PLK4 drives centriole amplification and apical surface area expansion in multiciliated cells
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Ependymal cells lacking PLK4 differentiate but fail to recruit STIL to deuterosomes.
(A) Schematic overview of the experiment to recombine the Plk4F/F allele in ependymal cells in vitro. Ependymal cells were harvested from Plk4F/F mice or Plk4F/+ controls and seeded onto coverslips. …
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Figure 1—source data 1
Values for biological and technical replicates for graphs in Figure 1.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Knockout of PLK4 leads to centriole loss in neural progenitor cells (NPCs).
(A) Diagram of the Plk4F allele showing LoxP sites surrounding exon 5 (top). Diagram of the Plk4Δ allele following Cre-mediated recombination (middle). Diagram of the mRNA product created from the Pl…
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Figure 1—figure supplement 1—source data 1
Values for biological and technical replicates for graphs in Figure 1—figure supplement 1, and pedigree information for Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Loss of PLK4 blocks centriole assembly in ependymal cells.
(A) Quantification of the number of centrioles per cell in disengagement (D) phase. Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP were analyzed at differentiation days 7 and 8. N=3, …
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Figure 2—source data 1
Values for biological and technical replicates for graphs in Figure 2.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Ependymal cells lacking PLK4 recruit CEP152 but fail centriole amplification.
(A) Representative images of Plk4F/F or Plk4F/+ ependymal cells transduced with AAV-Cre-GFP. Cells were stained with antibodies against DEUP1 (white) and CEP152 (magenta). Scale bar=5 μm. (B) …
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Figure 2—figure supplement 1—source data 1
Values for biological and technical replicates for graphs in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig2-figsupp1-data1-v2.xlsx
Figure 3
PLK4 is required for multicliogenesis in vivo.
Confocal images of the lateral ventricle from control or Plk4F/F;Usp28F/F;NestinCre mouse brains at P21. Sections were stained with antibodies against FOXJ1 (white), CEP164 (yellow), and …
Figure 4 with 1 supplement
PLK4 promotes centriole assembly in mTECs.
(A) Schematic of the experimental design to genetically deplete PLK4 in mTECs. Plk4F/F;R26Cre tracheal epithelial cells were seeded onto collagen-coated transwell filters and allowed to proliferate …
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Figure 4—source data 1
Values for biological and technical replicates for graphs in Figure 4.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
mTECs lacking PLK4 recruit CEP152 but fail centriole amplification.
(A) Representative confocal images of control and Plk4F/F;R26Cre mTECs at ALI 3. Cells were expressing Centrin-GFP and stained with antibodies against DEUP1 (magenta) and CEP152 (white). Scale bar=5 …
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Figure 4—figure supplement 1—source data 1
Values for biological and technical replicates for graphs in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 1 supplement
mTECs that fail centriole amplification form aberrant assemblies of centriole proteins.
(A) Quantification of the percent of control and Plk4F/F;R26Cre mTECs with Centrin aggregates at ALI 3. N=3, n>150. Two-tailed Welch’s t-test. (B) Confocal images of control and Plk4F/F;R26Cre mTECs …
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Figure 5—source data 1
Values for biological replicates and n for the graph in Figure 5.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Blocking centriole amplification in mTECs results in assemblies of some centriole proteins.
(A) Graph showing the percent of cells with Centrin aggregates in stages II-VI of centriole amplification in control mTECs at ALI 3. N=3, n>50. (B) Representative images of control and Plk4F/F;R26Cre…
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Figure 5—figure supplement 1—source data 1
Values for biological and technical replicates for graphs in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig5-figsupp1-data1-v2.xlsx
Figure 6 with 4 supplements
Plk4 kinase activity is critical for centriole amplification in MCCs.
(A) Quantification of the intensity of PLK4 on deuterosomes in mTECs at ALI 2. N=3 (Plk4cWT/F;R26Cre) or 4 (Plk4cKD/F;R26Cre), n=158 (Plk4cWT/F;R26Cre) or 187 (Plk4cKD/F;R26Cre). Two-tailed Welch’s …
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Figure 6—source data 1
Values for biological and technical replicates for graphs in Figure 6.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Inhibition of PLK4 kinase activity with centrinone delays centriole amplification in ependymal cells.
(A) Quantification of the intensity of PLK4 on deuterosomes in ependymal cells treated with DMSO or increasing concentrations of centrinone on differentiation day 5. Cells in all stages of centriole …
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Figure 6—figure supplement 1—source data 1
Values for biological and technical replicates for graphs in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Inhibition of PLK4 kinase activity with centrinone does not perturb centriole amplification in mTECs.
