Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4
2005; Springer Nature; Volume: 24; Issue: 12 Linguagem: Inglês
10.1038/sj.emboj.7600697
ISSN1460-2075
AutoresYoshifumi Okochi, Koutarou D. Kimura, Akane Ohta, Ikue Mori,
Tópico(s)Circadian rhythm and melatonin
ResumoArticle26 May 2005free access Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4 Yoshifumi Okochi Yoshifumi Okochi Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Search for more papers by this author Koutarou D Kimura Koutarou D Kimura Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, JapanPresent address: Structural Biology Center, National Institute of Genetics, Mishima 411-8540, Japan Search for more papers by this author Akane Ohta Akane Ohta Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Search for more papers by this author Ikue Mori Corresponding Author Ikue Mori Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Institute for Advanced Research, Nagoya University, Nagoya, Japan Search for more papers by this author Yoshifumi Okochi Yoshifumi Okochi Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Search for more papers by this author Koutarou D Kimura Koutarou D Kimura Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, JapanPresent address: Structural Biology Center, National Institute of Genetics, Mishima 411-8540, Japan Search for more papers by this author Akane Ohta Akane Ohta Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Search for more papers by this author Ikue Mori Corresponding Author Ikue Mori Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Institute for Advanced Research, Nagoya University, Nagoya, Japan Search for more papers by this author Author Information Yoshifumi Okochi1, Koutarou D Kimura1, Akane Ohta1 and Ikue Mori 1,2 1Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan 2Institute for Advanced Research, Nagoya University, Nagoya, Japan *Corresponding author. Department of Molecular Biology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan. Tel.: +81 52 789 4560; Fax: +81 52 789 4558; E-mail: [email protected] The EMBO Journal (2005)24:2127-2137https://doi.org/10.1038/sj.emboj.7600697 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling. Introduction Sensory neurons are mostly specialized to detect different environmental stimuli. For example, photoreceptor cells sense light exclusively and olfactory neurons sense odorants. In each sensory neuron, different molecules are involved in primary sensory signal transduction. In mammals, light is received by the G protein-coupled receptor rhodopsin, which in turn activates Gα transducin and phosphodiesterase (PDE), and leads to the closure of a cGMP-gated cation (CNG) channel (Ebrey and Koutalos, 2001). Activation of an odorant receptor by an odorant stimulates Golf protein, which increases the level of cAMP via activation of adenylyl cyclase, resulting in the opening of a cAMP-gated cation (CNG) channel (Pace et al, 1985; Firestein et al, 1991). Nociceptive signals directly activate TRP channels, TRPV1 and TRPV2 (Caterina et al, 1997, 1999). Given the numerous specific sensory neurons, we are led to ask how the activation or inactivation of the signaling molecules is regulated to respond properly to environmental stimuli. Molecular mechanisms for the highly tuned regulation of these signaling pathways still remain largely unclear. The protein kinase C (PKC) family is a group of serine/threonine protein kinases that play important roles in intracellular signal transduction. The PKC family comprises at least 10 isozymes, which are divided into three subgroups based on their structures: conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC) (Nishizuka, 1992). cPKCs require Ca2+ and diacylglycerol (DAG) for activation, nPKCs require DAG but not Ca2+, and aPKCs require neither Ca2+ nor DAG. Extensive pharmacological analyses show that PKC members are involved in such cellular events as cellular proliferation and differentiation (Nishizuka, 