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The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans.

Schouteden C, Serwas D, Palfy M, Dammermann A - J. Cell Biol. (2015)

Bottom Line: Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links.Co-inhibition of all three modules completely disrupted transition zone structure.Surprisingly, axoneme assembly was only mildly perturbed.

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Affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), A-1030 Vienna, Austria.

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Ciliogenesis is largely normal in transition zone mutants. (A) Phasmid cilia in wild-type and transition zone mutants visualized by the IFT marker CHE-11:GFP (arrowheads). (B and C) Quantitation of phasmid cilia number per animal (B, n > 50 animals) and length (C, n > 25 cilia) based on CHE-11:GFP. Error bars indicate the 95% confidence intervals. Asterisks indicate statistically significant difference to wild-type (t test, P < 0.01). (D) Stills and kymographs from time-lapse sequences of IFT in phasmids of wild-type and transition zone triple mutants expressing CHE-11:GFP. (E) Anterograde IFT rates in the middle segment of wild-type and transition zone mutant phasmids (n > 40 particles per strain). Error bars indicate the 95% confidence interval. (F) Transmission electron micrographs of amphid cilia at the level of the middle segment of the axoneme in wild-type, ccep-290Δ, ccep-290Δ;nphp-4, and ccep-290;mksr-2;nphp-4 mutants. Axonemes were essentially normal except for occasional displaced doublet microtubules (arrowheads). Doublet microtubule number was not significantly affected (8.7 ± 1.3 2×MTs/cilium, n = 38 wild type; 7.7 ± 1.8 2×MTs/cilium, n = 27 triple mutant; t test, NS). Bars: (A and D) 5 µm; (F) 200 nm.
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fig2: Ciliogenesis is largely normal in transition zone mutants. (A) Phasmid cilia in wild-type and transition zone mutants visualized by the IFT marker CHE-11:GFP (arrowheads). (B and C) Quantitation of phasmid cilia number per animal (B, n > 50 animals) and length (C, n > 25 cilia) based on CHE-11:GFP. Error bars indicate the 95% confidence intervals. Asterisks indicate statistically significant difference to wild-type (t test, P < 0.01). (D) Stills and kymographs from time-lapse sequences of IFT in phasmids of wild-type and transition zone triple mutants expressing CHE-11:GFP. (E) Anterograde IFT rates in the middle segment of wild-type and transition zone mutant phasmids (n > 40 particles per strain). Error bars indicate the 95% confidence interval. (F) Transmission electron micrographs of amphid cilia at the level of the middle segment of the axoneme in wild-type, ccep-290Δ, ccep-290Δ;nphp-4, and ccep-290;mksr-2;nphp-4 mutants. Axonemes were essentially normal except for occasional displaced doublet microtubules (arrowheads). Doublet microtubule number was not significantly affected (8.7 ± 1.3 2×MTs/cilium, n = 38 wild type; 7.7 ± 1.8 2×MTs/cilium, n = 27 triple mutant; t test, NS). Bars: (A and D) 5 µm; (F) 200 nm.

Mentions: We next examined the consequences of transition zone perturbations on cilia assembly. A commonly used method to assess cilia integrity in C. elegans is the dye-fill assay, which monitors uptake of the lipophilic dye DiI (Inglis et al., 2007). This dye is taken up by a subset of ciliated neurons (12 amphid neurons in the head, 4 phasmid neurons in the tail) through their exposed ciliated endings and accumulates in their cell bodies. Defective dye-filling (Dyf) is often indicative of compromised cilia structure. As previously reported for other transition zone mutants (Williams et al., 2008, 2011), ccep-290 mutants individually did not display significant dye-fill defects. However, double mutants with either nphp-1 or nphp-4 were strongly Dyf. In contrast, no genetic interactions were detected with mksr-2. Dye-filling was restored by expression of the appropriate GFP transgene, confirming specificity of the mutant phenotypes and functionality of our GFP reporters (Fig. S1 H; Fig. S2, D and E; and not depicted). These results would appear to support a role for the transition zone in cilia assembly. However, direct visualization of phasmid cilia using the IFT marker CHE-11:GFP failed to reveal any major defects. Most strikingly, ∼83% of cilia were found to remain in ccep-290;mksr-2;nphp-4 triple mutants in which all three transition zone modules are inhibited (Fig. 2, A and B). Cilia lengths (Fig. 2 C) and IFT rates (Fig. 2 D, E) were also largely normal. Ciliary ultrastructure was also unaffected with the exception of occasional displaced doublet microtubules, likely reflecting disorganization within the transition zone (Fig. 2 F). We conclude that loss of transition zone structures has only mild effects on axoneme assembly and organization in C. elegans.


The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans.

