The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans.
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.
Affiliation: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), A-1030 Vienna, Austria.
- Caenorhabditis elegans/cytology/metabolism*
- Caenorhabditis elegans Proteins/physiology*
- Cell Adhesion*
- Microtubule-Associated Proteins/physiology*
- Microscopy, Video
- Protein Multimerization
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fig1: CCEP-290 belongs to a third transition zone module required for assembly of the central cylinder. (A) Transition zone modules in C. elegans, based on genetic interactions and localization interdependencies (Huang et al., 2011; Williams et al., 2011; this study). (B) Modules in vertebrates, based on proteomic studies (green, red, blue: Sang et al., 2011; dark green: Chih et al., 2012; light green: Garcia-Gonzalo et al., 2011). *, C. elegans names used for ease of comparison. HGNC names: B9D1/MKSR1, B9D2/MKSR2, TMEM216/MKS2, TMEM67/MKS3, RPGRIP1L/MKS5, CC2D2A/MKS6. (C) Immunofluorescence micrographs of phasmid (tail) cilia of worms expressing GFP:CCEP-290 and stained for HYLS-1 and glutamylated tubulin. (D and E) Localization interdependencies between CCEP-290 and MKS/NPHP module components MKS-5, NPHP-4, and MKSR-2. Panels show phasmid cilia in worms coexpressing mCherry:HYLS1 and GFP:CCEP-290/MKSR-2/NPHP-4 in wild-type and transition zone mutants, as indicated. (F) MKSR-2 at transition zones is reduced due to dispersal along the ciliary axoneme. A still image and kymograph from a time-lapse sequence of GFP:MKSR-2 in ccep-290Δ mutant phasmids is shown (see also Video 1). The line indicates the kymograph axis. (G) Transmission electron micrographs of amphid transition zones in wild-type, ccep-290Δ, ccep-290Δ;nphp-4, and ccep-290;mksr-2;nphp-4 mutants. While a central cylinder is not apparent and transition zones are fragmented, Y-links (arrowheads) are still occasionally present in ccep-290Δ mutants. Inner singlet microtubule numbers are reduced (3.8 ± 1.5, n = 25 wild-type; 1.6 ± 1.4, n = 21 ccep-290Δ; t test; P < 0.0001), potentially due to loss of the central cylinder to which they normally attach. Transition zone structures are completely lost in ccep-290;mksr-2;nphp-4 triple mutants. Bars: (C, D, and E) 1 µm; (F) 5 µm; (G) 200 nm.
Increasing evidence points to the transition zone being organized into multiple protein complexes or modules with distinct functions. Best characterized are the MKS and NPHP modules, which are composed of proteins mutated in the ciliopathies Meckel syndrome and nephronophthisis. Previous work in C. elegans showed these to be recruited independently of each other and function redundantly in assembly of transition zone Y-links (Fig. 1 A; Williams et al., 2011). Proteomic analysis in vertebrates identified a potential third module including the key ciliopathy protein CEP290 (Fig. 1 B; Sang et al., 2011). Reciprocal BLAST searches identified a C. elegans homologue encoded by the predicted gene Y47G6A.17, which we named CCEP-290 for C. elegans CEP290 (Fig. S1, A and B). Phylogenetic analysis found CEP290 to be conserved across all major eukaryotic phyla, supporting a central role for this protein at the transition zone (Fig. S1 C; Hodges et al., 2010).