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The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans.

Warburton-Pitt SR, Silva M, Nguyen KC, Hall DH, Barr MM - PLoS Genet. (2014)

Bottom Line: The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes.NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC.We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the "Inversin compartment" (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules-nphp-2+klp-11 and arl-13+unc-119-which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

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The EF hand is necessary for proper NPHP-2 localization and function.(A) Diagram of the domains present in Inversin, NPHP-2, and each of the NPHP-2 domain deletion constructs. (B) Localization of GFP-tagged NPHP-2 domain deletion constructs. In the head, full length NPHP-2::GFP localizes to the InvC of amphid channel cilia, to the base of IL2 cilia, and to the base of either OLQ or CEP cilia. In the tail (outlined by dashes), NPHP-2::GFP localizes to the InvC of phasmid cilia. Localization of both NPHP-2-NLS1Δ::GFP and NPHP-2-NLS2Δ::GFP appeared roughly wild-type in all cell types, though both constructs appeared enriched in AWC wing cilia. NPHP-2-EFΔ::GFP is present in IL2 and CEP cilia but fails to localize to amphid channel and phasmid cilia. (C) NPHP-2-NLS1Δ::GFP, NPHP-2-NLS2Δ::GFP, and NPHP-2-EFΔ::GFP can rescue nphp-2 nphp-4 amphid and phasmid dye-filling defects, but NPHP-2-EFΔ::GFP rescue is significantly worse than full length NPHP-2::GFP in both amphid and phasmid neurons. Yellow arrowheads - IL2 cilia, red arrowheads - OLQ, blue arrowheads - CEP cilia, white bar - amphid/phasmid bundle. Letters indicate statistically distinct groups. Data was analyzed with pairwise Mann-Whitney U-test against both positive and negative controls, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Non-transgene expressing siblings were used as negative controls in rescue experiments.
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pgen-1004866-g004: The EF hand is necessary for proper NPHP-2 localization and function.(A) Diagram of the domains present in Inversin, NPHP-2, and each of the NPHP-2 domain deletion constructs. (B) Localization of GFP-tagged NPHP-2 domain deletion constructs. In the head, full length NPHP-2::GFP localizes to the InvC of amphid channel cilia, to the base of IL2 cilia, and to the base of either OLQ or CEP cilia. In the tail (outlined by dashes), NPHP-2::GFP localizes to the InvC of phasmid cilia. Localization of both NPHP-2-NLS1Δ::GFP and NPHP-2-NLS2Δ::GFP appeared roughly wild-type in all cell types, though both constructs appeared enriched in AWC wing cilia. NPHP-2-EFΔ::GFP is present in IL2 and CEP cilia but fails to localize to amphid channel and phasmid cilia. (C) NPHP-2-NLS1Δ::GFP, NPHP-2-NLS2Δ::GFP, and NPHP-2-EFΔ::GFP can rescue nphp-2 nphp-4 amphid and phasmid dye-filling defects, but NPHP-2-EFΔ::GFP rescue is significantly worse than full length NPHP-2::GFP in both amphid and phasmid neurons. Yellow arrowheads - IL2 cilia, red arrowheads - OLQ, blue arrowheads - CEP cilia, white bar - amphid/phasmid bundle. Letters indicate statistically distinct groups. Data was analyzed with pairwise Mann-Whitney U-test against both positive and negative controls, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Non-transgene expressing siblings were used as negative controls in rescue experiments.

Mentions: NPHP-2::GFP signal length is similar in wild-type and nphp-2 backgrounds (1.77±0.23 um vs 1.74±0.22 um, st. dev.) and NPHP-2::GFP rescues the SynDyf phenotype of the nphp-2 nphp-4 double mutant (Fig. 4B) [17]. These results indicate that this reporter reflects NPHP-2 functional and endogenous localization. In both nphp-4 and mks-3 single mutants, NPHP-2::GFP was properly targeted to and imported into the cilium. Mislocalized NPHP-2::GFP puncta in the periciliary region were sometimes visible (Fig. 3A). In mks-3; nphp-4 double mutants, there were severe ciliogenic and dendritic extension errors, as previously reported [17], [42]; in phasmid cilia that were visible and placed properly, NPHP-2::GFP localization appeared as in mks-3 and nphp-4 single mutants (S2A Figure). In a wild-type background, ARL-13::GFP localized exclusively to the doublet region in amphid channel and phasmid neurons (Fig. 3C). Like NPHP-2::GFP, ARL-13::GFP was targeted to and imported into the cilium properly in mks-3 and nphp-4 mutants. Similar to published reports, we also observed ARL-13::GFP mislocalization to the periciliary membrane, as judged by a fluorescent “fringe” surrounding the periciliary region where the membrane lies (Fig. 3C, enhanced contrast in S2B Figure), which has been suggested to be due to a failure of the TZ diffusion barrier [34]. In amphid cilia, like in phasmid cilia, both NPHP-2 and ARL-13 were targeted to the cilium and imported properly, and both infrequently exhibited mild mislocalization (S3 Figure).


The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans.

