Silva E et al. (2016), Ccdc11 is a novel centriolar satellite protein ...

XB-ART-51522

Mol Biol Cell 2016 Jan 01;271:48-63. doi: 10.1091/mbc.E15-07-0474.

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Ccdc11 is a novel centriolar satellite protein essential for ciliogenesis and establishment of left-right asymmetry.

Silva E , Betleja E , John E , Spear P , Moresco JJ , Zhang S , Yates JR , Mitchell BJ , Mahjoub MR .

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The establishment of left-right (L-R) asymmetry in vertebrates is dependent on the sensory and motile functions of cilia during embryogenesis. Mutations in CCDC11 disrupt L-R asymmetry and cause congenital heart disease in humans, yet the molecular and cellular functions of the protein remain unknown. Here we demonstrate that Ccdc11 is a novel component of centriolar satellites-cytoplasmic granules that serve as recruitment sites for proteins destined for the centrosome and cilium. Ccdc11 interacts with core components of satellites, and its loss disrupts the subcellular organization of satellite proteins and perturbs primary cilium assembly. Ccdc11 colocalizes with satellite proteins in human multiciliated tracheal epithelia, and its loss inhibits motile ciliogenesis. Similarly, depletion of CCDC11 in Xenopus embryos causes defective assembly and motility of cilia in multiciliated epidermal cells. To determine the role of CCDC11 during vertebrate development, we generated mutant alleles in zebrafish. Loss of CCDC11 leads to defective ciliogenesis in the pronephros and within the Kupffer's vesicle and results in aberrant L-R axis determination. Our results highlight a critical role for Ccdc11 in the assembly and function of motile cilia and implicate centriolar satellite-associated proteins as a new class of proteins in the pathology of L-R patterning and congenital heart disease.

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Species referenced: Xenopus
Genes referenced: cfap53 tgfbi
GO keywords: cilium assembly
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Phenotypes: Xla Wt + cfap53 MO (Fig. 4 B) [+]

