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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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klp-11 and unc-119 genetically interact with arl-13 and nphp-2 in an hdac-6 dependent manner.(A) klp-11 single mutants are not Dyf. klp-11 is SynDyf with arl-13, which is partially suppressed by deletion of hdac-6. (B) unc-119 single mutants are moderately Dyf. unc-119 is SynDyf with both klp-11 and nphp-2. unc-119; hdac-6; nphp-2 triple mutants exhibit suppression of the unc-119 Dyf phenotype. (C) Diagram of interactions between klp-11, arl-13, nphp-2, unc-119, and hdac-6, and between DR and TZ genes, based on SynDyf phenotypes presented in panels A and B and in Fig. 1 and S5 Figure. The T-bar indicates hdac-6 mediated suppression of SynDyf phenotypes. Data was analyzed with pairwise Mann-Whitney U-test between wild type, double mutants, and their respective single mutants, followed by the Holm-Bonferroni multiple comparison adjustment. *, significant versus single mutants at a total alpha of 0.05. **, significant versus single mutants at a total alpha of 0.01. nphp-2, arl-13, hdac-6, and arl-13; hdac-6 Dyf data is presented in Fig. 1.
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pgen-1004866-g006: klp-11 and unc-119 genetically interact with arl-13 and nphp-2 in an hdac-6 dependent manner.(A) klp-11 single mutants are not Dyf. klp-11 is SynDyf with arl-13, which is partially suppressed by deletion of hdac-6. (B) unc-119 single mutants are moderately Dyf. unc-119 is SynDyf with both klp-11 and nphp-2. unc-119; hdac-6; nphp-2 triple mutants exhibit suppression of the unc-119 Dyf phenotype. (C) Diagram of interactions between klp-11, arl-13, nphp-2, unc-119, and hdac-6, and between DR and TZ genes, based on SynDyf phenotypes presented in panels A and B and in Fig. 1 and S5 Figure. The T-bar indicates hdac-6 mediated suppression of SynDyf phenotypes. Data was analyzed with pairwise Mann-Whitney U-test between wild type, double mutants, and their respective single mutants, followed by the Holm-Bonferroni multiple comparison adjustment. *, significant versus single mutants at a total alpha of 0.05. **, significant versus single mutants at a total alpha of 0.01. nphp-2, arl-13, hdac-6, and arl-13; hdac-6 Dyf data is presented in Fig. 1.

Mentions: Horizontal panels indicated by 1, 2, and 3 correspond to singlet region, doublet region-to-singlet region transition, and TZ levels, respectively, and are comparable across the genotypes. The insets show an enlarged view of the region within the white box, and are diagrammed in the accompanying cartoon. All scale bars are 250 nm. (A1–3) In wild-type amphids, all doublet B-tubules within a cilium have similar spans. (A1) Distal microtubule singlets are devoid of B-tubules. B-tubule containing microtubule doublets are present in (A2) the doublet region and (A3) the TZ. (B1–3) In nphp-2 animals, amphid channel cilia are shifted lengthwise with respect to each other (cf. Fig. 6A). Within a cilium, spans of microtubule doublet B-tubules are asynchronous; arrows in insets indicate these microtubules. TZ Y-links are disorganized (C1–3) In arl-13; nphp-2 animals, most amphid channel cilia are absent. (C1) The distal end is filled with electron dense material with unresolvable microtubules. (C2) A single, stub-like cilium is visible, consisting of only microtubule singlets. (C3) TZ microtubules are abnormal with some missing microtubule doublets. We also observe vesicle-like structures at this level that are indicated by arrows. (D1–3) In arl-13; hdac-6; nphp-2 animals, most amphid cilia are visible. Ectopic singlets and doublets are still present. Cilia are shifted posteriorly towards the tail. (D1–2) insets show asynchronous microtubules within cilia.


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)

klp-11 and unc-119 genetically interact with arl-13 and nphp-2 in an hdac-6 dependent manner.(A) klp-11 single mutants are not Dyf. klp-11 is SynDyf with arl-13, which is partially suppressed by deletion of hdac-6. (B) unc-119 single mutants are moderately Dyf. unc-119 is SynDyf with both klp-11 and nphp-2. unc-119; hdac-6; nphp-2 triple mutants exhibit suppression of the unc-119 Dyf phenotype. (C) Diagram of interactions between klp-11, arl-13, nphp-2, unc-119, and hdac-6, and between DR and TZ genes, based on SynDyf phenotypes presented in panels A and B and in Fig. 1 and S5 Figure. The T-bar indicates hdac-6 mediated suppression of SynDyf phenotypes. Data was analyzed with pairwise Mann-Whitney U-test between wild type, double mutants, and their respective single mutants, followed by the Holm-Bonferroni multiple comparison adjustment. *, significant versus single mutants at a total alpha of 0.05. **, significant versus single mutants at a total alpha of 0.01. nphp-2, arl-13, hdac-6, and arl-13; hdac-6 Dyf data is presented in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g006: klp-11 and unc-119 genetically interact with arl-13 and nphp-2 in an hdac-6 dependent manner.(A) klp-11 single mutants are not Dyf. klp-11 is SynDyf with arl-13, which is partially suppressed by deletion of hdac-6. (B) unc-119 single mutants are moderately Dyf. unc-119 is SynDyf with both klp-11 and nphp-2. unc-119; hdac-6; nphp-2 triple mutants exhibit suppression of the unc-119 Dyf phenotype. (C) Diagram of interactions between klp-11, arl-13, nphp-2, unc-119, and hdac-6, and between DR and TZ genes, based on SynDyf phenotypes presented in panels A and B and in Fig. 1 and S5 Figure. The T-bar indicates hdac-6 mediated suppression of SynDyf phenotypes. Data was analyzed with pairwise Mann-Whitney U-test between wild type, double mutants, and their respective single mutants, followed by the Holm-Bonferroni multiple comparison adjustment. *, significant versus single mutants at a total alpha of 0.05. **, significant versus single mutants at a total alpha of 0.01. nphp-2, arl-13, hdac-6, and arl-13; hdac-6 Dyf data is presented in Fig. 1.
Mentions: Horizontal panels indicated by 1, 2, and 3 correspond to singlet region, doublet region-to-singlet region transition, and TZ levels, respectively, and are comparable across the genotypes. The insets show an enlarged view of the region within the white box, and are diagrammed in the accompanying cartoon. All scale bars are 250 nm. (A1–3) In wild-type amphids, all doublet B-tubules within a cilium have similar spans. (A1) Distal microtubule singlets are devoid of B-tubules. B-tubule containing microtubule doublets are present in (A2) the doublet region and (A3) the TZ. (B1–3) In nphp-2 animals, amphid channel cilia are shifted lengthwise with respect to each other (cf. Fig. 6A). Within a cilium, spans of microtubule doublet B-tubules are asynchronous; arrows in insets indicate these microtubules. TZ Y-links are disorganized (C1–3) In arl-13; nphp-2 animals, most amphid channel cilia are absent. (C1) The distal end is filled with electron dense material with unresolvable microtubules. (C2) A single, stub-like cilium is visible, consisting of only microtubule singlets. (C3) TZ microtubules are abnormal with some missing microtubule doublets. We also observe vesicle-like structures at this level that are indicated by arrows. (D1–3) In arl-13; hdac-6; nphp-2 animals, most amphid cilia are visible. Ectopic singlets and doublets are still present. Cilia are shifted posteriorly towards the tail. (D1–2) insets show asynchronous microtubules within cilia.

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