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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, arl-13, and hdac-6 regulate glutamylation in head and tail cilia.(A) Worms stained with GT335 anti-glutamylated tubulin antibody. In WT background, GT335 labels amphid and phasmid doublet region microtubules, and CEP doublet region and singlet region microtubules consistently, and OLQ cilia inconsistently. CEP distal microtubule singlets were also labelled by GT335. Inner labial cilia were also glutamylated, but specific IL1 and IL2 identification was not possible. nphp-2 mutants showed characteristic posterior shifted cilia, and glutamylation in the head looked similar to WT, with infrequent weak inner labial cilia staining. Phasmids exhibited an elongated glutamylation signal. arl-13 mutants exhibited elongated staining of amphid bundle, CEP, and OLQ cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. unc-119 mutants exhibited almost no staining of CEP, IL, and OLQ cilia. hdac-6 mutants displayed shorter amphid bundle staining, and reduced IL and OLQ staining. (B) In ttll-4 mutants, NPHP-2::GFP and GFP::UNC-119 localize similarly to WT. (C) In ccpp-1 mutants, both NPHP-2::GFP and GFP::UNC-119 localize similarly to in WT, and GFP::UNC-119 is present in the TZ and accumulates at the distal dendrite. Yellow arrowheads – IL2 cilia, red arrowheads - OLQ cilia, blue arrowheads – CEP cilia, white bar – amphid/phasmid bundle.
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pgen-1004866-g007: nphp-2, arl-13, and hdac-6 regulate glutamylation in head and tail cilia.(A) Worms stained with GT335 anti-glutamylated tubulin antibody. In WT background, GT335 labels amphid and phasmid doublet region microtubules, and CEP doublet region and singlet region microtubules consistently, and OLQ cilia inconsistently. CEP distal microtubule singlets were also labelled by GT335. Inner labial cilia were also glutamylated, but specific IL1 and IL2 identification was not possible. nphp-2 mutants showed characteristic posterior shifted cilia, and glutamylation in the head looked similar to WT, with infrequent weak inner labial cilia staining. Phasmids exhibited an elongated glutamylation signal. arl-13 mutants exhibited elongated staining of amphid bundle, CEP, and OLQ cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. unc-119 mutants exhibited almost no staining of CEP, IL, and OLQ cilia. hdac-6 mutants displayed shorter amphid bundle staining, and reduced IL and OLQ staining. (B) In ttll-4 mutants, NPHP-2::GFP and GFP::UNC-119 localize similarly to WT. (C) In ccpp-1 mutants, both NPHP-2::GFP and GFP::UNC-119 localize similarly to in WT, and GFP::UNC-119 is present in the TZ and accumulates at the distal dendrite. Yellow arrowheads – IL2 cilia, red arrowheads - OLQ cilia, blue arrowheads – CEP cilia, white bar – amphid/phasmid bundle.

Mentions: In wild-type animals, the anti-glutamylated tubulin antibody GT335 labeled the doublet region of amphid channel and phasmid cilia (Fig. 7A) [52]. nphp-2 mutants exhibited characteristic cilia displacement in the amphids, but no qualitative changes in head cilia glutamylation. The glutamylation signal in nphp-2 phasmid cilia ranged from wild-type-like to extremely elongated, which is consistent with the TEM observation of B-tubules extending into the distal axoneme (Fig. 2B). arl-13 mutants exhibited elongated staining in amphid channel cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. hdac-6 mutants appeared to have shortened GT335 staining of amphid channel cilia. We also observed significant differences in the length of the phasmid GT335 ciliary signal. Both arl-13 (3.95±0.25 µm) and nphp-2 (4.30±0.32 µm) mutants had phasmid staining significantly longer than in wild type (2.79±0.07 µm), while unc-119 (2.40±0.05 µm) mutants had staining significantly shorter (S7E Figure). The length of GT335 staining in amphid cilia was not quantified due to the difficulty of unbiased measurement of a single cilium within the amphid bundle.