1995), suggesting roles of PKCs in various intracellular signal transductions. In sensory neurons, PKCs mediate the sensitization of the heat response induced by the inflammatory peptide bradykinin in rat nociceptive neurons (Cesare and McNaughton, 1996), inhibit the phototransduction cascade in the photoreceptor of horseshoe crabs (Dabdoub and Payne, 1999), and are required for the response to synthetic sweeteners by mammalian taste cells (Varkevisser and Kinnamon, 2000). Nevertheless, the in vivo function of most members of the PKC family in sensory neurons has remained unknown. The nematode Caenorhabditis elegans can sense various environmental stimuli mainly by means of sensory neurons in the head (amphid neurons). The sensory neurons for each specific stimulus have been identified by a series of laser ablation experiments. For example, temperature stimulus is sensed by thermosensory neurons AFD (Mori and Ohshima, 1995), attractive odorants are sensed by olfactory neurons AWA and AWC (Bargmann et al, 1993), and aversive high osmolality is sensed by nociceptive neurons ASH (Hart et al, 1995; Maricq et al, 1995). Genetic analyses of mutants that are defective in sensory behaviors to these stimuli have identified the several molecules in sensory signaling pathways: tax-4 and tax-2 encode the alpha and beta subunits of cyclic nucleotide-gated (CNG) channel, respectively, which are required for thermosensation in AFD neurons and olfaction in AWC neurons (Coburn and Bargmann, 1996; Komatsu et al, 1996, 1999), osm-9 and ocr-2 encode TRPV channel homologs, which are required for olfaction in AWA neurons and nociception in ASH neurons (Colbert et al, 1997; Tobin et al, 2002), odr-3 encodes Gα protein, which is required for olfaction in AWA and AWC neurons and nociception in ASH neurons (Roayaie et al, 1998), and odr-1 and daf-11 encode guanylyl cyclases, which are required for olfaction in AWC neurons (Birnby et al, 2000; L'Etoile and Bargmann, 2000). These molecules are homologs of the molecules that function in similar types of sensory neurons of other animals, implicating evolutionarily conserved roles for these molecular pathways in each type of sensory signaling. In this study, we found that nPKC-epsilon/eta plays essential and different roles in many different types of sensory neurons of C. elegans. We isolated thermotaxis-defective mutants from a genetic screen. Of these, a novel mutant, ttx-4, showed a thermophilic phenotype in thermotaxis, and other behavioral abnormalities in osmotic avoidance and chemotaxis to odorants and NaCl. We revealed that ttx-4 encodes nPKC-epsilon/eta and is expressed and functions in many head sensory neurons responsible for the behaviors that are abnormal in the mutant. The loss-of-function (lf) mutation in TTX-4 caused hyperactivation of the AFD neurons, whereas the gain-of-function (gf) form led to their inactivation. In contrast, TTX-4 lf caused inactivation of the ASH neurons, whereas TTX-4 gf led to their hyperactivation. Further, pharmacological and genetic analyses showed that sensory signaling in AFD thermosensory neurons is regulated exclusively by TTX-4, which likely functions in the downstream of diacylglycerol kinase (DGK)-1 and in the upstream of TAX-4 CNG channel, while sensory signalings in AWA and AWC olfactory neurons are regulated by TTX-4 and nPKC-delta/theta TPA-1. Our results suggest that TTX-4 nPKC-epsilon/eta plays diverse roles in various sensory neurons to regulate positively or negatively different cell signaling pathways. Results ttx-4 mutants show defects in thermotaxis and other sensory behaviors C. elegans senses temperature mainly by a pair of head sensory neurons AFD, and the sensation of temperature is observed as a characteristic thermotaxis behavior (Hedgecock and Russell, 1975; Mori and Ohshima, 1995; Mori, 1999). In the thermotaxis behavior, wild-type animals migrate to their previously cultivated temperature on a radial temperature gradient from 17 to 25°C (Hedgecock