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

Abstract

Cilia are cellular projections that perform sensory and motile functions. A key ciliary subdomain is the transition zone, which lies between basal body and axoneme. Previous work in Caenorhabditis elegans identified two ciliopathy-associated protein complexes or modules that direct assembly of transition zone Y-links. Here, we identify C. elegans CEP290 as a component of a third module required to form an inner scaffolding structure called the central cylinder. Co-inhibition of all three modules completely disrupted transition zone structure. Surprisingly, axoneme assembly was only mildly perturbed. However, dendrite extension by retrograde migration was strongly impaired, revealing an unexpected role for the transition zone in cell adhesion.

Figure 1.

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.

Figure 2.

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.

Figure 3.

A role for the transition zone in dendrite attachment. (A) Schematic of dendrite formation by retrograde extension in C. elegans amphids. (B) Stills from time-lapse movie of embryo expressing myristoylated GFP in amphid neurons undergoing retrograde migration. Overlay of GFP signal is shown on transmitted light images of the embryo. Insets show GFP only. Note that the position of dendritic tips (arrowhead) appears fixed as cell bodies (asterisk) move. See also Video 3. (C) Quantitation of phasmid dendrite lengths in wild-type and transition zone mutants at the L4 stage, using CHE-11:GFP. Each dot on the scatter plot represents a single dendrite, with bars indicating average and 95% confidence intervals. n > 20 dendrites per condition. Asterisks indicate statistically significant differences to wild-type (t test; ***, P < 0.001). (D) Tail of a ccep-290;nphp-1 mutant animal expressing CHE-11:GFP. While dendrite extension has failed in both phasmids on the left hand side, right-hand-side phasmids have formed normally. Note that cell bodies are positioned similarly on both sides (broken line). (E) Immunofluorescence micrographs of amphids and phasmids of late embryos expressing myristoylated GFP and LAP:CCEP-290 stained for CCEP-290 with antibody to S-tag. CCEP-290 is located at the tip of the elongating dendrite (arrowheads). (F) Phasmid dendrite lengths in combinations of dex-1/dyf-7 and nphp-4 mutants, quantified as in C. Wild-type data are shown for comparison. ns42 and ns117 are hypomorphic alleles of dex-1 and dyf-7, dyf-7(m537) closer to a loss of function (Heiman and Shaham, 2009). Asterisks indicate statistically significant differences to dex-1/dyf-7 single mutants (t test; ***, P < 0.001; **, P < 0.01). (G) Still images of wild-type and ccep-290;nphp-4 mutant embryos expressing DYF-7:GFP. Overlay of GFP signal on transmitted light images of the embryo. Note the enrichment of DYF-7:GFP at the tips of elongating dendrites (arrowheads). Bars: (D) 5 µm; (B, E, and G) 10 µm. Insets in E are magnified 2×.

Figure 4.

Molecular architecture of the transition zone and function in cell adhesion. (A) 3D reconstruction model of the transition zone in C. elegans, highlighting key features. See also Video 2. Based on protein localization and mutant phenotypes, CCEP-290 is an essential component of the central cylinder (this study), whereas MKS and NPHP module components function in assembly of Y-links (Williams et al., 2011). Transition zone mutants display no apparent defects in axoneme assembly. However, ciliary gating and cell adhesion are compromised. (B) The dendrite of C. elegans amphid and phasmid neurons forms by retrograde extension, with the cell body moving backward while the dendritic tip remains in place. The transition zone mediates tip anchorage via interactions with the extracellular matrix. (C) Positioning of the primary cilium determines cell fate in vertebrate neuroepithelium. Cells with apically positioned cilia maintain their position at the apical adherens junction belt, whereas cells with basolateral cilia delaminate (Wilsch-Bräuninger et al., 2012; Paridaen et al., 2013). Differential signaling, but also mechanical anchorage by the cilium, could explain this result.