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, arl-13, and hdac-6 regulate glutamylation in head and tail cilia.(A) Worms stained with GT335 anti-glutamylated tubulin antibody. In WT background, GT335 labels amphid and phasmid doublet region microtubules, and CEP doublet region and singlet region microtubules consistently, and OLQ cilia inconsistently. CEP distal microtubule singlets were also labelled by GT335. Inner labial cilia were also glutamylated, but specific IL1 and IL2 identification was not possible. nphp-2 mutants showed characteristic posterior shifted cilia, and glutamylation in the head looked similar to WT, with infrequent weak inner labial cilia staining. Phasmids exhibited an elongated glutamylation signal. arl-13 mutants exhibited elongated staining of amphid bundle, CEP, and OLQ cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. unc-119 mutants exhibited almost no staining of CEP, IL, and OLQ cilia. hdac-6 mutants displayed shorter amphid bundle staining, and reduced IL and OLQ staining. (B) In ttll-4 mutants, NPHP-2::GFP and GFP::UNC-119 localize similarly to WT. (C) In ccpp-1 mutants, both NPHP-2::GFP and GFP::UNC-119 localize similarly to in WT, and GFP::UNC-119 is present in the TZ and accumulates at the distal dendrite. Yellow arrowheads – IL2 cilia, red arrowheads - OLQ cilia, blue arrowheads – CEP cilia, white bar – amphid/phasmid bundle.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004866-g007: nphp-2, arl-13, and hdac-6 regulate glutamylation in head and tail cilia.(A) Worms stained with GT335 anti-glutamylated tubulin antibody. In WT background, GT335 labels amphid and phasmid doublet region microtubules, and CEP doublet region and singlet region microtubules consistently, and OLQ cilia inconsistently. CEP distal microtubule singlets were also labelled by GT335. Inner labial cilia were also glutamylated, but specific IL1 and IL2 identification was not possible. nphp-2 mutants showed characteristic posterior shifted cilia, and glutamylation in the head looked similar to WT, with infrequent weak inner labial cilia staining. Phasmids exhibited an elongated glutamylation signal. arl-13 mutants exhibited elongated staining of amphid bundle, CEP, and OLQ cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. unc-119 mutants exhibited almost no staining of CEP, IL, and OLQ cilia. hdac-6 mutants displayed shorter amphid bundle staining, and reduced IL and OLQ staining. (B) In ttll-4 mutants, NPHP-2::GFP and GFP::UNC-119 localize similarly to WT. (C) In ccpp-1 mutants, both NPHP-2::GFP and GFP::UNC-119 localize similarly to in WT, and GFP::UNC-119 is present in the TZ and accumulates at the distal dendrite. Yellow arrowheads – IL2 cilia, red arrowheads - OLQ cilia, blue arrowheads – CEP cilia, white bar – amphid/phasmid bundle.
Mentions: In wild-type animals, the anti-glutamylated tubulin antibody GT335 labeled the doublet region of amphid channel and phasmid cilia (Fig. 7A) [52]. nphp-2 mutants exhibited characteristic cilia displacement in the amphids, but no qualitative changes in head cilia glutamylation. The glutamylation signal in nphp-2 phasmid cilia ranged from wild-type-like to extremely elongated, which is consistent with the TEM observation of B-tubules extending into the distal axoneme (Fig. 2B). arl-13 mutants exhibited elongated staining in amphid channel cilia. Amphid staining in unc-119 mutants was extremely shortened and cilia were angled inwards. hdac-6 mutants appeared to have shortened GT335 staining of amphid channel cilia. We also observed significant differences in the length of the phasmid GT335 ciliary signal. Both arl-13 (3.95±0.25 µm) and nphp-2 (4.30±0.32 µm) mutants had phasmid staining significantly longer than in wild type (2.79±0.07 µm), while unc-119 (2.40±0.05 µm) mutants had staining significantly shorter (S7E Figure). The length of GT335 staining in amphid cilia was not quantified due to the difficulty of unbiased measurement of a single cilium within the amphid bundle.

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