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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 and ARL-13 do not require TZ-, doublet region-, and InvC-associated genes for ciliary targeting.(A) In wild type (WT), NPHP-2 is restricted to the proximal cilium. In both nphp-4 and mks-3 mutants NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. Several NPHP-2::GFP puncta were visible in the periciliary compartment. (B) In doublet region and InvC mutants, NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium in klp-11, arl-13, and unc-119 mutants. arl-13 and klp-11 mutants exhibited periciliary NPHP-2::GFP puncta, and unc-119 mutants exhibited distal dendritic accumulation of NPHP-2::GFP. (C) In WT, ARL-13::GFP localizes to the proximal cilium. In both nphp-4 and mks-3 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. ARL-13::GFP also mislocalized to the periciliary membrane compartment in TZ mutants. (D) In nphp-2 and klp-11 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. In these mutants, ARL-13::GFP also mislocalized to the periciliary membrane compartment, and in unc-119 mutants, ARL-13::GFP mislocalized to the distal dendrite. Periciliary puncta – arrowheads, periciliary membrane – white arc, distal dendrite/periciliary accumulation – white bar. Periciliary membrane localization was judged by a visible enrichment of ARL-13::GFP on the edges of the periciliary compartment without a concomitant enrichment in the interior lumen of the periciliary region.
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pgen-1004866-g003: NPHP-2 and ARL-13 do not require TZ-, doublet region-, and InvC-associated genes for ciliary targeting.(A) In wild type (WT), NPHP-2 is restricted to the proximal cilium. In both nphp-4 and mks-3 mutants NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. Several NPHP-2::GFP puncta were visible in the periciliary compartment. (B) In doublet region and InvC mutants, NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium in klp-11, arl-13, and unc-119 mutants. arl-13 and klp-11 mutants exhibited periciliary NPHP-2::GFP puncta, and unc-119 mutants exhibited distal dendritic accumulation of NPHP-2::GFP. (C) In WT, ARL-13::GFP localizes to the proximal cilium. In both nphp-4 and mks-3 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. ARL-13::GFP also mislocalized to the periciliary membrane compartment in TZ mutants. (D) In nphp-2 and klp-11 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. In these mutants, ARL-13::GFP also mislocalized to the periciliary membrane compartment, and in unc-119 mutants, ARL-13::GFP mislocalized to the distal dendrite. Periciliary puncta – arrowheads, periciliary membrane – white arc, distal dendrite/periciliary accumulation – white bar. Periciliary membrane localization was judged by a visible enrichment of ARL-13::GFP on the edges of the periciliary compartment without a concomitant enrichment in the interior lumen of the periciliary region.

Mentions: Localization of InvC and doublet region components can be broken down into three steps: first the protein is targeted to the cilia base, second, the protein is imported into the cilium, and third, the protein is restricted to a subdomain of the cilium [23]. The factors required for the initial establishment of the InvC and doublet region cilia targeting and localization restriction are unknown. The TZ functions as a regulator of ciliary protein import (Reviewed in [39]), and has been implicated in the import of InvC and doublet region components in mammalian cilia [40], [41]. As both nphp-2 and arl-13 genetically interact with TZ-associated genes [17], [37], we wanted to determine if NPHP-2 and ARL-13 ciliary targeting and import requires TZ components. In C. elegans, TZ genes are organized into two genetic and physical modules—the mks module and the nphp-1+nphp-4 module [8], [17], [25], [42]. We examined the localization of NPHP-2 and ARL-13 in mutants missing a component of each module (Fig. 3A,C).


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 and ARL-13 do not require TZ-, doublet region-, and InvC-associated genes for ciliary targeting.(A) In wild type (WT), NPHP-2 is restricted to the proximal cilium. In both nphp-4 and mks-3 mutants NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. Several NPHP-2::GFP puncta were visible in the periciliary compartment. (B) In doublet region and InvC mutants, NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium in klp-11, arl-13, and unc-119 mutants. arl-13 and klp-11 mutants exhibited periciliary NPHP-2::GFP puncta, and unc-119 mutants exhibited distal dendritic accumulation of NPHP-2::GFP. (C) In WT, ARL-13::GFP localizes to the proximal cilium. In both nphp-4 and mks-3 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. ARL-13::GFP also mislocalized to the periciliary membrane compartment in TZ mutants. (D) In nphp-2 and klp-11 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. In these mutants, ARL-13::GFP also mislocalized to the periciliary membrane compartment, and in unc-119 mutants, ARL-13::GFP mislocalized to the distal dendrite. Periciliary puncta – arrowheads, periciliary membrane – white arc, distal dendrite/periciliary accumulation – white bar. Periciliary membrane localization was judged by a visible enrichment of ARL-13::GFP on the edges of the periciliary compartment without a concomitant enrichment in the interior lumen of the periciliary region.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g003: NPHP-2 and ARL-13 do not require TZ-, doublet region-, and InvC-associated genes for ciliary targeting.(A) In wild type (WT), NPHP-2 is restricted to the proximal cilium. In both nphp-4 and mks-3 mutants NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. Several NPHP-2::GFP puncta were visible in the periciliary compartment. (B) In doublet region and InvC mutants, NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium in klp-11, arl-13, and unc-119 mutants. arl-13 and klp-11 mutants exhibited periciliary NPHP-2::GFP puncta, and unc-119 mutants exhibited distal dendritic accumulation of NPHP-2::GFP. (C) In WT, ARL-13::GFP localizes to the proximal cilium. In both nphp-4 and mks-3 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. ARL-13::GFP also mislocalized to the periciliary membrane compartment in TZ mutants. (D) In nphp-2 and klp-11 mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. In these mutants, ARL-13::GFP also mislocalized to the periciliary membrane compartment, and in unc-119 mutants, ARL-13::GFP mislocalized to the distal dendrite. Periciliary puncta – arrowheads, periciliary membrane – white arc, distal dendrite/periciliary accumulation – white bar. Periciliary membrane localization was judged by a visible enrichment of ARL-13::GFP on the edges of the periciliary compartment without a concomitant enrichment in the interior lumen of the periciliary region.
Mentions: Localization of InvC and doublet region components can be broken down into three steps: first the protein is targeted to the cilia base, second, the protein is imported into the cilium, and third, the protein is restricted to a subdomain of the cilium [23]. The factors required for the initial establishment of the InvC and doublet region cilia targeting and localization restriction are unknown. The TZ functions as a regulator of ciliary protein import (Reviewed in [39]), and has been implicated in the import of InvC and doublet region components in mammalian cilia [40], [41]. As both nphp-2 and arl-13 genetically interact with TZ-associated genes [17], [37], we wanted to determine if NPHP-2 and ARL-13 ciliary targeting and import requires TZ components. In C. elegans, TZ genes are organized into two genetic and physical modules—the mks module and the nphp-1+nphp-4 module [8], [17], [25], [42]. We examined the localization of NPHP-2 and ARL-13 in mutants missing a component of each module (Fig. 3A,C).

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