Massive centriole production can occur in the absence of deuterosomes in multiciliated cells

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Abstract

Multiciliated cells (MCCs) amplify large numbers of centrioles that convert into basal bodies, which are required for producing multiple motile cilia. Most centrioles amplified by MCCs grow on the surface of organelles called deuterosomes, whereas a smaller number grow through the centriolar pathway in association with the two parent centrioles. Here, we show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. Therefore, deuterosomes may have evolved to relieve, rather than supplement, the centriolar pathway during multiciliogenesis. Remarkably, MCCs lacking parent centrioles and deuterosomes also amplify the appropriate number of centrioles inside a cloud of pericentriolar and fibrogranular material. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.

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Fig. 1: Deup1-knockout mice lack deuterosomes.
Fig. 2: Deuterosomes are dispensable for centriole amplification during multiciliogenesis.
Fig. 3: Live imaging of differentiating Deup1/ ependymal cells reveals normal step-wise kinetics of centriole amplification.
Fig. 4: Centriole amplification in the absence of deuterosomes occurs on and in proximity to pre-existing centrioles.
Fig. 5: The loss of Deup1 does not compromise basal-body symmetry or cilia function.
Fig. 6: Centriole amplification can occur without deuterosomes and parent centrioles.

Data availability

Source data for Figs. 1a, 2a,c, 3a, 5a,c,e–g, 6b,d and Extended Data Figs. 1b,c, 2a,c,d, 3a–c,e,f, 5a–d,f,h, 6a,c, 7b–d have been provided online. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank all of the members of the Spassky laboratory for their comments and discussions, M. Legendre for identifying the DEUP1 mutation on the gnomAD control database, K. Oegema and A.K. Shiau (Ludwig Institute for Cancer Research) for sharing centrinone, A.-K. Konate and R. Nagalingum for administrative support and the IBENS Animal Facility for animal care. We thank the IBENS Imaging Facility, with grants from Région Ile-de-France (NERF 2011-45), Fondation pour la Recherche Médicale (FRM; DGE 20111123023), and Fédération pour la Recherche sur le Cerveau–Rotary International France (2011). We thank Y. Wang of the Nathans laboratory for technical assistance. The authors would like to thank the Genome Aggregation Database (gnomAD) and the groups that provided the exome and genome variant data to this resource. We are grateful to J. Nathans, A. Rosen and the Johns Hopkins Institute for Basic Biomedical Sciences for providing research support to A.J.H. This work was supported by grants from the National Institutes of Health (R01GM114119 and R01GM133897) and an American Cancer Society Scholar (RSG-16-156-01-CCG) to A.J.H., National Institutes of Health (R01GM089970) to B.M., Agence Nationale de la Recherche (ANR) Investissements d’Avenir (ANR-10-LABX-54 MEMO LIFE and ANR-11-IDEX-0001-02 PSL Research University) to IBENS. The Spassky laboratory is supported by INSERM, CNRS, EcoleNormale Supérieure (ENS), FRM (EquipeFRM grant 20140329547), a European Research Council (ERC Consolidator grant 647466) to N.S. and ANR (ANRJC JC-15- CE13-0005-01) to A.M.

Author information

O.M. and M.S.L. designed, performed and analysed the majority of the experiments, with O.M. performing and analysing the live imaging, super-resolution microscopy and CLEM, and M.S.L. generating and validating the Deup1/ and Deup1/; Cep63T/T mice, quantifying the basal body number in all of the genotypes and performing the siRNA experiments. G.M.L quantified the cilia beat frequency and cilia length. V.G. and A.K. assisted with the image quantification and P.R. performed the electron microscopy sample preparation. E.B. and B.J.M. performed and analysed the Xenopus multiciliated cell data. N.S., A.M. and A.J.H. analysed the data. A.J.H and A.M. conceived and co-supervised the study. A.J.H., A.M., O.M. and M.S.L. co-wrote the manuscript.

