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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 encodes a novel protein with a zf-MYND motif.(A) Genetic and physical map of the chb-3 locus. Y50C1, Y2F4 (C. elegans genomic YAC clone), and a Y48G1A.3 PCR fragment (6.5 kb) which contained the Y48G1A.3 predicted gene and 1 kb of upstream sequence, rescued the Daf-c phenotype of chb-3(eg52). The other clones are all YAC clones that failed to rescue the chb-3 phenotypes. (B) Gene structure and mutation sites of chb-3/Y48G1A.3. Exons are boxed. (C) chb-3 encodes a novel protein with four consecutive ankyrin repeats (ANK) close to the N terminus and a MYND-type Zn-finger motif (zf-MYND) close to the C terminus. Homologues from D. melanogaster (NP 648406), Homo sapiens (NP 064715), and Mus musculus (BAC36860) are included in the alignment. The amino-acid identities (and similarities in parentheses) to CHB-3 are shown for each ANK and zf-MYND domain and the region between the domains. (D) GFP expression from chb-3p::CHB-3::GFP in the head and tail regions of a wild-type animal (young adult stage). Projection of confocal microscopic sectioning images. Anterior is to the right. The scale bar represents 20 µm. (E) Phenotype rescue by chb-3 expressed under the control of the indicated sensory promoters. Sensory neurons in which each promoter drives expression are also shown. To examine rescuing of the Chb phenotype (suppression of che-2-small-body size), each construct was introduced into chb-3(eg52);che-2(e1033) animals as an extrachromosomal (Ex) array. Decrease of the size with the Ex array means rescuing of the Chb phenotype. To examine rescuing of the chemotaxis defect and Daf-c phenotype, each construct was introduced into chb-3(eg52) animals as an Ex array. Chemotaxis assays were performed with isoamyl alcohol (1/100 dilution). Animals with (light gray bars) and without (dark gray bars) the Ex array were compared. The mark (*) indicates the significant difference from control (p<0.01, t test).
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pgen-1001211-g003: chb-3 encodes a novel protein with a zf-MYND motif.(A) Genetic and physical map of the chb-3 locus. Y50C1, Y2F4 (C. elegans genomic YAC clone), and a Y48G1A.3 PCR fragment (6.5 kb) which contained the Y48G1A.3 predicted gene and 1 kb of upstream sequence, rescued the Daf-c phenotype of chb-3(eg52). The other clones are all YAC clones that failed to rescue the chb-3 phenotypes. (B) Gene structure and mutation sites of chb-3/Y48G1A.3. Exons are boxed. (C) chb-3 encodes a novel protein with four consecutive ankyrin repeats (ANK) close to the N terminus and a MYND-type Zn-finger motif (zf-MYND) close to the C terminus. Homologues from D. melanogaster (NP 648406), Homo sapiens (NP 064715), and Mus musculus (BAC36860) are included in the alignment. The amino-acid identities (and similarities in parentheses) to CHB-3 are shown for each ANK and zf-MYND domain and the region between the domains. (D) GFP expression from chb-3p::CHB-3::GFP in the head and tail regions of a wild-type animal (young adult stage). Projection of confocal microscopic sectioning images. Anterior is to the right. The scale bar represents 20 µm. (E) Phenotype rescue by chb-3 expressed under the control of the indicated sensory promoters. Sensory neurons in which each promoter drives expression are also shown. To examine rescuing of the Chb phenotype (suppression of che-2-small-body size), each construct was introduced into chb-3(eg52);che-2(e1033) animals as an extrachromosomal (Ex) array. Decrease of the size with the Ex array means rescuing of the Chb phenotype. To examine rescuing of the chemotaxis defect and Daf-c phenotype, each construct was introduced into chb-3(eg52) animals as an Ex array. Chemotaxis assays were performed with isoamyl alcohol (1/100 dilution). Animals with (light gray bars) and without (dark gray bars) the Ex array were compared. The mark (*) indicates the significant difference from control (p<0.01, t test).

Mentions: By SNP mapping and germline transformation experiments, we identified a corresponding gene for the chb-3(eg52) mutation, Y48G1A.3 (Figure 3A, also see Materials and Methods). A genomic DNA fragment containing a single predicted gene (Y48G1A.3) restored the small body and the confined tracking pattern which are characteristic of the che-2 single mutant, when introduced into the chb-3(eg52);che-2(e1033) double mutant (Figure 1B and data not shown). The same fragment also rescued the Daf-c phenotype and the chemotaxis defect to diacetyl, when introduced into the chb-3(eg52) mutant (Figure 1E and data not shown). BLAST searches and conserved domain analysis (CDART) were performed with the predicted protein sequence of 388 amino acids. We found highly homologous proteins (∼30% identity/∼50% similarity throughout the protein) in species from flies to humans (Figure 3C), although none have defined functions. CDART revealed that CHB-3/Y48G1A.3 contains 4 consecutive ankyrin repeats in the N-terminal portion (amino acids 9-127). Ankyrin repeats are known to mediate protein-protein interactions in a diverse range of protein families. CHB-3/Y48G1A.3 also contains a motif termed a MYND type Zn-finger (zf-MYND) in the region close to the C terminus (amino acids 321-357). The zf-MYND motif is found in several different types of protein, including transcription factors and scaffolding proteins, and is thought to also mediate protein-protein interactions [23], [24].


