17 Papers found (0.019 seconds)

Adachi, Takeshi et al. (2015) International Worm Meeting "mRNA decapping enzyme DCAP-2 is involved in morphological diversification of cilia."

Iwasaki, Syunsuke, Adachi, Takeshi, Tanabe, Syoichi, Izumi, Susumu, Nagahama, Keigo

[

International Worm Meeting,

2015]

Cilia are microtubule-based protruding organelles observed in cell surfaces. In C. elegans, non-motile cilia are associated with amphid and phasmid sensory neurons to sense external stimuli. These cilia differ in morphology and the number between cell types. ASE has a single rod-like cilium, whereas ADL has dual cilia. Cilia of AWB and AWC are shaped like a wing. These characteristics of cilia are strictly determined according to cell fates. To understand molecular mechanisms that regulate the number of cilia, we screened mutants with the reduced number of ADL cilia. We revealed that loss of dcap-2, which encodes a putative mRNA decapping enzyme, causes reduction of the number of ADL cilia. mRNA decapping enzymes catalyze hydrolysis of mRNA caps. Shapes of AWB and AWC cilia are also affected in dcap-2 mutants, whereas ASE cilium morphology appears normal. The results suggest that dcap-2 is involved in morphological diversification of cilia rather than general ciliogenesis mechanisms. To evaluate chemosensory sensitivity of AWC in dcap-2 mutants, we analyzed intracellular localization of EGL-4/cGMP-dependent protein kinase. In dcap-2 mutants, EGL-4::GFP fusion protein is localized in AWC nuclear as well as cilia malformed mutants. These results suggest that AWC chemosensation is affected in dcap-2 mutants.

Takeshi Adachi et al. (2005) International Worm Meeting "A Suppressor Screen for Genes that Regulate Salt Chemotaxis Learning in C. elegans."

Takeshi Adachi, Masahiro Tomioka, Yuichi Iino, Hirofumi Kunitomo

[

International Worm Meeting,

2005]

C. elegans shows plasticities of chemotaxis in response to changing environmental conditions. Wild-type animals are attracted to NaCl, but show avoidance behaviors after a conditioning with NaCl and starvation (food-/NaCl+). This behavioral plasticity, salt chemotaxis learning, is not induced under the food-/NaCl- or food+/NaCl+ conditions, and is therefore likely to be a form of associative learning (1). We have shown that the insulin-like signaling pathway, which is known to regulate dauer formation, longevity, and fat metabolism, regulates salt chemotaxis learning. Several mutants of ins-1, daf-2 and age-1, which encode homologues of insulin peptide, insulin/IGF-I receptor and PI 3 kinase (PI3K), respectively, showed learning defects; these mutants showed positive chemotaxis to salt even after pre-exposure to the salt (2). The loss-of-function mutants of daf-18, which encodes PTEN phosphatase, an enzyme that catalyzes a reverse reaction of PI3K, showed reduced chemotaxis to salt even without pre-exposure to salt. age-1 mutations suppressed the reduced chemotaxis of daf-18 mutants, and conversely, daf-18 mutations suppressed the learning defect of age-1 mutants. This suggests that the balance between AGE-1 PI 3 kinase and DAF-18 PTEN phosphatase regulates the extent and orientation of salt chemotaxis. To identify genes that regulate salt chemotaxis learning, we screened for suppressors of the reduced chemotaxis phenotype of daf-18(e1375) mutants. We mutagenized daf-18(e1375) with EMS and screened about 15,000 genomes. Through this screening, we isolated 23 mutants with improved chemotaxis. Of these suppressors, 11 mutants also showed learning defects in the daf-18(e1375) background. When tested in the daf-18(+) background, one mutant, JN914, showed a strong defect and several others showed weak defects in learning. We mapped JN914 to the left arm of chromosome I using snip-SNPs. JN914 exhibited hyperactive locomotion and constitutive egg-laying phenotype in a dominant fashion. These phenotypes are similar to gain-of-function mutants and overexpression strains of egl-30, which maps to this interval. Furthermore, the egl-30 overexpression strain, syIs36[egl-30(+)], showed a defect in salt chemotaxis learning. We are currently testing the prediction that JN914 carries a gain-of-function mutation in egl-30 and also mapping the other mutants isolated in this screening. (1) Saeki et al. 2001 J. Exp. Biol. 204, 1757-1764. (2) Tomioka et al., this meeting.