Correspondence to Alice Meunier or Andrew J. Holland.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Deup1/ mice lack Deup1 mRNA and detectable protein.

a, A Deup1 knockout was generated by replacing a region from within exon 2 to within exon 7 of the Deup1 gene with a LacZ reporter followed by a polyA sequence. (top) Schematic representation of the Deup1 gene and (bottom) mRNA structure in Deup1+/+ and Deup1/ mice. b, RT-qPCR analysis of Deup1 mRNA levels in postnatal day 5 brain tissue. Deleted (Del.) exons denote primers designed to amplify exons 2–3 that are deleted in Deup1/ mice. The average of Deup1+/+ samples was normalized to 1. n = 3 mice/genotype. Bars represent mean ± s.d. c, RT-qPCR analysis of testes in 2–5-month-old mice using 3 different primer sets. The average of Deup1+/+ samples was normalized to 1. n ≥ 3 mice/genotype. Bars represent mean ± s.d. d, Immunofluorescence images of mTECs at air–liquid interface (ALI) day 4. Scale bars represent 10 μm. e, Western blot of lysates from mTECs at ALI day 3 and 5, and two different ependymal cell cultures differentiated for 8 days. Membranes were probed with antibodies against DEUP1 and α-tubulin was used as a loading control. Full blot shown in Source Data. f, Immunoblot of DLD-1 cells expressing doxycycline (dox) inducible mmDEUP1-Myc transgene. Membranes were probed with antibodies against DEUP1 or Myc. Arrow denotes the mmDEUP1-Myc protein and the asterisk shows endogenous Myc. Full blot shown in Source Data. g, Immunoblot of HEK293 cells expressing either full-length (F.L.) or exons 8–12 (Ex 8–12) of mmDEUP1 in the presence or absence of the proteasome inhibitor, MG132. MG132 was used to enable the detection of unstable protein fragments. Membranes were probed with antibodies against DEUP1 or Myc. α-tubulin was used as a loading control. Arrow denotes the full-length mmDEUP1-Myc protein, arrowhead denotes the exon 8–12 mmDEUP1-Myc protein and the asterisk shows endogenous Myc. Full blot shown in Source Data. Source data Source data

Extended Data Fig. 2 DEUP1 is not required for centriole amplification.

a, Quantification of CEP164 foci which marks the mature basal bodies in control or Deup1/ ependymal cells. The total number of cells analysed per genotype is indicated. n = 3 mice/genotype. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). Bars represent mean ± s.d. b, Representative images of mature centrioles in Deup1+/+ or Deup1/ ependymal cells stained with an antibody against CEP164. Scale bars represent 10 μm. c, Quantification of Deup1–GFP intensity in control or Deup1 morpholino-treated Xenopus epithelial cells. Note, the Deup1 morpholino oligonucleotide efficiently silenced expression of an mRNA encoding a morpholino-targetable fragment of Deup1 fused to GFP in Xenopus MCCs. n ≥ 5 embryos per genotype. The total number of cells analysed per condition is indicated. P values, unpaired, two-tailed, Welch’s t-test. * = p ≤ 0.05. Bars represent mean ± s.d. d, Quantification of centriole number in control or Deup1 morpholino-treated Xenopus epithelial cells. Points represent the average number of centrioles per embryo. n ≥ 5 embryos per genotype. The total number of cells analysed per condition is indicated. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). Bars represent mean ± s.d. e, Representative immunofluorescence images from control or Deup1 morpholino-treated Xenopus epithelial cells stained with tight junction marker, ZO-1, and centriole marker, Centrin. Scale bars represent 10 μm. Source data

Extended Data Fig. 3 Deup1 siRNA does not suppress centriole amplification in MCCs.