Warburton-Pitt SR, Silva M, Nguyen KC, Hall DH, Barr MM - PLoS Genet. (2014)

The EF hand is necessary for proper NPHP-2 localization and function.(A) Diagram of the domains present in Inversin, NPHP-2, and each of the NPHP-2 domain deletion constructs. (B) Localization of GFP-tagged NPHP-2 domain deletion constructs. In the head, full length NPHP-2::GFP localizes to the InvC of amphid channel cilia, to the base of IL2 cilia, and to the base of either OLQ or CEP cilia. In the tail (outlined by dashes), NPHP-2::GFP localizes to the InvC of phasmid cilia. Localization of both NPHP-2-NLS1Δ::GFP and NPHP-2-NLS2Δ::GFP appeared roughly wild-type in all cell types, though both constructs appeared enriched in AWC wing cilia. NPHP-2-EFΔ::GFP is present in IL2 and CEP cilia but fails to localize to amphid channel and phasmid cilia. (C) NPHP-2-NLS1Δ::GFP, NPHP-2-NLS2Δ::GFP, and NPHP-2-EFΔ::GFP can rescue nphp-2 nphp-4 amphid and phasmid dye-filling defects, but NPHP-2-EFΔ::GFP rescue is significantly worse than full length NPHP-2::GFP in both amphid and phasmid neurons. Yellow arrowheads - IL2 cilia, red arrowheads - OLQ, blue arrowheads - CEP cilia, white bar - amphid/phasmid bundle. Letters indicate statistically distinct groups. Data was analyzed with pairwise Mann-Whitney U-test against both positive and negative controls, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Non-transgene expressing siblings were used as negative controls in rescue experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263411&req=5

pgen-1004866-g004: The EF hand is necessary for proper NPHP-2 localization and function.(A) Diagram of the domains present in Inversin, NPHP-2, and each of the NPHP-2 domain deletion constructs. (B) Localization of GFP-tagged NPHP-2 domain deletion constructs. In the head, full length NPHP-2::GFP localizes to the InvC of amphid channel cilia, to the base of IL2 cilia, and to the base of either OLQ or CEP cilia. In the tail (outlined by dashes), NPHP-2::GFP localizes to the InvC of phasmid cilia. Localization of both NPHP-2-NLS1Δ::GFP and NPHP-2-NLS2Δ::GFP appeared roughly wild-type in all cell types, though both constructs appeared enriched in AWC wing cilia. NPHP-2-EFΔ::GFP is present in IL2 and CEP cilia but fails to localize to amphid channel and phasmid cilia. (C) NPHP-2-NLS1Δ::GFP, NPHP-2-NLS2Δ::GFP, and NPHP-2-EFΔ::GFP can rescue nphp-2 nphp-4 amphid and phasmid dye-filling defects, but NPHP-2-EFΔ::GFP rescue is significantly worse than full length NPHP-2::GFP in both amphid and phasmid neurons. Yellow arrowheads - IL2 cilia, red arrowheads - OLQ, blue arrowheads - CEP cilia, white bar - amphid/phasmid bundle. Letters indicate statistically distinct groups. Data was analyzed with pairwise Mann-Whitney U-test against both positive and negative controls, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Non-transgene expressing siblings were used as negative controls in rescue experiments.
Mentions: NPHP-2::GFP signal length is similar in wild-type and nphp-2 backgrounds (1.77±0.23 um vs 1.74±0.22 um, st. dev.) and NPHP-2::GFP rescues the SynDyf phenotype of the nphp-2 nphp-4 double mutant (Fig. 4B) [17]. These results indicate that this reporter reflects NPHP-2 functional and endogenous localization. In both nphp-4 and mks-3 single mutants, NPHP-2::GFP was properly targeted to and imported into the cilium. Mislocalized NPHP-2::GFP puncta in the periciliary region were sometimes visible (Fig. 3A). In mks-3; nphp-4 double mutants, there were severe ciliogenic and dendritic extension errors, as previously reported [17], [42]; in phasmid cilia that were visible and placed properly, NPHP-2::GFP localization appeared as in mks-3 and nphp-4 single mutants (S2A Figure). In a wild-type background, ARL-13::GFP localized exclusively to the doublet region in amphid channel and phasmid neurons (Fig. 3C). Like NPHP-2::GFP, ARL-13::GFP was targeted to and imported into the cilium properly in mks-3 and nphp-4 mutants. Similar to published reports, we also observed ARL-13::GFP mislocalization to the periciliary membrane, as judged by a fluorescent “fringe” surrounding the periciliary region where the membrane lies (Fig. 3C, enhanced contrast in S2B Figure), which has been suggested to be due to a failure of the TZ diffusion barrier [34]. In amphid cilia, like in phasmid cilia, both NPHP-2 and ARL-13 were targeted to the cilium and imported properly, and both infrequently exhibited mild mislocalization (S3 Figure).

Bottom Line: The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes.NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC.We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the "Inversin compartment" (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules-nphp-2+klp-11 and arl-13+unc-119-which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

Show MeSH
Related in: MedlinePlus