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	FIGURE 1:. Ccdc11 is a component of centriolar satellites. (A) Immunoblot of lysates from tetracycline-inducible GFP-Ccdc11–expressing RPE-1 stable cell line (RPE::GFP-Ccdc11) probed with anti-Ccdc11 antibody. Numbers on the left indicate molecular mass of markers in kilodaltons. (B) Localization of endogenous Ccdc11 throughout the cell cycle. Asynchronously growing MEF cells were fixed and stained for Ccdc11 (green), centrin to identify centrioles (red), acetylated tubulin to mark cilia (red), and DNA (4′,6-diamidino-2-phenylindole [DAPI]; blue). The centriolar satellite distribution of Ccdc11 is conserved through G1, S phase, G2, and mitosis. After serum starvation to induce ciliogenesis, the satellite pool of Ccdc11 is lost, and the protein is enriched at the transition zone (distal end of each centriole, arrowheads). Insets, magnified images of the centrosome region. Scale bar, 5 μm. (C) RPE::GFP-Ccdc11 cells immunostained with antibodies against GFP (green), PCM-1 (red), or Cep290 (red), as well as centrin (magenta) or acetylated tubulin (light blue) to mark centrioles and cilia, respectively. Ccdc11 colocalizes with PCM-1 and Cep290 at satellites in G1 and with Cep290 at the transition zone in G0. Scale bar, 5 μm. (D, E) RPE-1 cells were treated with nocodazole for 1 h to depolymerize microtubules and then allowed to recover for up to 30 min. Samples were fixed at the indicated times and stained for Ccdc11 (green), centrioles (centrin, magenta), microtubules (α-tubulin, red), and DNA (DAPI, blue). Ccdc11 localization to centriolar satellites is microtubule dependent, but the centriolar pool of Ccdc11 does not require microtubules. Scale bar, 10 μm. Arrowhead indicates the satellite pool of Ccdc11. We scored 300 cells for each sample, from three independent experiments; **p < 0.05. (F) Ccdc11 interacts with satellite proteins PCM-1 and Cep290. Immunoprecipitation was performed on extracts from RPE::GFP-Ccdc11 cells using anti-GFP antibody or control IgG and then probed for GFP and endogenous PCM-1, Cep290, and γ-tubulin (negative control).
	FIGURE 2:. Depletion of Ccdc11 causes dispersal of centriolar satellites and disrupts primary cilium assembly. (A) RPE-1 cells transfected with either nontargeting control siRNA or siRNA against Ccdc11. Cells were fixed and immunostained with antibodies against Ccdc11, PCM-1, or Cep290 (green). Cells were also stained for centrioles (centrin, red) and nuclei (DAPI, blue). Knockdown of Ccdc11 results in loss of satellites surrounding the centrioles. Insets, magnified images of the centrosome region. Scale bar, 10 μm. (B) Immunoblot analysis of extracts from RPE-1 cells transfected with control or Ccdc11 siRNA oligonucleotides and probed with antibodies targeting Ccdc11, PCM-1, Cep290, and actin (as a loading control). Depletion of Ccdc11 does not affect the expression levels of endogenous PCM-1 or Cep290. (C, D) siRNA-mediated depletion of Ccdc11 causes a significant reduction in the percentage of cells with organized satellites. Total fluorescence intensities (D) for PCM-1 and Cep290 were determined within a 2-μm circle centered on the centrosome in cells treated with the indicated siRNAs. The histogram presents the mean total intensities relative to that in the control siRNA-treated samples. We scored 450 cells for each sample from three independent experiments; p < 0.05. (E, F) Control and Ccdc11-depleted RPE-1 cells were serum starved, fixed, and immunostained for Ccdc11 (green) and acetylated tubulin (red) to label primary cilia. Depletion of Ccdc11 significantly reduced the percentage of cells with cilia, which is rescued by cotransfection with siRNA-resistant, full-length GFP-Ccdc11 but not with the truncated protein GFP-Ccdc11(PM). We scored 300 cells for each sample from three independent experiments; p < 0.05. Scale bar, 10 μm. (G) Schematic of Ccdc11 deletion constructs and summary of their localizations to centrioles and/or satellites. Colocalization was determined by analyzing RPE-1 cells transfected with Myc-tagged Ccdc11 deletion constructs and immunostaining of fixed cells with anti-Myc and antibodies targeting centrin, PCM-1, and Cep290. CC, coiled-coil domains. (H) Ccdc11-depleted RPE-1 cells were transfected with either Myc-Ccdc11(FL) or Myc-Ccdc11(PM), fixed, and immunostained with antibodies against Myc, centrin, PCM-1, and nuclei (DAPI). Scale bar, 10 μm. We scored 300 cells for each sample from three independent experiments; **p < 0.05. (I) Immunoprecipitation using anti-Myc antibody was performed on extracts of RPE-1 cells expressing either Myc-Ccdc11(FL) or Myc-Ccdc11(PM). Immunoprecipitates were probed for Myc and endogenous PCM-1.
	FIGURE 3:. Ccdc11 is required for motile cilia formation in human multiciliated epithelial cells. (A) Schematic of the differentiation steps of cultured multiciliated respiratory epithelia. The key phases of the culture are depicted in cartoon form and include the progenitor phase (pre-ALI), centriologenesis (ALI day 7), initiation of ciliogenesis (ALI day 14), and mature multiciliated cells (ALI day 21). (B) Expression levels of endogenous Ccdc11 in hTECs. Cells were grown on Transwell permeable filter supports, and ALI was established 2 d after cells reached confluence. Cells were harvested on the indicated days, and lysates were analyzed by immunoblotting with antibodies against Ccdc11 and actin. Graph shows quantification of Ccdc11 levels normalized to actin for each stage. (C, D) Localization of endogenous Ccdc11 in hTEC at different stages of differentiation, viewed en face (C) or longitudinally (D). Progenitor cells were grown until confluent, and differentiation was induced by creating