(A) Quantification of the percent of MBB stage mTECs at ALI 7 treated with either DMSO or 10 μM of centrinone. N=3, n>300. Two-tailed Welch’s t-test. (B) Quantification of the number of centrioles …
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Figure 6—figure supplement 2—source data 1
Values for biological and technical replicates for graphs in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig6-figsupp2-data1-v2.xlsx
Figure 6—figure supplement 3
A mouse model for the conditional expression of kinase-dead PLK4.
(A, B) Diagram of the Plk4cKD (A) and Plk4cWT (B) alleles showing LoxP sites surrounding a stop cassette between exons 4 and 5 (top). The stop cassette prevents transcription of either the wild-type …
Figure 6—figure supplement 4
PLK4 kinase activity is required for centriole amplification in ependymal cells.
(A) Percent of AAV-Cre transduced ependymal cells with PLK4 on deuterosomes. N=3, n>70. Two-tailed Welch’s t-test. (B) Representative images of AAV-Cre transduced Plk4cWT/F and Plk4cKD/F ependymal …
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Figure 6—figure supplement 4—source data 1
Values for biological and technical replicates for graphs in Figure 6—figure supplement 4.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig6-figsupp4-data1-v2.xlsx
Figure 7
Centriole amplification promotes apical surface area expansion in mTECs.
(A) Representative confocal images of control mTECs in stages II-VI of centriole amplification. Cells were expressing Centrin-GFP and stained with antibodies against DEUP1 (magenta) and ZO1 (white). …
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Figure 7—source data 1
Values for biological and technical replicates for graphs in Figure 7.
- https://cdn.elifesciences.org/articles/80643/elife-80643-fig7-data1-v2.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit polyclonal CEP164 | EMD Millipore | ABE2621 | (IF) use 1:1000 |
| Antibody | Mouse monoclonal acetylated-α-tubulin | Cell Signaling Technology | 12152 | (IF) use 1:1000, (IHC) use 1:500 |
| Antibody | Rat polyclonal ZO-1 | Thermo Fisher Scientific | 14-9776-82 | (IF) use 1:1000 |
| Antibody | Rabbit polyclonal DEUP1 | Mercey et al., 2019. https://doi.org/10.1038/s41556-019-0427-x | (IF) use 1:1000 | |
| Antibody | Mouse monoclonal SAS6 | Santa Cruz Biotechnology | sc-81431 | (IF) use 1:1000 |
| Antibody | Rabbit polyclonal PCNT | Abcam | ab4448 | (IF) use 1:1000 |
| Antibody | Mouse monoclonal FOXJ1 | Thermo Fisher Scientific | 14-9965-82 | (IF) use 1:1000 |
| Antibody | Rabbit polyclonal PLK4 | Moyer et al., 2015. https://doi.org/10.1083/jcb.201502088 | (IF) use 1:1000 | |
| Antibody | Rabbit polyclonal STIL | Moyer et al., 2015.https://doi.org/10.1083/jcb.201502088 | (IF) use 1:1000 | |
| Antibody | Rabbit polyclonal CEP135 | This study | Homemade, raised against a.a. 649–1140. (IF) use 1:1000 | |
| Antibody | Rabbit polyclonal acetylated-α-Tubulin | Cell Signaling Technology | 5335 | (IF) use 1:1000, (IHC) use 1:500 |
| Antibody | Rabbit polyclonal CNTROB | Atlas Antibodies | HPA023321 | (IF) use 1:1000 |
| Antibody | Rabbit polyclonal CEP152 | This study | Homemade, raised against a.a. 491–810. (IF) use 1:1000 | |
| Antibody | Rabbit polyclonal CDK5RAP2 | Bethyl | BETIHC-00063 | (IF) use 1:1000 |
| Antibody | Goat polyclonal CEP192 | Moyer and Holland, 2019. https://doi.org/10.7554/eLife.46054 | (IF) use 1:1000 | |
| Antibody | Goat polyclonal g-Tubulin | Levine et al., 2017. https://doi.org//10.1016/j.devcel.2016.12.022 | (IF) use 1:1000 | |
| Chemical compound, drug | Centrinone | Tocris Bioscience | Cat. no. 5687 | Varying concentrations |
| Chemical compound, drug | Verapamil | Cytoskeleton, Inc | Cat. #CY-SC002 | Use at 10 µM |
| Other | DAPI Stain | MilliporeSigma | Cat. no. 10236276001 | (IF) use 1:1000 |
