C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals

Abstract

The primary cilium plays critical roles in vertebrate development and physiology, but the mechanisms underlying its biogenesis remain poorly understood. We investigated the molecular function of C2 calcium-dependent domain containing 3 (C2cd3), an essential regulator of primary cilium biogenesis. We show that C2cd3 is localized to the centriolar satellites in a microtubule- and Pcm1-dependent manner; however, C2cd3 is dispensable for centriolar satellite integrity. C2cd3 is also localized to the distal ends of both mother and daughter centrioles and is required for the recruitment of five centriolar distal appendage proteins: Sclt1, Ccdc41, Cep89, Fbf1, and Cep164. Furthermore, loss of C2cd3 results in failure in the recruitment of Ttbk2 to the ciliary basal body as well as the removal of Cp110 from the ciliary basal body, two critical steps in initiating ciliogenesis. C2cd3 is also required for recruiting the intraflagellar transport proteins Ift88 and Ift52 to the mother centriole. Consistent with a role in distal appendage assembly, C2cd3 is essential for ciliary vesicle docking to the mother centriole. Our results suggest that C2cd3 regulates cilium biogenesis by promoting the assembly of centriolar distal appendages critical for docking ciliary vesicles and recruiting other essential ciliogenic proteins.

Keywords: Bbs4; Ofd1; centrosome; ciliopathy.

Fig. 1.

C2cd3 is localized to centriolar satellites. (A) In interphase, C2cd3 is present in punctae around centrosomes, whereas in mitosis, C2cd3 is localized to two punctae at each spindle pole. The lack of staining in C2cd3^GT mutant cells indicates the specificity of the C2cd3 antibody, and GFP–C2cd3 exhibits similar localization to endogenous C2cd3. (B) The centriolar satellite C2cd3 is dispersed upon nocodazole treatment, revealing its centriolar localization. DMSO-treated cells served as a control. (C) The overexpression of p50/Dynamitin disrupts the centriolar satellite localization of C2cd3. (D) C2cd3 is colocalized with Pcm1 and Bbs4. (E) Overexpressed C2cd3 coimmunoprecipitates with Pcm1 and Bbs4. (F) C2cd3 is dispersed from centriolar satellites in cells treated with a Pcm1 siRNA. The lack of Pcm1 staining indicates the effectiveness of the knockdown. Both Pcm1 and C2cd3 are localized to centriolar satellites in the control cells treated with a scramble siRNA. Centrosomes and spindle poles are labeled with γ-tubulin. The nuclei are visualized with DAPI. For quantitative analyses, SD is indicated. n = 3 independent experiments. IP, immunoprecipitation; WB, Western blot.

Fig. 2.

C2cd3 is localized to the distal end of centrioles and is required for distal appendage assembly. (A) C2cd3 is localized distal to ninein on centrioles. On the mother centriole, ninein is localized to both the proximal end and subdistal appendages (SAs), appearing as three dots on a lateral view. Ninein is also localized to the proximal end of the daughter centriole, appearing as a single dot. The illustration schematizes the localization of C2cd3 and ninein on centrioles. (B) C2cd3 is at the same distal level as Cep164 on the centriole. Upper shows a lateral view of the mother centriole in which Cep164 appears as a bar. Lower shows a top view of the mother centriole in which Cep164 appears as a ring. Note that Cep164 is only localized to the distal appendages (DAs) of mother centriole, whereas C2cd3 is localized to both centrioles. The diagram shows the localization of C2cd3 and Cep164 on the centrioles. Endogenous and overexpressed (C) Cep164, (D) GFP–Sclt1, (E) GFP–Ccdc41, (F) GFP–Cep89, and (G) GFP–Fbf1 fail to be recruited to the mother centriole in the absence of C2cd3. The centrosomes are labeled with γ-tubulin, and the nuclei are visualized with DAPI. For quantitative analyses, SD is indicated. n = 3 independent experiments.

Fig. 3.

Ttbk2 recruitment and Cp110 removal are compromised in the absence of C2cd3. (A) GFP–Ttbk2 is localized to the centriole in wild-type cells but not in C2cd3^GT mutant cells. (B) Cp110 is removed from the mother centriole in wild-type cells but not in C2cd3^GT mutant cells. Note that Cp110 staining is dynamic through the cell cycle. Before centrosome duplication, Cp110 was observed as one dot in wild-type cells, representing the daughter centriole (“1”). In contrast, two dots were observed in C2cd3^GT mutant cells, representing both the mother and daughter centrioles (“2”). After centrosome duplication, only one centrosome is positive for Cp110 (“1+0”) in wild-type cells, but both centrosomes are positive for Cp110 (“1+1”) in C2cd3^GT mutant cells. In the G2 phase, the procentrioles are capped with Cp110 such that Cp110 appears as two dots in the centrosome containing the daughter centriole, and one dot representing the procentriole in the centrosome containing the mother centriole (“2+1”) in wild-type cells. In contrast, C2cd3 mutant cells exhibit two Cp110 dots in each centrosome in the G2 phase (“2+2”). The quantitative analysis includes interphase cells. The centrosomes are labeled with γ-tubulin, and the nuclei are visualized with DAPI. For quantitative analyses, SD is indicated. n = 3 independent experiments.

Fig. 4.

C2cd3 is essential for the recruitment of Ift88 and Ift52 to the mother centriole. (A) Ift88 is localized to the basal body and along the cilium in ciliated wild-type cells and to the mother centriole in nonciliated wild-type cells. The centriolar staining of Ift88 is absent in C2cd3^GT mutant cells. (B) Western blot analysis reveals comparable levels of Ift88 protein in C2cd3^GT mutant and wild-type cells. (C) Coimmunoprecipitation analysis reveals the physical interaction between overexpressed C2cd3 and Ift88. (D) GFP–Ift52 is localized to the basal body and along the cilium in ciliated wild-type cells and to the mother centriole in nonciliated wild-type cells. The centriolar staining of GFP–Ift52 is absent in C2cd3^GT mutant cells. The centrosomes are labeled with γ-tubulin, and the nuclei are visualized with DAPI. The quantitative analyses of Ift88 and Ift52 centriolar recruitment only include nonciliated cells. For quantitative analyses, SD is indicated. n = 3 independent experiments.

Fig. 5.

C2cd3 is important for ciliary vesicle docking during ciliogenesis. (A) GFP–Smo is present in the cilia and ciliary vesicles docked to the mother centriole in wild-type cells, but is not present at the mother centriole in C2cd3^GT mutant cells, suggesting a defect in ciliary vesicle docking. The quantitative analysis only includes nonciliated cells. (B) TEMs of centrioles in E10.5 wild-type and C2cd3^GT mutant mouse embryos. Longitudinal sections (Left) and cross-sections (Right) are presented. Arrowheads indicate subdistal appendages and asterisks indicate docked ciliary vesicles. (C) A model based on our data and published results (9, 10) suggests that C2cd3 is critical for the assembly of centriolar distal appendages, which in turn underlie the recruitment of Ttbk2, Ift88, and Ift52 as well as the removal of Cp110 and the docking of ciliary vesicles. C2cd3 may play a more direct role in recruiting Ift88 to the distal appendages. For quantitative analyses, SD is indicated. n = 3 independent experiments.