and Russell, 1975; Mori and Ohshima, 1995); For example, the wild-type animals migrate to 20°C and stay there isothermally on a radial temperature gradient after cultivation at 20°C with sufficient food (Figure 1A and B). To elucidate the molecular mechanism of thermosensation, a genetic screen was performed for mutants defective in thermotaxis (ttx). Two mutants, nj1 and nj3, defined a novel gene ttx-4. Another mutant, ttx-4(nj4), was isolated in a different screen (A Mohri, M Koike, and I Mori, unpublished). All three ttx-4 alleles were genetically recessive (data not shown). The ttx-4 mutants showed a thermophilic phenotype, in which the animals always migrate to a temperature higher than the cultivation temperature (Figure 1A and B, and Supplementary data). The ttx-4(nj1) and ttx-4(nj3) mutants were severely defective and the ttx-4(nj4) mutants were partially defective. Figure 1.Phenotypes of ttx-4. (A) Tracks of wild-type and ttx-4 mutant animals showing thermotaxis behaviors on a radial temperature gradient ranging from 17°C (center) to 25°C (periphery). Animals were grown at 20°C. (B) The spectra of thermotaxis phenotypes of wild type and ttx-4 mutants. The phenotypic categories are described in Materials and methods. For each genotype, 100–160 animals were individually assayed on thermotaxis assay plates. (C) Chemotaxis of wild type and ttx-4 mutants for odorants. The concentration of odorants is described in Materials and methods. For each assay, 50–100 animals were analyzed. The bars represent the means of three independent assays with an error bar showing s.e.m. (D) Chemotaxis to NaCl. For each genotype, 100–170 animals were individually assayed. The phenotypic categories are described in Materials and methods. (E) Time-course assay for osmotic avoidance against 8 M glycerol. For each assay, 10 animals were placed inside of a high osmotic strength ring made from the given concentrations of 8 M glycerol solution, and the numbers of animals remaining within the ring were scored after 3, 6, 9, 12, and 15 min. ‘Wild type with 0 M glycerol’ shows the animals' free movement without the osmotic barrier. The bars represent the means of more than three independent assays with an error bar showing s.e.m. Download figure Download PowerPoint All ttx-4 mutants also showed severe defects in chemotactic responses to odorants sensed by AWA or AWC olfactory neurons, except that they were capable of partially responding to pyrazine sensed by AWA neurons (Figure 1C). In addition, these mutants were defective in chemotaxis to the water-soluble compound NaCl sensed mainly by ASE gustatory neurons (Figure 1D and Supplementary data). The ttx-4(nj1) mutants were severely defective in chemotaxis to NaCl, and ttx-4(nj3) and ttx-4(nj4) were partially defective. Furthermore, ttx-4 mutants showed a defect in osmotic avoidance behavior mediated by ASH nociceptive neurons (Figure 1E). A time-course assay was performed to observe how animals respond to osmotic strength that is decreasing over time probably due to diffusion. The ttx-4(nj1) mutants showed a defect as severe as that of the osm-9 mutants (Figure 1E). osm-9 encodes a TRPV channel homolog, and its null mutation completely abolishes the function of ASH neurons (Colbert et al, 1997). ttx-4(nj3) and ttx-4(nj4) mutants were less defective than ttx-4(nj1) mutants in osmotic avoidance behavior (Figure 1E). ttx-4 gene encodes an nPKC-epsilon/eta ortholog The ttx-4 gene was identified by a series of genetic mapping and subsequent rescue experiments with cloned genomic fragments (Figure 2A). Behavioral defects in the ttx-4 mutants were rescued by introduction of the cosmid C38F2 as well as the cloned genomic fragment containing two predicted genes, F57F5.5 and F10C2.3. The cloned genomic fragment without F10C2.3 also rescued the defects in ttx-4 mutants, indicating that F57F5.5 is the ttx-4 gene. Figure 2.Genetic and molecular analyses of ttx-4. (A) Genetic map position of the ttx-4 gene on