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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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nphp-2 single, arl-13; nphp-2 double, and arl-13; hdac-6; nphp-2 triple mutants exhibit defects in ciliary ultrastructure.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.
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pgen-1004866-g002: nphp-2 single, arl-13; nphp-2 double, and arl-13; hdac-6; nphp-2 triple mutants exhibit defects in ciliary ultrastructure.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.

Mentions: To gain a better understanding of the defects present in nphp-2 single and arl-13; nphp-2 double mutants, and to examine the effects of hdac-6 mediated suppression of the double mutant defects, we used serial-section transmission electron microscopy (TEM) to examine the ultrastructure of amphid channel cilia. Wild-type amphid cilia are divided into three segments based on microtubule ultrastructure: the TZ, the doublet region, and the singlet region (Fig. 2A). One of the two microtubules within a doublet, the A-tubule, extends to form the microtubule singlet seen in the distal cilium (Fig. 2A1); the second tubule of the doublet, the B-tubule, terminates at the distal end of the doublet region. The doublet microtubules of the TZ and doublet region are arranged in a circular pattern in close proximity to the ciliary membrane (Fig. 2A2, 2A3). Within a particular cilium, microtubule ultrastructural characteristics—the location in the lumen, membrane association, and singlet/doublet architecture—are similar across all nine outer microtubule doublets.


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)

nphp-2 single, arl-13; nphp-2 double, and arl-13; hdac-6; nphp-2 triple mutants exhibit defects in ciliary ultrastructure.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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g002: nphp-2 single, arl-13; nphp-2 double, and arl-13; hdac-6; nphp-2 triple mutants exhibit defects in ciliary ultrastructure.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.
Mentions: To gain a better understanding of the defects present in nphp-2 single and arl-13; nphp-2 double mutants, and to examine the effects of hdac-6 mediated suppression of the double mutant defects, we used serial-section transmission electron microscopy (TEM) to examine the ultrastructure of amphid channel cilia. Wild-type amphid cilia are divided into three segments based on microtubule ultrastructure: the TZ, the doublet region, and the singlet region (Fig. 2A). One of the two microtubules within a doublet, the A-tubule, extends to form the microtubule singlet seen in the distal cilium (Fig. 2A1); the second tubule of the doublet, the B-tubule, terminates at the distal end of the doublet region. The doublet microtubules of the TZ and doublet region are arranged in a circular pattern in close proximity to the ciliary membrane (Fig. 2A2, 2A3). Within a particular cilium, microtubule ultrastructural characteristics—the location in the lumen, membrane association, and singlet/doublet architecture—are similar across all nine outer microtubule doublets.

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