Takeshi Adachi et al. (2007) International Worm Meeting "A Suppressor Screen for Genes that Regulate Salt Chemotaxis Learning in C. elegans."

Ikue Mori, Takeshi Adachi, Yoshifumi Okochi, Hirofumi Kunitomo, Masahiro Tomioka, Yuichi Iino

[

International Worm Meeting,

2007]

C. elegans is attracted to NaCl, but starts to avoid it after a conditioning with NaCl and starvation. We have previously reported that several mutants of DAF-2 (insulin receptor) and AGE-1 (PI 3 kinase) showed defects in this form of learning; these mutants showed attractive behavior to the salt even after pre-exposure to salt. We have also shown that insulin-like signaling regulates the learning in the ASER salt-sensing neuron. These findings raised an important question: how does insulin-like signaling regulate the function of ASER ? daf-18 encodes the phosphatase, PTEN, which catalyzes a reverse reaction of PI 3 kinase. Loss-of-function mutants of daf-18 showed reduced chemotaxis to salt even without pre-exposure to salt, presumably because the activity of insulin-like signaling is upregulated. To identify genes that regulate salt chemotaxis learning, we screened for suppressors of the reduced chemotaxis of daf-18 mutants. Through this screen, we isolated 23 mutants with improved chemotaxis. One mutant showed a strong learning defect and was found to have a missense mutation, pe914, in egl-30, which encodes the a subunit of Gq. This mutation causes the Y61N amino acid substitution in EGL-30 Gq. Previously known gain-of-function mutation of egl-30 also suppressed the chemotaxis defect of daf-18 mutants, and expression of EGL-30(Y61N) in ASER was sufficient to restore the reduced chemotaxis. In neuromuscular junctions, Gq positively regulates production of presynaptic diacylglycerol (DAG), which facilitates neurotransmitter release. It was also reported that DAG regulates the function of various sensory neurons by protein kinase C (PKC)-dependent mechanisms. We found that expression of a gain-of-function form of TTX-4 nPKC-e/h in ASER restored the reduced chemotaxis of daf-18 mutants. We propose a hypothetical model that insulin-like signaling negatively regulates synaptic transmission of ASER by antagonizing the EGL-30/DAG/TTX-4 pathway. Another allele, pe917, also suppressed the reduced chemotaxis of daf-18 mutants. pe917 was mapped to the right arm of chromosome V and identified as a nonsense mutation in gcy-22 receptor-type guanylyl cyclase. Although cGMP signaling is proposed to transduce chemosensory signals, little is known about functions of guanylyl cyclases. We are currently trying to determine the site of action of GCY-22 and its role in salt chemotaxis learning.

Takeshi Adachi et al. (2006) East Asia C. elegans Meeting "A Suppressor Screen for Genes that Regulate Salt Chemotaxis Learning in C. elegans."

Masahiro Tomioka, Takeshi Adachi, Yuichi Iino, Ikue Mori, Yoshifumi Okochi, Hirofumi Kunitomo

[

East Asia C. elegans Meeting,

2006]

