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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::GFP, ARL-13::GFP, and GFP::UNC-119 colabel with GT335 staining.In amphid channel and phasmid cilia, NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 signals overlap with GT335 staining. ARL-13::GFP and GT335 staining overlap completely. NPHP-2::GFP does not overlap completely with GT335. TZ staining by GT335 is frequently visible, as in the NPHP-2::GFP overlay. Though the signal of GFP::UNC-119 is dim due to the staining procedure, GFP::UNC-119 appears not to overlap completely with GT335. Data is quantified in S6A Figure.
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pgen-1004866-g008: NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 colabel with GT335 staining.In amphid channel and phasmid cilia, NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 signals overlap with GT335 staining. ARL-13::GFP and GT335 staining overlap completely. NPHP-2::GFP does not overlap completely with GT335. TZ staining by GT335 is frequently visible, as in the NPHP-2::GFP overlay. Though the signal of GFP::UNC-119 is dim due to the staining procedure, GFP::UNC-119 appears not to overlap completely with GT335. Data is quantified in S6A Figure.

Mentions: To allow for a more direct comparison of subciliary localization, we stained transgenic NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 strains with GT335 (Fig. 8). NPHP-2::GFP did not fully overlap with GT335, only colabelling the proximal portion of the doublet region of amphid and phasmid cilia. However, ARL-13::GFP colabelled with a greater portion of the GT335 doublet region signal in amphid cilia than did NPHP-2::GFP, and completely colabelled with GT335 in phasmid cilia (Fig. 8, S6A Figure); this suggests that ARL-13 is associated with the microtubule doublets that define the doublet region. GFP::UNC-119 colabelled with either a significant fraction of the length of the GT335 signal. Additionally, GFP::UNC-119 did not extend beyond the GT335 labelled doublet region, indicating that GFP::UNC-119 is excluded from the TZ, which is not labelled by GT335.


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::GFP, ARL-13::GFP, and GFP::UNC-119 colabel with GT335 staining.In amphid channel and phasmid cilia, NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 signals overlap with GT335 staining. ARL-13::GFP and GT335 staining overlap completely. NPHP-2::GFP does not overlap completely with GT335. TZ staining by GT335 is frequently visible, as in the NPHP-2::GFP overlay. Though the signal of GFP::UNC-119 is dim due to the staining procedure, GFP::UNC-119 appears not to overlap completely with GT335. Data is quantified in S6A Figure.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g008: NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 colabel with GT335 staining.In amphid channel and phasmid cilia, NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 signals overlap with GT335 staining. ARL-13::GFP and GT335 staining overlap completely. NPHP-2::GFP does not overlap completely with GT335. TZ staining by GT335 is frequently visible, as in the NPHP-2::GFP overlay. Though the signal of GFP::UNC-119 is dim due to the staining procedure, GFP::UNC-119 appears not to overlap completely with GT335. Data is quantified in S6A Figure.
Mentions: To allow for a more direct comparison of subciliary localization, we stained transgenic NPHP-2::GFP, ARL-13::GFP, and GFP::UNC-119 strains with GT335 (Fig. 8). NPHP-2::GFP did not fully overlap with GT335, only colabelling the proximal portion of the doublet region of amphid and phasmid cilia. However, ARL-13::GFP colabelled with a greater portion of the GT335 doublet region signal in amphid cilia than did NPHP-2::GFP, and completely colabelled with GT335 in phasmid cilia (Fig. 8, S6A Figure); this suggests that ARL-13 is associated with the microtubule doublets that define the doublet region. GFP::UNC-119 colabelled with either a significant fraction of the length of the GT335 signal. Additionally, GFP::UNC-119 did not extend beyond the GT335 labelled doublet region, indicating that GFP::UNC-119 is excluded from the TZ, which is not labelled by GT335.

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