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A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

Fujiwara M, Teramoto T, Ishihara T, Ohshima Y, McIntire SL - PLoS Genet. (2010)

Bottom Line: By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals.Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans.Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. fujiwara.manabi.734@m.kyushu-u.ac.jp

ABSTRACT
Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

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chb-3 is a che-2 small-body-size suppressor.(A) Time course of changes in body size indicated by the length of the perimeter of the lateral image of the animals. Body size was measured at the indicated time points from the L4 stage. Error bars indicate the standard error of the mean (s.e.m). (B) Body size indicated by the length of the animals 48 hours after the L4 stage. “n.s.” indicates that the difference is not significant (p>0.05, t test). (C) Typical tracks left by a single animal on an E. coli lawn during an 18-hours period. Scale bar, 10 mm. (D) Chemotaxis and osmotic avoidance assays. Each data point represents an average of at least 3 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference (p<0.01, t test). (E) The rate of dauer formation at 20°C. The Daf-c phenotype of chb-3 was suppressed by che-2(e1033) but not by daf-3(e1376) or daf-16(m26). Rescue of the chb-3(eg52) Daf-c phenotype by the Y48G1A.3 transgene (Ex[Y48G1A.3/myo-3::gfp]) is also shown. Each data point represents an average of at least 5 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference from chb-3(eg52) (p<0.01, t test). #The dauer formation rate of chb-3(qj2) was determined by gathering eggs from the chb-3(qj2); Ex[tax-4p::CHB-3::GFP] rescued line, and observing hatched animals that lost the Ex array.
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pgen-1001211-g001: chb-3 is a che-2 small-body-size suppressor.(A) Time course of changes in body size indicated by the length of the perimeter of the lateral image of the animals. Body size was measured at the indicated time points from the L4 stage. Error bars indicate the standard error of the mean (s.e.m). (B) Body size indicated by the length of the animals 48 hours after the L4 stage. “n.s.” indicates that the difference is not significant (p>0.05, t test). (C) Typical tracks left by a single animal on an E. coli lawn during an 18-hours period. Scale bar, 10 mm. (D) Chemotaxis and osmotic avoidance assays. Each data point represents an average of at least 3 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference (p<0.01, t test). (E) The rate of dauer formation at 20°C. The Daf-c phenotype of chb-3 was suppressed by che-2(e1033) but not by daf-3(e1376) or daf-16(m26). Rescue of the chb-3(eg52) Daf-c phenotype by the Y48G1A.3 transgene (Ex[Y48G1A.3/myo-3::gfp]) is also shown. Each data point represents an average of at least 5 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference from chb-3(eg52) (p<0.01, t test). #The dauer formation rate of chb-3(qj2) was determined by gathering eggs from the chb-3(qj2); Ex[tax-4p::CHB-3::GFP] rescued line, and observing hatched animals that lost the Ex array.

Mentions: chb-3(eg52) was previously isolated as a mutant which suppresses the small body size of che-2, but not the cilium structural defect of che-2, in a screen that also identified egl-4 [6]. As shown in Figure 1A and 1B, the chb-3(eg52) mutation caused an increase in body size of che-2(e1033). Compared with wild-type animals (WT, N2 strain), the chb-3(eg52) single mutant were longer and larger overall (Figure 2). Suppression of the che-2 small-body phenotype by chb-3, however, is unlikely a result of simple additive effects of the mutation, because a che-2 transgene did not increase the size of the chb-3(eg52);che-2(e1033) mutant (chb-3(eg52);che-2(e1033);Ex[che-2] in Figure 1A). Moreover, the chb-3(eg52) single mutant and the chb-3(eg52);che-2(e1033) double mutant were similar in body size (Figure 1B). We defined the group of Chb mutants displaying body sizes that were not affected by the che-2 mutation as class I mutants, whereas the other Chb mutants were designated as class II mutants [6]. On the whole, the class I Chb genes are required for animals to make the body size small when animals do not perceive sensory inputs (e.g., cilium-defective mutants). The epistasis of chb-3 to che-2 indicates that chb-3 is a class I Chb mutant, suggesting a role for the chb-3 gene in the sensory processing-mediated regulation of body size.