ALI. Samples were fixed on the indicated days and stained with antibodies against Ccdc11, centrin, acetylated tubulin, and ZO1 (to mark cell boundaries). Scale bar, 5 μm. (E) Colocalization of GFP-Ccdc11 and PCM-1 in hTEC. Progenitor cells were infected with lentivirus expressing GFP-Ccdc11 and grown until confluent, and differentiation was induced by creating ALI. Samples were fixed on the indicated days and stained with antibodies against GFP, centrin, and PCM-1. Scale bar, 5 μm. (F–H) Quantification of ciliogenesis defects upon loss of Ccdc11 in hTEC. shRNA-mediated depletion of Ccdc11 results in significant reduction of primary cilium assembly in pre-ALI progenitor cells (F), reduced number of multiciliated cells at ALI day 21 (G), and decreased number of cells expressing FoxJ1 at ALI day 21 (H). We scored 300 cells for each sample from three independent experiments; **p < 0.05.
	FIGURE 4:. Ccdc11 is essential for motile cilia assembly and function in Xenopus. (A) Localization of RFP-Ccdc11 (red) in a multiciliated cell from Xenopus embryo shows accumulation near centrioles at the apical surface, marked with centrin-GFP (green). Arrow indicates the majority of Ccdc11 signal being present in a layer just beneath the centrioles. Scale bar, 5 μm. (B) Measurement of fluid flow across the surface of Xenopus embryos injected with a control MO (n = 12 embryos), CCDC11 MO (n = 10), or CCDC11 MO + CCDC11 mRNA (n = 9). Rates of fluid flow were recorded by visualizing the movement of fluorescent microbeads over the surface of the embryos (see Supplemental Videos S1 and S2). p < 0.05. (C) Multiciliated cells from stage 28 embryos injected with control or CCDC11 MO. Cilia were stained with antibodies against acetylated tubulin (green) and actin stained with phalloidin to mark cell boundaries (purple). The abundance and morphology of cilia appear abnormal in CCDC11 morphant embryos. Scale bar, 5μm. (D) Quantification of ciliary regrowth after deciliation. Cilia were stained using antibodies against acetylated tubulin, and length was measured in untreated cells and in cells at 1, 2, and 4 h after chemical deciliation (28 cells taken from five embryos). p < 0.05. (E, F) Quantification of cilia orientation and polarity in multiciliated cells. Embryos were injected with either control or CCDC11 MO, and orientation of basal bodies was determined using immunofluorescence staining of centrin-RFP and GFP-CLAMP (see Materials and Methods). White arrows in E show examples of the axis of basal body-rootlet orientation. Control MO, 10 cells from three embryos represented by color; CCDC11 MO, 16 cells from three embryos. CSD, circular SD.
	FIGURE 5:. Loss-of-function of CCDC11 causes defective ciliogenesis and L-R asymmetry in zebrafish. (A) CCDC11 mutant embryos display pericardial edema at 2 dpf. We scored 117 (wild-type) and 163 (mutant) embryos. p < 0.05. (B) Whole-mount in situ hybridization showing the expression of laterality markers in wild-type and CCDC11 mutants. Cmlc2 expression shows the heart tube looping to the left in control embryos at 48 hpf, whereas looping was reversed or absent in CCDC11 mutant embryos (A, atrium; V, ventricle). Spaw is expressed in the left lateral plate mesoderm in control embryos at the 18-somite stage (white arrow), whereas its expression is bilateral (black and white arrows), reversed (black arrow), or absent in a significant number of CCDC11 mutants. (C) Quantification of laterality defects in CCDC11 mutant embryos. For Cmlc2 analysis, 89 embryos (WT) and 167 embryos (CCDC11 mutants) were scored. For Spaw analysis, 154 embryos (WT) and 231 embryos (CCDC11 mutants) were scored. p < 0.05. (D) Immunofluorescence images of cilia in the KV of wild-type and CCDC11 mutant embryos. Embryos were fixed at the 8- 10-somite stages and stained with anti–acetylated α-tubulin. Scale bar, 10 μm. (E) Quantification of cilia abundance and length in KV. There was a significant reduction in cilia number and length in CCDC11 mutants compared with control embryos. Fifteen embryos from each genotype. p < 0.05. (F) Immunofluorescence staining of cilia in the posterior segment of the pronephros from wild-type and CCDC11 mutant embryos at 24 hpf stained with anti–acetylated tubulin. Scale bar, 10 μm. (G) Quantification of cilia length in the pronephros. Fifteen embryos from each genotype. p < 0.05.
	Supplementary Figure 5. (A) Validation of the Ccdc11 translation (ATG)-blocking morpholino in Xenopus embryos. 100 pg of GFP-CCDC11 or GFP-Centrin mRNA were injected into 2-cell stage embryos, together with 25 ng of either control or CCDC11 morpholino. Lysates from embryos was prepared at the 15-cell stage and analyzed by immunoblotting with anti-GFP antibody. (B) Overview of CCDC11 knockout strategy in zebrafish, depicting the position of TALEN binding regions relative to the BsmA1 restriction site within exon 2. The location of sequencing primers used for genotyping is also indicated. (C) Genotyping analysis of zebrafish mutants by PCR and restriction enzyme analysis. Each lane represents a BsmA1-digested amplicon encompassing CCDC11 TALEN target sequence from genomic DNA. The uncut amplicon is 576 bp in size, while BsmA1 digestion yields fragments of 388 bp and 188 bp. (D) Sequencing of independent strains of TALEN-mutagenized zebrafish, indicating the deleted base pairs and the position of the resulting stop codons. (E) RT-PCR of cDNA (transcribed from mRNA) from wild-type and CCDC11 mutant embryos. Primers targeting exons 3-4 and exons 4- 5 of CCDC11 were used. Primers targeting the β-actin gene were employed as loading control.

References [+] :

Anderson, The formation of basal bodies (centrioles) in the Rhesus monkey oviduct. 1971, Pubmed