Abstract
Dense multicilia in higher vertebrates are important for luminal flow and the removal of thick mucus. To generate hundreds of basal bodies for multiciliogenesis, specialized terminally differentiated epithelial cells undergo massive centriole amplification. In proliferating cells, however, centriole duplication occurs only once per cell cycle. How cells ensure proper regulation of centriole biogenesis in different contexts is poorly understood. We report that the centriole amplification is controlled by two duplicated genes, Cep63 and Deup1. Cep63 regulates mother-centriole-dependent centriole duplication. Deup1 governs deuterosome assembly to mediate large-scale de novo centriole biogenesis. Similarly to Cep63, Deup1 binds to Cep152 and then recruits Plk4 to activate centriole biogenesis. Phylogenetic analyses suggest that Deup1 diverged from Cep63 in a certain ancestor of lobe-finned fishes during vertebrate evolution and was subsequently adopted by tetrapods. Thus, the Cep63 gene duplication has enabled mother-centriole-independent assembly of the centriole duplication machinery to satisfy different requirements for centriole number.
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Acknowledgements
The authors thank Y. Zheng, Y. Shen and J. Ten for helpful suggestions; E. Nigg for Cep152 antibody; S. Li and Z. Li for technical support on 3D-SIM and H. Dai, T. Zhang, F. Qin and X. Zhu for technical assistance. This work was supported by the Chinese Academy of Sciences (XDA01010107), the National Basic Research Program of China (2012CB945003 and 2010CB912102), the National Science Foundation of China (31330045, 31271427 and 91129000) and Shanghai Municipal Science and Technology Commission (12JC1409900). X.Y. acknowledges the support of the SA-SIBS Scholarship Program.
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X.Y. and X.Z. conceived and directed the project. H.Z. carried out most experimental work. L.Z. carried out the frog and transmission EM experiments. Y.Z did the experiments in bacteria and drew all illustrative models. J.C. established the culture and infection conditions for MTECs. S.L. found that CEP152 RNAi downregulated CEP63. Q.H. generated the homemade antibodies. T.X. and X.H. provided the 3D-SIM and frog systems. X.Z., X.Y. and H.Z. designed experiments, interpreted data, and wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Sequence alignment of Deup1 and Cep63.
The protein sequence alignment was generated by the Clustal X 2.0 multiple sequence alignment program using default parameters. The protein sequences were from GenBank accessions numbers NM_181645 (human DEUP1), KC211186 (mouse Deup1), NM_025180 (human CEP63), and KC211185 (mouse Cep63). The histogram depicts conservation between the two proteins. Related to Fig. 1.
Supplementary Figure 2 The aggregates formed by GFP–Cep63 or GFP–Cep152 do not support de novo procentriole formation.
(a) Expression of GFP-tagged Deup1, Cep63 or Cep152 in U2OS cells. (b,c) Analyses for procentriole formation. The arrowheads and arrows point to typical mother centrioles and GFP-positive aggregates, respectively. The bright SAS-6 foci, which mark the cartwheels, are used as procentriole markers in the quantification analyses. Only interphase cells containing MCD procentrioles, thus in the S or G2 phase, were scored (n = 30 cells from triplicates). GFP–Deup1-expressing cells served as the positive control for de dovo procentriole biogenesis. (d) Endogenous CEP152 exhibits punctate distributions in the GFP–Cep63 aggregates. Such a pattern is in sharp contrast to the ring-shaped distributions of CEP152 in the deuterosomes (arrow in the GFP–Deup1-positive cell) and MCD cradles (arrowheads) (also see Fig. 1h), suggesting that the GFP–Cep63 aggregates are disorganised. Apparently, the punctate CEP152 is unable to induce the cartwheel assembly, not to mention de novo centriole formation. Related to Fig. 1.
Supplementary Figure 3 Immunoblotting results for domain mapping.