chromosome V and results of rescue. The ttx-4(nj1) gene was mapped onto the region between sma-1 and vab-8, which is covered by about 10 cosmids. The numbers in parentheses indicate the fraction of rescued lines. For each genotype, 100–170 animals were individually assayed for thermotaxis or for chemotaxis to NaCl. Chemotaxis to odorants was evaluated based on more than three independent assays for each transgenic line. (B) Structure and sequence of TTX-4. Upper panel: Predicted structure of TTX-4. Lower panel: Alignment of TTX-4 with human nPKC-epsilon and nPKC-eta, Drosophila PKC98-PA and C. elegans nPKC-delta/theta TPA-1A, and mutation sites of ttx-4. Prefix: d, Drosophila; h, human. TTX-4 shares 56% overall identity with human nPKC-epsilon and 52% identity with human nPKC-eta. Black highlights identical residues, and gray highlights similar residues. The bar patterns above the amino-acid alignment correspond to those marking domains in the structure of TTX-4. The alanine in the pseudosubstrate (PS) region was changed to aspartate to construct the ttx-4 gain-of-function form (ttx-4gf) (Dekker et al, 1993). (C) Unrooted dendrogram of two C. elegans nPKCs, TTX-4 and TPA-1A, Drosophila PKC98-PA, and four human PKCs. Human cPKC-alpha and aPKC-zeta were used as the representation of cPKC and aPKC, respectively. The dendrogram was generated by bootstrap analysis and neighbor-joining method using a freely available program, Clustal W (Thompson et al, 1994) (http://www.ddbj.nig.ac.jp/search/clustalw-j.html). The numbers at the branches indicate the percentage bootstrap values from 1000 replicates. Download figure Download PowerPoint F57F5.5/ttx-4 gene encodes an nPKC-epsilon/eta ortholog, which has been designated as kin-13 (Figure 2B and C; Land et al, 1994). nj1 and nj4 have a missense mutation of a conserved amino acid in the catalytic loop (D502N) and the ATP-binding site (G390D) in the kinase domain, respectively. nj3 has a nonsense mutation in the C1 domain (W218stop) (Figure 2B). Since the nj3 mutation probably causes a truncated protein, the kinase activity should be completely lost in nj3 mutants. nj1 mutants exhibit a severer phenotype than do nj3 mutants, which suggests that the missense mutation nj1 might have a dominant negative effect. However, the nj1 mutation was recessive in our behavioral analysis (data not shown). TTX-4 nPKC-epsilon/eta is expressed and functions in sensory neurons Cells expressing the ttx-4 gene were identified by using a reporter construct in which the green fluorescence protein (GFP) gene was fused to the 3′ end of the wild-type ttx-4 genomic fragment (Figure 2A). A previous immunohistochemical study reported that KIN-13/TTX-4 protein was expressed mainly in sensory neurons and interneurons, although the specific cells had not been identified (Land et al, 1994). We found that the ttx-4::GFP reporter construct was expressed in many sensory neurons, including neurons that sense temperature (AFD), odorants (AWA and AWC), NaCl (ASE), or high osmolality (ASH) (Figure 3A and data not shown). The reporter construct was also expressed in many interneurons including AIY and AIZ, which play critical roles in thermotaxis (Figure 3A; Mori and Ohshima, 1995). Figure 3.Expression and cell-autonomous functions of TTX-4 in sensory neurons. (A) TTX-4 expression in sensory neurons and interneurons. Lateral view of L2 larva showing the cell body of several neurons. Anterior is to the left and dorsal is up. TTX-4 protein was localized to not only cell body but also dendrite and axon (data not shown). TTX-4 protein was also expressed in other posterior neurons and some motor neurons (data not shown). (B) Rescue of the thermophilic phenotype by specific expression of ttx-4 cDNA in AFD thermosensory neurons and the thermophilic phenotype of ttx-4 mutants was suppressed by the ttx-3 mutation, which leads to the inactivation of AIY interneurons. The gcy-8 promoter was used for AFD expression (Yu et al, 1997). gcy-8p::ttx-4 