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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 synthetic dye-filling defective phenotype of arl-13; nphp-2 mutants is modulated by hdac-6.hdac-6 deletion suppresses arl-13; nphp-2 double mutant phenotypes. In phasmids, nphp-2 and arl-13 single mutants are mildly Dyf, which is not suppressed by hdac-6 deletion. In both amphids and phasmids, arl-13; nphp-2 is severely SynDyf and was suppressed by hdac-6. Data in both panels was analyzed with pairwise Mann-Whitney U-test between all groups, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Groups from either panel sharing a capital letter are not significantly different, whereas groups from either panel with different capital letters do differ significantly.
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pgen-1004866-g001: The synthetic dye-filling defective phenotype of arl-13; nphp-2 mutants is modulated by hdac-6.hdac-6 deletion suppresses arl-13; nphp-2 double mutant phenotypes. In phasmids, nphp-2 and arl-13 single mutants are mildly Dyf, which is not suppressed by hdac-6 deletion. In both amphids and phasmids, arl-13; nphp-2 is severely SynDyf and was suppressed by hdac-6. Data in both panels was analyzed with pairwise Mann-Whitney U-test between all groups, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Groups from either panel sharing a capital letter are not significantly different, whereas groups from either panel with different capital letters do differ significantly.

Mentions: nphp-2 and arl-13 single mutants have statistically similar, moderate ciliogenic defects (Fig. 1). As hdac-6 and arl-3 modulate several arl-13 phenotypes [35], we sought to determine if nphp-2 and arl-13 genetically interact and if hdac-6 and arl-3 modulate nphp-2 phenotypes. We examined double, triple, and quadruple mutant combinations using “dye filling” of ciliated neurons as a gross indicator of ciliogenesis and cilia integrity [32]. Properly formed and placed cilia are environmentally exposed and take up fluorescent DiI dye, whereas stunted and misplaced cilia are not exposed and cannot take up DiI. Unlike the mild dye-filling defects (Dyf) of nphp-2 and arl-13 single mutants, arl-13; nphp-2 double mutants were severely synthetic dye-filling defective (SynDyf) in both the amphids and phasmids (Fig. 1). hdac-6 deletion did not suppress nphp-2 or arl-13 Dyf; this is contrary to previously published data indicating that hdac-6 can partially suppress the weak arl-13 single mutant Dyf, and may be due to a difference in dye filling or scoring method [35] (Materials and Methods). However, hdac-6 suppressed the arl-13; nphp-2 severe SynDyf phenotype to the mild Dyf severity of the single mutants in both amphids and phasmids (Fig. 1). hdac-6 may function by suppressing defects arising from one of the pathways or at a point where the two pathways converge. Combined, this data indicates that arl-13 and nphp-2 act in partially redundant parallel pathways antagonized by hdac-6.


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 synthetic dye-filling defective phenotype of arl-13; nphp-2 mutants is modulated by hdac-6.hdac-6 deletion suppresses arl-13; nphp-2 double mutant phenotypes. In phasmids, nphp-2 and arl-13 single mutants are mildly Dyf, which is not suppressed by hdac-6 deletion. In both amphids and phasmids, arl-13; nphp-2 is severely SynDyf and was suppressed by hdac-6. Data in both panels was analyzed with pairwise Mann-Whitney U-test between all groups, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Groups from either panel sharing a capital letter are not significantly different, whereas groups from either panel with different capital letters do differ significantly.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g001: The synthetic dye-filling defective phenotype of arl-13; nphp-2 mutants is modulated by hdac-6.hdac-6 deletion suppresses arl-13; nphp-2 double mutant phenotypes. In phasmids, nphp-2 and arl-13 single mutants are mildly Dyf, which is not suppressed by hdac-6 deletion. In both amphids and phasmids, arl-13; nphp-2 is severely SynDyf and was suppressed by hdac-6. Data in both panels was analyzed with pairwise Mann-Whitney U-test between all groups, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Groups from either panel sharing a capital letter are not significantly different, whereas groups from either panel with different capital letters do differ significantly.
Mentions: nphp-2 and arl-13 single mutants have statistically similar, moderate ciliogenic defects (Fig. 1). As hdac-6 and arl-3 modulate several arl-13 phenotypes [35], we sought to determine if nphp-2 and arl-13 genetically interact and if hdac-6 and arl-3 modulate nphp-2 phenotypes. We examined double, triple, and quadruple mutant combinations using “dye filling” of ciliated neurons as a gross indicator of ciliogenesis and cilia integrity [32]. Properly formed and placed cilia are environmentally exposed and take up fluorescent DiI dye, whereas stunted and misplaced cilia are not exposed and cannot take up DiI. Unlike the mild dye-filling defects (Dyf) of nphp-2 and arl-13 single mutants, arl-13; nphp-2 double mutants were severely synthetic dye-filling defective (SynDyf) in both the amphids and phasmids (Fig. 1). hdac-6 deletion did not suppress nphp-2 or arl-13 Dyf; this is contrary to previously published data indicating that hdac-6 can partially suppress the weak arl-13 single mutant Dyf, and may be due to a difference in dye filling or scoring method [35] (Materials and Methods). However, hdac-6 suppressed the arl-13; nphp-2 severe SynDyf phenotype to the mild Dyf severity of the single mutants in both amphids and phasmids (Fig. 1). hdac-6 may function by suppressing defects arising from one of the pathways or at a point where the two pathways converge. Combined, this data indicates that arl-13 and nphp-2 act in partially redundant parallel pathways antagonized by hdac-6.

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