(a) Mapping of the Deup1- and CEP63-interactiing domains of Cep152. The numbers in the diagrams indicate amino acid positions. Exogenous Cep152, its mutants, or firefly luciferase (Luc) was expressed alone or together with Flag-Deup1 in HEK293T cells, as indicated. Coimmunoprecipitation was then performed using anti-Flag resin. Luciferase served as the negative control. The asterisks indicate the positions of full-length fusion proteins. (b) Mapping of the CEP152-interacting domain of Deup1. Flag-tagged Deup1 or mutants were expressed in HEK293T cells. Coimmunoprecipitation was then performed. (c) Mapping of the CEP152-interacting domain of Cep63. Flag-tagged Cep63 or mutants were expressed in HEK293T cells. Coimmunoprecipitation was then performed. These results are summarised in Fig. 1j.
Supplementary Figure 4 Effect of Deup1 or Cep63 RNAi in MTECs at ALI d 3.
(a) Cep152 failed to exhibit the ring-shaped deuterosome localization upon Deup1 RNAi. The arrows indicate MCD procentriole. The insets are magnified 2× to show typical de novo procentrioles (arrowheads). (b-d) Ablation of Cep63 alone in MTECs does not block deuterosome formation and centriole amplification. MTECs were infected with lentivirus to express control (Ctrl-i) or Cep63-specific (63-i1 or 63-i2) shRNA together with GFP–Cetn1. The GFP-positive cells at ALI d 3 were sorted out using FACS and subjected to immunoblotting to show RNAi efficiency. α-Tubulin served as the loading control. The insets are magnified to show MCD procentrioles. The statistical results for centriole numbers are presented in Fig. 6d.
Supplementary Figure 5 Validation of the specificity of Dp-MO1 and Dp-MO2 using a GFP reporter.
(a) Diagrams ofin vitro-transcribed mRNAs. The red bars indicate the locations of the MO-targeting sites. (b) Dp-MO1 and Dp-MO2 specifically blocked the translation of the xDpATG-GFP mRNA. Xenopus embryos at the two- or four-cell stage were microinjected with a mixture of 10 ng MO, 100 pg GFP or xDpATG-GFP mRNA, and 50 pg RFP-F mRNA (as an injection marker). The autofluorescence of GFP or RFP was visualised at approximately the developmental stage 26. Related to Fig. 8d.
Supplementary Figure 6 Full scans of original blots.
The boxed regions indicate the areas shown in the figures.
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3D model of a part of a cell in stage II immunostained for Deup1 (green), Cep152 (blue), and Centrin (red).
The top and side views are represented in Fig. 4c. (MOV 5587 kb)
3D model of a part of a cell in stage III immunostained for Deup1 (green), Cep152 (blue), and Centrin (red).
The top and side views are represented in Fig. 4c. (MOV 8021 kb)
3D model of a part of a cell in stage IV immunostained for Deup1 (green), Cep152 (blue), and Centrin (red).
The top and side views are represented in Fig. 4c. (MOV 9609 kb)
3D model of a part of a cell in stage V immunostained for Deup1 (green), Cep152 (blue), and Centrin (red).
The top and side views are represented in Fig. 4c. (MOV 9753 kb)
3D model of a part of a cell in stage VI immunostained for Deup1 (green), Cep152 (blue), and Centrin (red).
The top and side views are represented in Fig. 4c. (MOV 7096 kb)
EM images from four consecutive sections of a control (Ctrl-i) and a Deup1-knockdown (Dp-i1) MTEC at ALI d3.
The thickness of each section is 70 nm. Deuterosomes (d), mother centrioles (m), and daughter procentrioles around mother (p) are numbered according to the sequence of appearance. The overlaid images are shown in Fig. 7a. (MOV 1085 kb)
3D model for deuterosome-like structures formed by His-Deup1 (green) in E. coli.
The protein expression was induced with 10 μM IPTG. See Fig. 7d for the original 3D-SIM image. (MOV 4525 kb)
3D model for deuterosome-like structures formed by His-Deup1 (green) in E. coli.
The protein expression was induced with 50 μM IPTG. See Fig. 7d for the original 3D-SIM image. (MOV 5332 kb)
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Zhao, H., Zhu, L., Zhu, Y. et al. The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis. Nat Cell Biol 15, 1434–1444 (2013). https://doi.org/10.1038/ncb2880
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DOI: https://doi.org/10.1038/ncb2880
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