cDNA was injected at 2 ng/μl. For each genotype, 60–100 animals were individually assayed for thermotaxis. (C) Rescue of the chemotaxis phenotype by specific expression of TTX-4 in AWC olfactory neurons. The odr-3 promoter was used for AWC expression (Roayaie et al, 1998). odr-3p::ttx-4 cDNA was injected at 20 ng/μl. A total of 10 animals were placed on each assay plate in this behavioral analysis. The bars represent the means of more than three independent assays. (D) Rescue of defective osmotic avoidance by specific expression of TTX-4 in ASH nociceptive neurons. The sra-6 promoter was used for ASH expression (Troemel et al, 1995). sra-6p::ttx-4 cDNA was injected at 20 ng/μl. A total of 10 animals were placed on each assay plate in this time-course assay for osmotic avoidance. The bars represent the means of more than three independent assays with an error bar showing s.e.m. Download figure Download PowerPoint To identify the cells that require TTX-4 activity for their appropriate sensory behavior, the ttx-4 cDNA was expressed by using cell-specific promoters. Specific expression of the ttx-4 cDNA in the AFD thermosensory neurons of ttx-4(nj1) mutants rescued the thermophilic phenotype (Figure 3B), but not other behavioral defects such as chemotaxis to NaCl in ttx-4 mutants (data not shown). Furthermore, specific expression of ttx-4 cDNA in the AWC olfactory neurons or in the ASH nociceptive neurons rescued the abnormal chemotaxis to AWC-sensed odorants (Figure 3C) or the abnormal avoidance response to high osmolality (Figure 3D), respectively. These results suggest that TTX-4 nPKC-epsilon/eta acts cell-autonomously in the sensory neurons. Genetic epistasis analysis also indicates that the thermophilic phenotype of the ttx-4 mutant is caused by abnormal AFD function. The AIY interneurons are thought to receive signals directly from the AFD neurons (White et al, 1986). Laser ablation of the AIY interneurons or mutations in ttx-3, a transcription factor that specifies AIY cell differentiation, lead to cryophilic phenotypes in which the animals always migrate to a temperature lower than the cultivation temperature (Mori and Ohshima, 1995; Hobert et al, 1997; Altun-Gultekin et al, 2001). We examined ttx-4;ttx-3 double mutants to show that the inactivation of AIY by the ttx-3 mutation suppresses the thermophilic phenotype of ttx-4 mutants. The majority of ttx-4;ttx-3 double mutants showed a cryophilic phenotype (Figure 3B), which resembles the phenotype of ttx-3 mutants. This result is consistent with the idea that the thermophilic phenotype of ttx-4 mutants is due to abnormal function of the AFD neurons (Figure 3B). Thus, we conclude that TTX-4 functions in the AFD thermosensory neurons. The incomplete suppression of the thermophilic phenotype of ttx-4 by the ttx-3 mutation might, however, implicate other neuron(s), where TTX-4 nPKC-epsilon/eta plays a minor role in thermosensation. TTX-4 nPKC-epsilon/eta negatively regulates the function of AFD neurons We asked how TTX-4 nPKC-epsilon/eta regulates thermosensory signal transduction in the AFD neurons. Inactivation of the AFD thermosensory neurons by a mutation in the AFD-specific transcription factor ttx-1 gene causes a cryophilic phenotype (Satterlee et al, 2001), which is opposite to the thermophilic phenotype of the ttx-4 mutants (Figure 1A). Laser ablation of the AFD neurons also causes a cryophilic, athermotactic, or cryophilic/athermotactic synthetic phenotype on a radial temperature gradient (Mori and Ohshima, 1995). To elucidate whether AFD neurons are hyperactivated in ttx-4 mutants, a gain-of-function form (TTX-4 gf) (Dekker et al, 1993) was expressed specifically in the AFD neurons of the wild type and ttx-4 mutants. Wild-type animals and ttx-4 mutants expressing TTX-4 gf in AFD neurons showed the cryophilic phenotype (Figure 4A), suggesting that abnormal activation of TTX-4 inactivates AFD neurons. Considering this result and the thermophilic phenotype in ttx-4 mutants, the