C. elegans is attracted to NaCl, but starts to avoid it after a conditioning with NaCl and starvation. We have previously reported that several mutants of DAF-2 (insulin receptor) and AGE-1 (PI 3 kinase) showed defects in this form of learning; these mutants showed attractive behavior to the salt even after pre-exposure to salt. We have also shown that insulin-like signaling regulates the learning in the ASER salt-sensing neuron. These findings raised an important question: how does insulin-like signaling regulate the function of ASER ? daf-18 encodes PTEN phosphatase, which catalyzes a reverse reaction of PI 3 kinase. Loss-of-function mutants of daf-18 showed reduced chemotaxis to salt even without pre-exposure to salt, presumably because the activity of insulin-like signaling is upregulated. To identify genes that regulate salt chemotaxis learning, we screened for suppressors of the reduced chemotaxis of daf-18 mutants. Through this screening, we isolated 23 mutants with improved chemotaxis. One mutant, pe914, showed a strong learning defect and was found to have a missense mutation (Y61N) in egl-30, which encodes the α subunit of Gq. Previously known gain-of-function mutants of egl-30 also showed learning defects and expression of EGL-30(Y61N) in ASER was sufficient to cause a learning defect. In neuromuscular junctions, Gq positively regulates production of presynaptic diacylglycerol (DAG), which facilitates neurotransmitter release. It was also reported that DAG regulates the funtion of various seosory neurons by protein kinase C (PKC)-dependent mechanisms. We found that exogenous DAG analogue phorbol ester disrupted salt chemotaxis learning and also found that animals expressing a gain-of-function form of TTX-4 nPKC-ε/η in ASER showed a severe learning defect. We currently propose a hypothetical model that insulin-like signaling negatively regulates synaptic transmission of ASER by antagonizing the EGL-30/DAG/TTX-4 pathway. We also mapped another allele pe917 isolated in this screening to the right arm of chromosome V.

Ohno, Hayao et al. (2017) International Worm Meeting "Diacylglycerol encodes differences between past and current stimulus intensity."

Ohno, Hayao, Adachi, Takeshi, Iino, Yuichi, Sakai, Naoko

[

International Worm Meeting,

2017]

C. elegans can memorize external salt concentrations and is attracted to the salt concentration at which it has been fed, whereas it avoids the previous salt concentration if it has been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). An important question is how worms compare the intensity of the past and current sensory stimuli and execute appropriate chemotactic behaviors. The plasticity of salt chemotaxis is regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway and the insulin/PI3K pathway, both of which act in the ASER taste receptor neuron. Genetic manipulations that activate or inactivate the DAG/PKC pathway result in worm migration to higher or lower salt concentrations, respectively. The insulin/PI3K pathway is essential for the avoidance of the salt concentrations associated with starvation. We have monitored the abundance of DAG in the ASER presynaptic regions, where the nPKC-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in a manner dependent on the TAX-4 CNG channel subunit and the EGL-8 phospholipase C beta . These DAG responses are largely diminished after starvation. An unbiased genetic screen and mutant analyses show that the DAG pathway acts downstream of the insulin/PI3K/Akt pathway. Our results suggest that the abundance of DAG in the ASER presynaptic region can encode differences between past and current salt concentrations and the insulin/PI3K pathway regulates the change of salt concentration preferences through modulation of the DAG dynamics.

Ohno, H. et al. (2015) International Worm Meeting "In vivo imaging of diacylglycerol signaling in a taste receptor neuron involved in salt-concentration memory."

Iino, Y., Ohno, H.

[

International Worm Meeting,

2015]

Adaptation to environmental conditions by modifying behavior often involves simple forms of learning and memory. We have previously reported that C. elegans worms can memorize ambient salt concentrations and are attracted to the previous salt concentration at which they have been fed, whereas they avoid it if they have been starved (Kunitomo et al., Nat. Commun., 2013; Ohno et al., Science, 2014). This behavioral plasticity has been suggested to be regulated by the diacylglycerol (DAG)/protein kinase C (PKC) pathway acting in a taste receptor neuron, ASER. Genetic manipulations that activate or inactivate this pathway in ASER result in worm migration to higher or lower salt concentrations, respectively (Adachi et al., Genetics, 2010; Kunitomo et al., Nat. Commun., 2013). To explore the role of DAG signaling in salt concentration memory, we have monitored the abundance of DAG in ASER of living worms. When the external salt concentration is increased or decreased from the concentration that has been experienced with food, DAG level is reduced or elevated, respectively, in presynaptic regions or regions adjacent to presynapses, where a novel protein kinase C (nPKC)-epsilon/eta ortholog TTX-4 (a.k.a. PKC-1) is localized. These DAG responses are largely diminished after starvation. These results raise the possibility that the activity of DAG signaling in the presynaptic region of ASER encodes the differences between past and present salt concentrations and thereby forms an integral component of the food-associated memory.