a, Quantification of control or Deup1 siRNA-treated cells undergoing centriole amplification that contained (DEUP1+ ) or lacked (DEUP1-) DEUP1 foci. To identify cells in the process of centriole amplification we immunostained for STIL, which localizes to immature procentrioles but is absent from mature basal bodies. n = 3 cultures/genotype. b, Quantification of the intensity of DEUP1 signal in STIL+ /DEUP1+ controls or STIL+ /DEUP1- siRNA-treated cells. n = 3 cultures/genotype. The average DEUP1 intensity for the control siRNA sample was normalized to 1 for each experiment. P values, one sample t-test compared to a mean of 1. **** = p ≤ 0.0001. c, Quantification of centriole number in control and Deup1 siRNA-treated cells. Only STIL+ cells were quantified so that DEUP1 depletion could be monitored, and only cells depleted for DEUP1 (assessed by immunostaining) in the siRNA condition were quantified. n = 3 cultures/genotype. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). d, Representative images of control ependymal cells and cells with siRNA-mediated depletion of DEUP1 undergoing centriole amplification. Scale bars represent 10 μm. Source data

Extended Data Fig. 4 Procentrioles form in the vicinity of parent centrioles in cells that lack deuterosomes.

a, Quantification of the ratio of SAS6 intensity at the younger parent centriole (YPC) compared to the older parent centriole (OPC) in ependymal cells in vivo (P2–P6) during A-phase. GT335 staining was used to mark the cilium that forms from the older parent centriole. n = 3 mice/genotype. P values, one sample t-test compared to the value of 1, which represents identical SAS6 intensity levels at the YPC and OPC. *** = p ≤ 0.001, **** = p ≤ 0.0001. b, Quantification of SAS6 intensity on parent centrioles in Deup1+/+ and Deup1/ ependymal cells in amplification phase in vivo (P2–P6). The SAS6 signal associated with the two parent centrioles was summed together and normalized to background SAS6 staining of the same cell. n = 3 mice/genotype. P values, unpaired, two-tailed, Welch’s t-test. **** = p ≤ 0.0001. All bars represent mean ± s.d. c, Serial EM images of a Deup1/ ependymal cell in the growth phase from basal (Z1) to apical (Z10). Note the important centriole amplification on, and in the vicinity of, the parental centrioles. Bottom right shows a schematic representation of the relative position of procentrioles formed by the parent centrioles. Scale bar represents 500 nm. Source data

Extended Data Fig. 5 CEP63 modestly affects centriole amplification in Deup1/ MCCs.

a, RT-qPCR analysis of Cep63 mRNA levels in Deup1+/+ and Deup1/ mTECs. n = 3 cultures/genotype. The average of Deup1+/+ samples was normalized to 1. b, Quantification of CEP63 protein levels in ependymal cells in the amplification phase by immunostaining. To account for differences in the number of procentrioles present in each cell, CEP63 intensity levels were normalized to the intensity of the procentriole marker SAS6. n = 3 cultures/genotype. c, RT-qPCR analysis of Cep63 mRNA levels in testes and mTECs from Cep63+/+ and Cep63T/T mice using two different primer sets. The average of Cep63+/+ samples was normalized to 1. n = 3 mice or cultures/genotype. d, Quantification of CEP164 foci which marks basal bodies in cultured mature ependymal cells. Data from Deup1+/+ and Deup1/ cultures are from Extended Data 2a and shown for comparison. n = 3 cultures/genotype. e, Representative images of mature basal bodies using an antibody against CEP164 in mature ependymal cells in vitro. f, Quantification of CEP164 foci in mature mTECs. Data from Deup1+/+ and Deup1/ cultures are from Fig. 2a and shown for comparison. n = 3 cultures/genotype. g, Representative images from mature mTECs. DAPI marks the nuclei, acetylated-tubulin (AcTub) marks cilia, ZO-1 marks tight junctions and CEP164 stains basal bodies. h, Quantification of the basal body marker CEP164 foci in adult-brain sections. Data from Deup1+/+ and Deup1/ mice are from Fig. 2c and shown for comparison. n = 3 mice/genotype. i, Representative images of ependymal cells in adult-brain sections. DAPI marks the nuclei, ZO-1 marks tight junctions and CEP164 stains basal bodies. j, Scanning electron micrographs of trachea from control or Deup1/; Cep63T/T adult mice. All scale bars represent 10 μm. All bars represent mean ± s.d. All P values are from unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05), * = p ≤ 0.05. Source data