AFD neurons may be hyperactivated in ttx-4 mutants. Figure 4.Effect of TTX-4 gf in AFD neurons and PMA treatment in thermotaxis. (A) Expression of the TTX-4 gf form caused inactivation of the AFD neurons, and TTX-4 and TAX-6 interact genetically in the AFD neurons. Gain-of-function forms of ttx-4 or tax-6 (Kuhara et al, 2002) were expressed in wild type and tax-6 and ttx-4 mutants using the AFD-specific gcy-8 promoter. The expression of gcy-8p::ttx-4gf did not affect morphology of the AFD neurons (data not shown). gcy-8p::ttx-4gf or gcy-8p::tax-6gf was injected at 2 ng/μl. For each genotype, 60–140 animals were individually assayed. (B) Schematic view of PMA treatment. (C) PMA inactivated AFD neurons through TTX-4 activation. gcy-8p::ttx-4 cDNA was injected at 2 ng/μl. For each genotype, 30–90 animals were individually assayed. Download figure Download PowerPoint In tax-6 lf mutants, the AFD neurons are also hyperactivated, resulting in the thermophilic phenotype (Kuhara et al, 2002). TAX-6 encodes calcineurin, a calcium/calmodulin-dependent protein phosphatase (Kuhara et al, 2002). To investigate whether TTX-4 and TAX-6 interact in the AFD neurons, TTX-4 gf form or TAX-6 gf form was expressed specifically in the AFD neurons of tax-6 or ttx-4 mutants, respectively. AFD-specific expression of TTX-4 gf in tax-6 mutants partially suppressed the thermophilic phenotype (Figure 4A). The thermophilic phenotype in ttx-4 mutants was also suppressed partially by AFD-specific expression of TAX-6 gf that completely inactivates AFD neurons in wild-type animals (Figure 4A; Kuhara et al, 2002). These results did not show clear genetic epistatic dominance, but suggest that TTX-4 and TAX-6 interact genetically (see Discussion). Activation of TTX-4 by DAG in adult animals causes inactivation of the AFD neurons We addressed whether the activity of TTX-4 is required in the developed mature AFD neurons or in the developing AFD neurons. In this case, pharmacological analysis provides better temporal resolution than genetic analysis to understand the physiological role of the molecules. The mammalian nPKC subtype is activated by DAG. Therefore, TTX-4 nPKC-epsilon/eta in wild-type animals may be activated by exogenous phorbol-12 myristate 13-acetate (PMA), an analog of DAG, thereby leading to the cryophilic phenotype as observed in transgenic animals expressing TTX-4 gf in the AFD neurons. To investigate the effect of PMA on thermotaxis, we treated wild-type and ttx-4 mutant adult animals with PMA (1 μg/ml) for 2 h at 20°C, and then subjected them to the thermotaxis assay (Figure 4B). PMA-treated wild-type animals showed the cryophilic phenotype (Figure 4C), suggesting that PMA treatment in adulthood can behaviorally mimic the activation of TTX-4 in vivo. In contrast to the wild-type animals, PMA-treated ttx-4 mutants showed the thermophilic phenotype as did the PMA-untreated ttx-4 mutants (Figure 4C). This result suggests that the cryophilic phenotype of the PMA-treated wild-type animals is due to the activation of TTX-4, not to the side effect of the drug. We further asked whether TTX-4 in AFD neurons is the target of PMA. Most of the PMA-treated ttx-4 mutants expressing ttx-4 cDNA only in the AFD neurons showed the cryophilic phenotype (Figure 4C), which suggests that TTX-4 in the AFD neurons is the main target of PMA. Taken together, DAG likely activates TTX-4 to regulate thermosensation in the mature AFD neurons. TTX-4 nPKC-epsilon/eta may function in the downstream of DGK-1 and in the upstream of TAX-4 channel What molecules regulate the activation of TTX-4? Phospholipase C (PLC) produces DAG, which leads to various cellular responses probably through PKC activation (Nishizuka, 1995; Rhee, 2001). PLC-beta is required for taste response in mouse and phototransduction in Drosophila (Bloomquist et al, 1988; Zhang et al, 2003). To investigate whether PLC-beta supplies DAG to activate TTX-4, we tried to test the thermotaxis behavior of egl-8 mutants