Schouteden C, Serwas D, Palfy M, Dammermann A - J. Cell Biol. (2015)

Ciliogenesis is largely normal in transition zone mutants. (A) Phasmid cilia in wild-type and transition zone mutants visualized by the IFT marker CHE-11:GFP (arrowheads). (B and C) Quantitation of phasmid cilia number per animal (B, n > 50 animals) and length (C, n > 25 cilia) based on CHE-11:GFP. Error bars indicate the 95% confidence intervals. Asterisks indicate statistically significant difference to wild-type (t test, P < 0.01). (D) Stills and kymographs from time-lapse sequences of IFT in phasmids of wild-type and transition zone triple mutants expressing CHE-11:GFP. (E) Anterograde IFT rates in the middle segment of wild-type and transition zone mutant phasmids (n > 40 particles per strain). Error bars indicate the 95% confidence interval. (F) Transmission electron micrographs of amphid cilia at the level of the middle segment of the axoneme in wild-type, ccep-290Δ, ccep-290Δ;nphp-4, and ccep-290;mksr-2;nphp-4 mutants. Axonemes were essentially normal except for occasional displaced doublet microtubules (arrowheads). Doublet microtubule number was not significantly affected (8.7 ± 1.3 2×MTs/cilium, n = 38 wild type; 7.7 ± 1.8 2×MTs/cilium, n = 27 triple mutant; t test, NS). Bars: (A and D) 5 µm; (F) 200 nm.
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fig2: Ciliogenesis is largely normal in transition zone mutants. (A) Phasmid cilia in wild-type and transition zone mutants visualized by the IFT marker CHE-11:GFP (arrowheads). (B and C) Quantitation of phasmid cilia number per animal (B, n > 50 animals) and length (C, n > 25 cilia) based on CHE-11:GFP. Error bars indicate the 95% confidence intervals. Asterisks indicate statistically significant difference to wild-type (t test, P < 0.01). (D) Stills and kymographs from time-lapse sequences of IFT in phasmids of wild-type and transition zone triple mutants expressing CHE-11:GFP. (E) Anterograde IFT rates in the middle segment of wild-type and transition zone mutant phasmids (n > 40 particles per strain). Error bars indicate the 95% confidence interval. (F) Transmission electron micrographs of amphid cilia at the level of the middle segment of the axoneme in wild-type, ccep-290Δ, ccep-290Δ;nphp-4, and ccep-290;mksr-2;nphp-4 mutants. Axonemes were essentially normal except for occasional displaced doublet microtubules (arrowheads). Doublet microtubule number was not significantly affected (8.7 ± 1.3 2×MTs/cilium, n = 38 wild type; 7.7 ± 1.8 2×MTs/cilium, n = 27 triple mutant; t test, NS). Bars: (A and D) 5 µm; (F) 200 nm.
Mentions: We next examined the consequences of transition zone perturbations on cilia assembly. A commonly used method to assess cilia integrity in C. elegans is the dye-fill assay, which monitors uptake of the lipophilic dye DiI (Inglis et al., 2007). This dye is taken up by a subset of ciliated neurons (12 amphid neurons in the head, 4 phasmid neurons in the tail) through their exposed ciliated endings and accumulates in their cell bodies. Defective dye-filling (Dyf) is often indicative of compromised cilia structure. As previously reported for other transition zone mutants (Williams et al., 2008, 2011), ccep-290 mutants individually did not display significant dye-fill defects. However, double mutants with either nphp-1 or nphp-4 were strongly Dyf. In contrast, no genetic interactions were detected with mksr-2. Dye-filling was restored by expression of the appropriate GFP transgene, confirming specificity of the mutant phenotypes and functionality of our GFP reporters (Fig. S1 H; Fig. S2, D and E; and not depicted). These results would appear to support a role for the transition zone in cilia assembly. However, direct visualization of phasmid cilia using the IFT marker CHE-11:GFP failed to reveal any major defects. Most strikingly, ∼83% of cilia were found to remain in ccep-290;mksr-2;nphp-4 triple mutants in which all three transition zone modules are inhibited (Fig. 2, A and B). Cilia lengths (Fig. 2 C) and IFT rates (Fig. 2 D, E) were also largely normal. Ciliary ultrastructure was also unaffected with the exception of occasional displaced doublet microtubules, likely reflecting disorganization within the transition zone (Fig. 2 F). We conclude that loss of transition zone structures has only mild effects on axoneme assembly and organization in C. elegans.

Bottom Line: Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links.Co-inhibition of all three modules completely disrupted transition zone structure.Surprisingly, axoneme assembly was only mildly perturbed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), A-1030 Vienna, Austria.

Show MeSH
Related in: MedlinePlus