Extended Data Fig. 6 Deup1 and Cep63 are dispensable for centriole amplification in Xenopus.

a, Quantification of centriole number in Xenopus epithelial cells treated with Cep63 or Deup1 and Cep63 morpholinos. Data from control and Deup1 morpholinos are from Extended Data 2d and shown for comparison. Points represent the average number of centrioles per cell in one embryo. n ≥ 5 embryos/genotype. The total number of cells analysed per condition is indicated. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). Bars represent mean + s.d. b, Representative immunofluorescence images from control, Cep63 or Deup1 and Cep63 morpholino-treated Xenopus epithelial cells stained with tight junction marker, ZO-1, and centriole marker, Centrin. Scale bars represent 10 μm. c, Quantification of Cep63–GFP intensity in control and Cep63 morpholino-treated Xenopus epithelial cells. Note, the Cep63 morpholino oligonucleotide efficiently silenced expression of an mRNA encoding a morpholino-targetable Cep63 fused to GFP in Xenopus MCCs. n = 3 embryos/genotype. The total number of cells analysed per condition is indicated. P values, unpaired, two-tailed, Welch’s t-test. * = p ≤ 0.05. Bars represent mean + s.d. d, Quantification of the percent of centrioles with normal or abnormal structure in Deup1+/+, Deup1/ and Deup1/; Cep63T/T cultured ependymal cells. n > 30 cells/genotype. The total number of centrioles analysed per genotype is indicated. e, Representative TEM images of normal and abnormal centrioles in Deup1+/+, Deup1/ and Deup1/; Cep63T/T cultured ependymal cells. Scale bar represents 100 nm. Source data

Extended Data Fig. 7 Procentrioles are amplified within a cloud of PCNT in both Deup1+/+ and Deup1/ cells with or without parent centrioles.

a, Immunofluorescence images of CENT2–GFP-expressing Deup1+/+ and Deup1/ ependymal cells. Brain sections were stained with antibodies against the procentriole protein SAS6 and pericentriolar material protein PCNT. X marks CENT2–GFP aggregates. Arrowheads point to parent centrioles. Insets show the same images with only the SAS6 staining and the PCNT cloud border generated through the semi-automatic segmentation process. Scale bars represent 1 μm. b, Quantification of the percent of SAS6+ foci observed within the PCNT cloud in Deup1+/+ and Deup1/ ependymal cells during the amplification phase in vivo. n= 3 mice/genotype. Each point represents a single cell. The total number of cells analysed per genotype is indicated. P values, unpaired, two-tailed, Welch’s t-test. * = p ≤ 0.05. Bars represent mean ± s.d. c, Quantification of the intensity of the PCNT cloud in Deup1+/+ ependymal cells with 2 parent centrioles and in Deup1/ ependymal cells with 0 parent centrioles during the amplification phase in vitro. n = 3 cultures/genotype. The total number of cells analysed per genotype is indicated. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). Bars represent mean ± s.d. d, Quantification of the area of the PCNT cloud in Deup1+/+ ependymal cells with 2 parent centrioles and in Deup1/ ependymal cells with 0 parent centrioles during the amplification phase in vitro. n = 3 cultures/genotype. The total number of cells analysed per genotype is indicated. P values, unpaired, two-tailed, Welch’s t-test. n.s. = not statistically significant (p > 0.05). Bars represent mean ± s.d. e, Model of centriole amplification in MCCs. (Blue) Wild type cells amplify centrioles on the surface of deuterosomes and the parent centrioles. (Orange) Deup1 knockout cells achieve the correct number of centrioles through the massive production of centrioles on the surface and in the vicinity of parent centrioles. (Red) MCCs that lack parent centrioles and deuterosomes amplify the correct number of centrioles within the confines of a PCM cloud. Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Live-cell imaging of centriole amplification in a CENT2–GFP Deup1+/+ ependymal cell shown in Fig. 3b. One frame captured every 40 min; displayed at 1 frame s−1. The A, G and D phases are depicted in grey, orange and green, respectively. Arrows indicate parent centrioles. ‘X’ marks a CENT2–GFP aggregate.

