysis are in progress. We also performed forward genetic screening to isolate new genes involved in AFD thermosensory signaling. The gene expression of nhr-38 is AFD specific and regulated by temperature stimulus. For example, the mutations of tax-4 or cmk-1 genes induce the downregulation of nhr-38::GFP (Satterlee et al., 2004; H.S., I.M., A.Kuhara, unpublished results). We utilized this phenomenon and isolated 12 suppressor strains that recover the gene expression of nhr-38::GFP in cmk-1 mutants. The behaviors of 11 strains are identical to athermotactic phenotype of cmk-1 mutants, while one suppressor strain IK673 exhibited neomorpholic, cryophilic phenotype. The gene responsible for this mutation was mapped on Ch I -0.69 to + 2.98. We thank T. Ishihara for nhr-38::GFP(H13::GFP), H. Inada for TMV-UV library, and CGC for cmk-1 mutants.

Sasakura, Hiroyuki et al. (2011) International Worm Meeting "Regulation of thermosensation by SRTX-1(GPCR) in AFD thermosensory neurons."

Suzuki, Keita, Mori, Ikue, Sasakura, Hiroyuki, Itoh, Hiroko

[

International Worm Meeting,

2011]

C. elegans senses the environmental temperature by AFD and AWC sensory neurons. Genetic analysis suggested that signal transduction of thermosensory system in AFD and AWC is similar to that of olfactory and visual system in C. elegans and other animals (Mori et al., 2007; Kuhara et al., 2008). Based on the analogy to olfactory and visual system, it is plausible to hypothesize that G protein-coupled receptors (GPCRs) sense the environmental temperature. Previous reports showed that srtx-1 encoding GPCR is specifically expressed in sensory ending of both AFD and AWC neurons (Colosimo et al., 2004; Biron et al., 2008). We evaluated the expression of srtx-1 in detail by comparing fluorescence intensities of srtx-1promoter::GFP in AFD and AWC. Our results suggest that srtx-1 is expressed strongly in AFD, very weakly in AWC OFF neuron, and not expressed in AWC ON neuron. We generated two deletion mutants, srtx-1(nj62) and srtx-1(nj63), and found that both mutants exhibited impaired thermotaxis. Wild type animals migrated to the previous cultivation temperature after cultivation at 17, 20 or 23 degree on a temperature gradient. Both srtx-1 mutants however migrated to colder region than the cultivation temperature after cultivated at 23 degree, and slightly but significantly migrated to colder region than the cultivation temperature after cultivation at 20 degree. By contrast, srtx-1 mutants migrated to higher region than the cultivation temperature after cultivation at 17 degree. The expression of strx-1cDNA only in AFD restored normal thermotactic behaviors. When strx-1cDNA was overexpressed in AFD of wild type animals, the distribution, on a temperature gradient, of the animals cultivated at 17 and 20 degree was shifted to higher region. Although srtx-1 mutants exhibited poorer Isothermal Tracking (IT) behavior, they retained the ability of IT behavior. Calcium imaging revealed that srtx-1 mutants showed lower response to temperature change but retained the response to temperature. These results suggest that thermosensory function of AFD is not completely but partially impaired in srtx-1 mutants. According to all these results, we propose that STRX-1 (GPCR) is a key component for thermosensation in AFD, somewhat regulates temperature sensing range. Given the recent finding that Rhodopsin, photoreceptor GPCR, functions in temperature discrimination in Drosophila larva (Shen et al., 2011), GPCR may be throughout evolution important for thermosensation. We thank D. Garbers for gcy-8p promoter, H. Oliver for ceh-36 partial promoter, C. Bargmann for str-1 and str-2 promoter H. Inada for TMV-UV library, T. Kohashi for improvement of assay systems.

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