lacking PLC-beta (Lackner et al, 1999). We, however, could not evaluate the thermotaxis behavior of egl-8 mutants, because they did not move on the assay plate owing to their locomotion defect (data not shown). Phospholipase D (PLD) is also involved in the supply of DAG indirectly (Liscovitch et al, 2000). pld-1 encodes the sole PLD gene in the C. elegans genome. We found that pld-1 mutants lacking a base sequence encoding a catalytic motif in the PLD-1 gene (N Hisamoto and K Matsumoto, personal communication) showed normal thermotaxis behavior (Figure 5). DGK, which exchanges DAG into phosphatidic acid (PA), is a possible candidate for the regulatory molecule of PKC (van Blitterswijk and Houssa, 2000). dgk-1 encodes DGK-theta (Nurrish et al, 1999), which is expressed in the neurons involved in thermotaxis (data not shown). If DGK-1 regulates TTX-4 activity in the AFD neuron, we expected that DAG would accumulate in the AFD neurons of dgk-1 mutants, which might lead to the excess activation of TTX-4, thereby causing the cryophilic phenotype. Consistent with our hypothesis, dgk-1 mutants showed the cryophilic phenotype (Figure 5). We then examined a ttx-4;dgk-1 double mutant to show that the ttx-4 mutation suppresses the cryophilic phenotype of dgk-1 mutants. Most ttx-4;dgk-1 double mutants showed a thermophilic phenotype (Figure 5). This is also consistent with the model that TTX-4 functions in the downstream of DGK-1. Figure 5.Thermotaxis behavior of pld-1, ttx-4;dgk-1 or tax-4;ttx-4 mutants. For each genotype, 60–90 animals were individually assayed on thermotaxis assay plates. Download figure Download PowerPoint We next addressed whether TTX-4 regulates primary sensory transduction in AFD neurons. tax-4 encodes the alpha subunit of the CNG channel that is required for thermosensation in AFD neurons, and tax-4 mutants show an athermotactic phenotype (Figure 5; Komatsu et al, 1996). We examined the tax-4;ttx-4 double mutant to see if the tax-4 mutation would suppress the thermophilic phenotype of the ttx-4 mutant. tax-4;ttx-4 double mutants showed an athermotactic phenotype, which is similar to the phenotype of the tax-4 mutants (Figure 5). This result is consistent with the possibility that TTX-4 functions in the upstream of TAX-4. Thus, one plausible molecular model is that TTX-4 nPKC-epsilon/eta may be under the negative regulation by DGK-1 and negatively regulate the thermosensory signal transduction pathway by inhibiting the activity of the TAX-4 channel directly or indirectly. TTX-4 nPKC-epsilon/eta positively regulates the function of ASH neurons Although the ttx-4 lf mutation leads to hyperactivation of AFD neurons, it causes defects in the sensation of high osmolality, implicating inactivation of ASH neurons. To elucidate whether ASH neurons are inactivated in ttx-4 mutants, the TTX-4 gf form was expressed specifically in ASH neurons of wild-type and ttx-4 mutant animals. ASH-specific expression of TTX-4 gf resulted in hypersensitivity to high osmolality in the transgenic animals (Figure 6A). More than 80% of transgenic animals expressing TTX-4 gf in the ASH neurons stayed within the glycerol ring for 21 min, while about 50% of the wild-type animals moved out of the ring within the same period (Figure 6A), suggesting that TTX-4 gf causes hyperactivation of ASH neurons. Thus, it is likely that TTX-4 positively regulates the sensation of high osmolality in the ASH neurons. Figure 6.Effects of TTX-4 gf in ASH neurons and PMA treatment in chemotaxis. (A) Expression of the TTX-4 gf form caused hyperactivation of the ASH neurons. Animals were grown at 23°C. A total of 10 animals were placed on the assay plate for osmotic avoidance. The bars represent the means of more than three independent assays with an error bar showing s.e.m. For each assay, the numbers of animals remaining within the ring were scored after 3, 6, 9, 12, 15, 18, and 21 min. (B, C) PMA treatment rescue
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