Ishiwa S et al. (2000) Japanese Worm Meeting "Analysis of nucleosome assembly factors, cia-1 and -2, in differentiation and proliferation during oogenesis and embryogenesis."

Ishiwa S, Horikoshi M, Chimura T

[

Japanese Worm Meeting,

2000]

A eukaryotic genome is packaged into a nucleoprotein complex known as chromatin. Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Histone chaperones are key components for alternation of chromatin as they stimulate assembly or disassembly of histone-DNA complexes (called nucleosomes), fundamental units of chromatin. Recently, we have reported that human CIA (CCG1-interacting factor A) is a novel histone chaperone which is most highly conserved among all the histone chaperones reported to date (1). Importantly, the genome of C. elegans and those of other multicellular organisms contain more than two putative genes for CIA, while the yeast genome contains one. Therefore, we expect that CIA family members play critical roles in developmental stage-specific alternation of chromatin structure in multicellular organisms. Using C. elegans as a model, we performed RNA interference experiment to investigate the in vivo biological functions of CIA family members and the underlying mechanisms through which they control transcription, cell growth, and differentiation. We found that two CIA-related genes, referred to as cia-1 and cia-2, have functionally redundant but distinct roles in promoting germ cell differentiation and embryogenesis. Different degree of embryonic lethality was caused by inactivation of each of the two family members, and in situ hybridization experiment indicated that both of them are expressed selectively in the gonad. Our findings suggest a model in which CIA-1 and CIA-2 regulate transcription of specific genes responsible for cell proliferation, by changing conformation of the chromatin structure. Munakata, T., Adachi, N., Yokoyama, N., Kuzuhara, T., and Horikoshi, M., Genes Cells 5, 221-233 (2000).

N Ishii et al. (1999) International C. elegans Meeting "Oxidative Stress and Aging in C.elegans: What lessons learned from mev-1?"

N Ishii, P Hartman

[

International C. elegans Meeting,

1999]

By measuring the life spans of various recombinant inbreds, Johnson and Wood estimated that genetic variability accounted for 20% to 50% of C. elegans life span. 1 We measured life span of animals over two successive generations and found that, while some variation exists in from generation to generation, the life span of a parent had little effect on that of its progeny. What are the environmental factors that control the balance of life span? Studies with the mev-1 gene provide evidence that reactive oxygen species (ROS) generated during respiration are key environmental factors in this process. Mev-1 mutants are hypersensitive to oxygen and methyl viologen (a singlet oxygen generator) and age precociously, particularly at high oxygen concentrations. 2 They also accumulate various aging markers more rapidly than does wild type. 3 The mev-1 mutation is in the cytochrome b subunit of succinate dehydrogenase ( cyt-1 ), which compromises enzymatic activity of the mitochondrial complex II. 4 It is likely that, as a result of this mutation, significantly higher levels of ROS are generated during respiration. Mev-1 animals shown hypermutability to oxygen, which represents the first example of a mitochrondrial dysfunction impacting nuclear mutation frequencies. In addition, temperature affects life span differently in mev-1 and wild type, likely through these metabolic differences. Electron microscopy reveals inclusions in the mitochondria of mev-1 animals, particularly under elevated oxygen concentrations. In addition, mev-1 embryos contain abnormally high numbers of ced-3 -dependent apoptotic cells. Collectively these data muster substantial support for the theory that ROS-induced damage is important in biological aging. 1 Johnson and Wood 1982 PNAS 79, 6603 2 Ishii et al., 1990 Mutat. Res. 237, 165. 3 Hosokawa et al., 1994. MAD 74, 161; Adachi et al., 1998 J. Geront. 53A, B240 4 Ishii et al., 1998. Nature 394, 6694

Kosuke Fujita et al. (2010) East Asia Worm Meeting "Identifying the site of dopamine action for regulating the enhancement of 2-nonanone avoidance"