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 encodes a novel protein with a zf-MYND motif.(A) Genetic and physical map of the chb-3 locus. Y50C1, Y2F4 (C. elegans genomic YAC clone), and a Y48G1A.3 PCR fragment (6.5 kb) which contained the Y48G1A.3 predicted gene and 1 kb of upstream sequence, rescued the Daf-c phenotype of chb-3(eg52). The other clones are all YAC clones that failed to rescue the chb-3 phenotypes. (B) Gene structure and mutation sites of chb-3/Y48G1A.3. Exons are boxed. (C) chb-3 encodes a novel protein with four consecutive ankyrin repeats (ANK) close to the N terminus and a MYND-type Zn-finger motif (zf-MYND) close to the C terminus. Homologues from D. melanogaster (NP 648406), Homo sapiens (NP 064715), and Mus musculus (BAC36860) are included in the alignment. The amino-acid identities (and similarities in parentheses) to CHB-3 are shown for each ANK and zf-MYND domain and the region between the domains. (D) GFP expression from chb-3p::CHB-3::GFP in the head and tail regions of a wild-type animal (young adult stage). Projection of confocal microscopic sectioning images. Anterior is to the right. The scale bar represents 20 µm. (E) Phenotype rescue by chb-3 expressed under the control of the indicated sensory promoters. Sensory neurons in which each promoter drives expression are also shown. To examine rescuing of the Chb phenotype (suppression of che-2-small-body size), each construct was introduced into chb-3(eg52);che-2(e1033) animals as an extrachromosomal (Ex) array. Decrease of the size with the Ex array means rescuing of the Chb phenotype. To examine rescuing of the chemotaxis defect and Daf-c phenotype, each construct was introduced into chb-3(eg52) animals as an Ex array. Chemotaxis assays were performed with isoamyl alcohol (1/100 dilution). Animals with (light gray bars) and without (dark gray bars) the Ex array were compared. The mark (*) indicates the significant difference from control (p<0.01, t test).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991246&req=5

pgen-1001211-g003: chb-3 encodes a novel protein with a zf-MYND motif.(A) Genetic and physical map of the chb-3 locus. Y50C1, Y2F4 (C. elegans genomic YAC clone), and a Y48G1A.3 PCR fragment (6.5 kb) which contained the Y48G1A.3 predicted gene and 1 kb of upstream sequence, rescued the Daf-c phenotype of chb-3(eg52). The other clones are all YAC clones that failed to rescue the chb-3 phenotypes. (B) Gene structure and mutation sites of chb-3/Y48G1A.3. Exons are boxed. (C) chb-3 encodes a novel protein with four consecutive ankyrin repeats (ANK) close to the N terminus and a MYND-type Zn-finger motif (zf-MYND) close to the C terminus. Homologues from D. melanogaster (NP 648406), Homo sapiens (NP 064715), and Mus musculus (BAC36860) are included in the alignment. The amino-acid identities (and similarities in parentheses) to CHB-3 are shown for each ANK and zf-MYND domain and the region between the domains. (D) GFP expression from chb-3p::CHB-3::GFP in the head and tail regions of a wild-type animal (young adult stage). Projection of confocal microscopic sectioning images. Anterior is to the right. The scale bar represents 20 µm. (E) Phenotype rescue by chb-3 expressed under the control of the indicated sensory promoters. Sensory neurons in which each promoter drives expression are also shown. To examine rescuing of the Chb phenotype (suppression of che-2-small-body size), each construct was introduced into chb-3(eg52);che-2(e1033) animals as an extrachromosomal (Ex) array. Decrease of the size with the Ex array means rescuing of the Chb phenotype. To examine rescuing of the chemotaxis defect and Daf-c phenotype, each construct was introduced into chb-3(eg52) animals as an Ex array. Chemotaxis assays were performed with isoamyl alcohol (1/100 dilution). Animals with (light gray bars) and without (dark gray bars) the Ex array were compared. The mark (*) indicates the significant difference from control (p<0.01, t test).
Mentions: By SNP mapping and germline transformation experiments, we identified a corresponding gene for the chb-3(eg52) mutation, Y48G1A.3 (Figure 3A, also see Materials and Methods). A genomic DNA fragment containing a single predicted gene (Y48G1A.3) restored the small body and the confined tracking pattern which are characteristic of the che-2 single mutant, when introduced into the chb-3(eg52);che-2(e1033) double mutant (Figure 1B and data not shown). The same fragment also rescued the Daf-c phenotype and the chemotaxis defect to diacetyl, when introduced into the chb-3(eg52) mutant (Figure 1E and data not shown). BLAST searches and conserved domain analysis (CDART) were performed with the predicted protein sequence of 388 amino acids. We found highly homologous proteins (∼30% identity/∼50% similarity throughout the protein) in species from flies to humans (Figure 3C), although none have defined functions. CDART revealed that CHB-3/Y48G1A.3 contains 4 consecutive ankyrin repeats in the N-terminal portion (amino acids 9-127). Ankyrin repeats are known to mediate protein-protein interactions in a diverse range of protein families. CHB-3/Y48G1A.3 also contains a motif termed a MYND type Zn-finger (zf-MYND) in the region close to the C terminus (amino acids 321-357). The zf-MYND motif is found in several different types of protein, including transcription factors and scaffolding proteins, and is thought to also mediate protein-protein interactions [23], [24].

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