A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

Fujiwara M, Teramoto T, Ishihara T, Ohshima Y, McIntire SL - PLoS Genet. (2010)

chb-3 is a che-2 small-body-size suppressor.(A) Time course of changes in body size indicated by the length of the perimeter of the lateral image of the animals. Body size was measured at the indicated time points from the L4 stage. Error bars indicate the standard error of the mean (s.e.m). (B) Body size indicated by the length of the animals 48 hours after the L4 stage. “n.s.” indicates that the difference is not significant (p>0.05, t test). (C) Typical tracks left by a single animal on an E. coli lawn during an 18-hours period. Scale bar, 10 mm. (D) Chemotaxis and osmotic avoidance assays. Each data point represents an average of at least 3 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference (p<0.01, t test). (E) The rate of dauer formation at 20°C. The Daf-c phenotype of chb-3 was suppressed by che-2(e1033) but not by daf-3(e1376) or daf-16(m26). Rescue of the chb-3(eg52) Daf-c phenotype by the Y48G1A.3 transgene (Ex[Y48G1A.3/myo-3::gfp]) is also shown. Each data point represents an average of at least 5 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference from chb-3(eg52) (p<0.01, t test). #The dauer formation rate of chb-3(qj2) was determined by gathering eggs from the chb-3(qj2); Ex[tax-4p::CHB-3::GFP] rescued line, and observing hatched animals that lost the Ex array.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991246&req=5

pgen-1001211-g001: chb-3 is a che-2 small-body-size suppressor.(A) Time course of changes in body size indicated by the length of the perimeter of the lateral image of the animals. Body size was measured at the indicated time points from the L4 stage. Error bars indicate the standard error of the mean (s.e.m). (B) Body size indicated by the length of the animals 48 hours after the L4 stage. “n.s.” indicates that the difference is not significant (p>0.05, t test). (C) Typical tracks left by a single animal on an E. coli lawn during an 18-hours period. Scale bar, 10 mm. (D) Chemotaxis and osmotic avoidance assays. Each data point represents an average of at least 3 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference (p<0.01, t test). (E) The rate of dauer formation at 20°C. The Daf-c phenotype of chb-3 was suppressed by che-2(e1033) but not by daf-3(e1376) or daf-16(m26). Rescue of the chb-3(eg52) Daf-c phenotype by the Y48G1A.3 transgene (Ex[Y48G1A.3/myo-3::gfp]) is also shown. Each data point represents an average of at least 5 independent assays. Error bars indicate s.e.m. The mark (*) indicates the significant difference from chb-3(eg52) (p<0.01, t test). #The dauer formation rate of chb-3(qj2) was determined by gathering eggs from the chb-3(qj2); Ex[tax-4p::CHB-3::GFP] rescued line, and observing hatched animals that lost the Ex array.
Mentions: chb-3(eg52) was previously isolated as a mutant which suppresses the small body size of che-2, but not the cilium structural defect of che-2, in a screen that also identified egl-4 [6]. As shown in Figure 1A and 1B, the chb-3(eg52) mutation caused an increase in body size of che-2(e1033). Compared with wild-type animals (WT, N2 strain), the chb-3(eg52) single mutant were longer and larger overall (Figure 2). Suppression of the che-2 small-body phenotype by chb-3, however, is unlikely a result of simple additive effects of the mutation, because a che-2 transgene did not increase the size of the chb-3(eg52);che-2(e1033) mutant (chb-3(eg52);che-2(e1033);Ex[che-2] in Figure 1A). Moreover, the chb-3(eg52) single mutant and the chb-3(eg52);che-2(e1033) double mutant were similar in body size (Figure 1B). We defined the group of Chb mutants displaying body sizes that were not affected by the che-2 mutation as class I mutants, whereas the other Chb mutants were designated as class II mutants [6]. On the whole, the class I Chb genes are required for animals to make the body size small when animals do not perceive sensory inputs (e.g., cilium-defective mutants). The epistasis of chb-3 to che-2 indicates that chb-3 is a class I Chb mutant, suggesting a role for the chb-3 gene in the sensory processing-mediated regulation of body size.

Bottom Line: By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals.Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans.Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan. fujiwara.manabi.734@m.kyushu-u.ac.jp

ABSTRACT
Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

Show MeSH
Related in: MedlinePlus