Kosuke Fujita, Kotaro Kimura

[

East Asia Worm Meeting,

2010]

Dopamine signaling plays significant roles in modulating behavior and in learning. Its molecular mechanisms in vivo, however, are complex and still unclear. We have found that dopamine signaling is required for a novel type of behavioral plasticity in C. elegans (Kimura, Fujita and Katsura, in revision). The avoidance behavior of animals to a repulsive odor 2-nonanone is significantly enhanced rather than reduced after 1 h preexposure to the odor, and this enhancement involves dopamine signaling. Unlike previously identified dopamine-regulated behavioral plasticities in C. elegans, which require the presence of food for dopamine action (Sawin et al., 2000; Hills et al., 2004; Sanyal et al., 2004; Kindt et al., 2007), the enhancement of 2-nonanone avoidance is food-independent, suggesting a new role of dopamine signaling in the regulation of behavioral responses. Genetic and pharmacological studies have revealed that one of the D2-like dopamine receptor homologs dop-3 is involved in the enhancement of 2-nonanone avoidance behavior. To identify the cellular site of action of dop-3, we are conducting neuron-specific rescue experiments with dop-3 mutants that exhibits the enhancement-defective phenotype. Various promoters for subsets of neurons were fused to dop-3 cDNA for the rescue experiments and to fluorescent protein to study the expression patterns. Expression of dop-3 cDNA in the neurons that are reportedly required to regulate locomotion (ventral nerve cord motor neurons with acr-2 or unc-47 promoters; Chase et al., 2004) or CREB expression (RIC interneurons with tdc-1 promoter or SIA with ceh-17; Suo et al., 2009) did not rescue the enhancement-defective phenotype while pan-neuronal expression (with the H20 promoter; a gift from Dr. Takeshi Ishihara) rescued the phenotype, suggesting that dop-3 functions at a new site of action to enhance the avoidance behavior. We continue the rescue experiment to identify neuron(s) for the dop-3 action. We thank Drs. Satoshi Suo (Mt. Sinai Hospital, Canada) for the dop-3 cDNA and Yuichi Iino and Masahiro Tomioka (U. Tokyo, Japan) for the promoter library.

Kotaro Kimura et al. (2003) International Worm Meeting "Transient response in AFD thermosensory neuron by rapid temperature change."

Ikue Mori, Kotaro Kimura

[

International Worm Meeting,

2003]

Little is known about how environmental signals are received, integrated and assessed in intact neuronal network to regulate behavior and/or adaptation of animals. To understand how actual signals are conveyed in the network, we are interested in monitoring neuronal activity of C. elegans with calcium imaging. The thermosensory circuit is of particular interest, because it is one of a few circuits whose sensory and interneurons are identified and little information is available as to how activity of AFD thermosensory neuron is regulated by temperature. Cameleon, a GFP-based calcium sensor, can substitute calcium concentration into fluorescence ratio between CFP and YFP in the macromolecule via flourescence resonance energy transfer (Ref. 1). Using cameleon, calcium imaging of worm touch and ASH sensory neurons have been reported by Schafer lab and other groups in recent worm meetings. To begin our analysis, we made an attempt to apply camelon system on calcium imaging of the thermosensory circuit. We found transient cameleon response associated with rapid temperature change in AFD thermosensory neuron. The fluorescence ratio in AFD cell body increases transiently when the temperature is increased from 15 deg to 25 deg within 20 seconds, and the ratio decreases when the temperature is decreased from 25 deg to 15 deg within same period. Magnitude of the ratio change during the temperature increase is larger than that of the decrease. However, the ratios at 15, 20 or 25 deg are at similar level when the worms have been kept at each temperature for minutes. These results suggest that activity of AFD may be dependent on temperature change and not on net temperature. We are currently working on obtaining a proof that the ratio change is due to calcium change specific to AFD. We thank Atsushi Miyawaki (RIKEN) for YC2.12 and Takeshi Ishihara (NIG) for AFD-specific promoter. Ref. 1: Miyawaki et al., Nature 388, 882 (1997)

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