Supplementary Video 2

Live imaging of centriole amplification in a CENT2–GFP Deup1/ ependymal cell shown in Fig. 3b. One frame captured every 40 min; displayed at 1 frame s−1. The A, G and D phases are depicted in grey, orange and green, respectively. Arrows indicate parent centrioles. ‘X’ marks CENT2–GFP aggregate.

Supplementary Video 3

Serial-section electron microcopy of a CENT2–GFP control ependymal cell shown in Fig. 1d. and Fig. 4b. The cell is in the growth phase. Images are taken in 70-nm sections; displayed at 4 frames s−1. Note that a reorientation occurs from the 14th section due to the imaging of a new EM grid.

Supplementary Video 4

Serial-section electron microcopy of a CENT2–GFP Deup1/ ependymal cell shown in Fig. 1d. and Fig. 4c. The cell is in the growth phase. Images are taken in 70-nm sections; displayed at 4 frames s−1. Note that reorientations occur at the 6th and 21st sections due to the imaging of new EM grids.

Supplementary Video 5

3D rendering of the serial-section electron microcopy data shown in Fig. 4c.

Supplementary Video 6

Live imaging of cilia beating in a control ependymal cell. Movies were imaged at 330 frames s−1; displayed at 20 frames s−1.

Supplementary Video 7

Live imaging of cilia beating in a Deup1/ ependymal cell. Movies were imaged at 330 frames s−1; displayed at 20 frames s−1.

Supplementary Video 8

Live imaging of cilia beating in a Deup1/; Cep63T/T ependymal cell. Movies imaged at 33 frames s−1; displayed at 20 frames s−1.

Supplementary Video 9

Live imaging of centriole amplification in a CENT2–GFP Deup1/ ependymal cell with two parent centrioles. Cell is shown in Fig. 6a. One frame captured every 40 min; displayed at 1 frame s−1. The A, G and D phases are depicted in grey, orange and green, respectively. Arrows indicate parent centrioles. ‘X’ marks a CENT2–GFP aggregate.

Supplementary Video 10

Live imaging of centriole amplification in a CENT2–GFP Deup1/ ependymal cell treated with centrinone to deplete both of the parent centrioles. Cell is shown in Fig. 6a. One frame captured every 40 min; displayed at 1 frame s−1. The amplification phases (A, G or D phases) cannot be identified without parent centrioles. ‘X’ marks a CENT2–GFP aggregate.

Supplementary Video 11

Serial-section electron microscopy of a CENT2–GFP Deup1/ ependymal cell treated with centrinone to deplete both of the parent centrioles. Cell is shown in Fig. 6e. The cell is in the amplification phase. Images are taken in 70-nm sections; displayed at 4 frames s−1. Note that reorientations occur at the 12th and the 30th sections due to the imaging of new EM grids.

Supplementary Video 12

Serial-section electron microscopy of a CENT2–GFP Deup1/ ependymal cell treated with centrinone to deplete both of the parent centrioles. Cell is shown in Fig. 6e. The cell is in the growth phase. Images are taken in 70-nm sections; displayed at 4 frames s−1. Note that reorientations occur at the 16th and the 35th sections due to the imaging of new EM grids.

Supplemental Table 1

Table showing the frequency of mice with the indicated genotype. Genotyping was performed between P4 and P7.

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Mercey, O., Levine, M.S., LoMastro, G.M. et al. Massive centriole production can occur in the absence of deuterosomes in multiciliated cells. Nat Cell Biol 21, 1544–1552 (2019) doi:10.1038/s41556-019-0427-x

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