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p53 protects against genome instability following centriole duplication failure.

Lambrus BG, Uetake Y, Clutario KM, Daggubati V, Snyder M, Sluder G, Holland AJ - J. Cell Biol. (2015)

Bottom Line: Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely.Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate.In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.

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Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

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Restoration of Plk4 levels in acentriolar cells leads to de novo centriole formation. (A) Immunoblot showing the level of immunoprecipitated endogenous Plk4-AID-FLAG at the indicated times after IAA washout in acentriolar, Plk4AID/AID;p53 shRNA cells. (B) Quantification of the number of Centrin foci per interphase cell at indicated times after IAA washout, in Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >200 cells from three independent experiments. (C, top) Quantification of the number of Centrin-marked de novo centrioles in interphase cells at 2 d after IAA washout (WO). Cells were transfected with the STIL siRNA 24 h before IAA washout. (bottom) Immunoblot showing depletion of STIL at 48 h after transfection with STIL siRNA. (D and E) Quantification of the fraction of bipolar and multipolar divisions in Plk4AID/AID;p53 shRNA at indicated times after IAA washout. Bars represent the mean of >120 cells from two independent experiments. All error bars in the figure represent the SEM. (F) Selected images of anaphase phenotypes. Both single and pairs of de novo centrioles clustered at the poles of the mitotic spindle. Cells were costained with Centrin and CEP192. (G) Selected images of Centrin-marked de novo centrioles at 1 d after IAA washout. Cells were costained with Plk4, STIL, SAS6, CPAP, CEP192, CEP152, and CEP135. Bars: (main) 5 µm; (inset) 1 µm.
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fig6: Restoration of Plk4 levels in acentriolar cells leads to de novo centriole formation. (A) Immunoblot showing the level of immunoprecipitated endogenous Plk4-AID-FLAG at the indicated times after IAA washout in acentriolar, Plk4AID/AID;p53 shRNA cells. (B) Quantification of the number of Centrin foci per interphase cell at indicated times after IAA washout, in Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >200 cells from three independent experiments. (C, top) Quantification of the number of Centrin-marked de novo centrioles in interphase cells at 2 d after IAA washout (WO). Cells were transfected with the STIL siRNA 24 h before IAA washout. (bottom) Immunoblot showing depletion of STIL at 48 h after transfection with STIL siRNA. (D and E) Quantification of the fraction of bipolar and multipolar divisions in Plk4AID/AID;p53 shRNA at indicated times after IAA washout. Bars represent the mean of >120 cells from two independent experiments. All error bars in the figure represent the SEM. (F) Selected images of anaphase phenotypes. Both single and pairs of de novo centrioles clustered at the poles of the mitotic spindle. Cells were costained with Centrin and CEP192. (G) Selected images of Centrin-marked de novo centrioles at 1 d after IAA washout. Cells were costained with Plk4, STIL, SAS6, CPAP, CEP192, CEP152, and CEP135. Bars: (main) 5 µm; (inset) 1 µm.

Mentions: In vertebrate somatic cells, de novo centriole assembly is initiated after the eradication of the existing centrioles by laser ablation or microsurgery (Khodjakov et al., 2002; La Terra et al., 2005; Uetake et al., 2007). In this case a variable number of de novo centrioles are spontaneously generated. We therefore examined the effect of restoring endogenous Plk4 levels in acentriolar cells. Endogenous Plk4 levels returned to normal within 12 h of IAA washout (Fig. 6 A) and promoted the penetrant formation of de novo centrioles: by 2 d after IAA washout, de novo centriole assembly occurred in 74% of cells and increased to >95% of cells by 4 d after IAA removal (Fig. 6 B and Fig. S4 H). Knockdown of the cartwheel component STIL prevented de novo centriole assembly, suggesting that, similar to canonical centriole duplication, de novo centrioles assemble through a cartwheel-dependent mechanism (Fig. 6 C). Collectively, these data show that Plk4 levels are rate limiting for both canonical and de novo centriole assembly.


p53 protects against genome instability following centriole duplication failure.

Lambrus BG, Uetake Y, Clutario KM, Daggubati V, Snyder M, Sluder G, Holland AJ - J. Cell Biol. (2015)

Restoration of Plk4 levels in acentriolar cells leads to de novo centriole formation. (A) Immunoblot showing the level of immunoprecipitated endogenous Plk4-AID-FLAG at the indicated times after IAA washout in acentriolar, Plk4AID/AID;p53 shRNA cells. (B) Quantification of the number of Centrin foci per interphase cell at indicated times after IAA washout, in Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >200 cells from three independent experiments. (C, top) Quantification of the number of Centrin-marked de novo centrioles in interphase cells at 2 d after IAA washout (WO). Cells were transfected with the STIL siRNA 24 h before IAA washout. (bottom) Immunoblot showing depletion of STIL at 48 h after transfection with STIL siRNA. (D and E) Quantification of the fraction of bipolar and multipolar divisions in Plk4AID/AID;p53 shRNA at indicated times after IAA washout. Bars represent the mean of >120 cells from two independent experiments. All error bars in the figure represent the SEM. (F) Selected images of anaphase phenotypes. Both single and pairs of de novo centrioles clustered at the poles of the mitotic spindle. Cells were costained with Centrin and CEP192. (G) Selected images of Centrin-marked de novo centrioles at 1 d after IAA washout. Cells were costained with Plk4, STIL, SAS6, CPAP, CEP192, CEP152, and CEP135. Bars: (main) 5 µm; (inset) 1 µm.
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fig6: Restoration of Plk4 levels in acentriolar cells leads to de novo centriole formation. (A) Immunoblot showing the level of immunoprecipitated endogenous Plk4-AID-FLAG at the indicated times after IAA washout in acentriolar, Plk4AID/AID;p53 shRNA cells. (B) Quantification of the number of Centrin foci per interphase cell at indicated times after IAA washout, in Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >200 cells from three independent experiments. (C, top) Quantification of the number of Centrin-marked de novo centrioles in interphase cells at 2 d after IAA washout (WO). Cells were transfected with the STIL siRNA 24 h before IAA washout. (bottom) Immunoblot showing depletion of STIL at 48 h after transfection with STIL siRNA. (D and E) Quantification of the fraction of bipolar and multipolar divisions in Plk4AID/AID;p53 shRNA at indicated times after IAA washout. Bars represent the mean of >120 cells from two independent experiments. All error bars in the figure represent the SEM. (F) Selected images of anaphase phenotypes. Both single and pairs of de novo centrioles clustered at the poles of the mitotic spindle. Cells were costained with Centrin and CEP192. (G) Selected images of Centrin-marked de novo centrioles at 1 d after IAA washout. Cells were costained with Plk4, STIL, SAS6, CPAP, CEP192, CEP152, and CEP135. Bars: (main) 5 µm; (inset) 1 µm.
Mentions: In vertebrate somatic cells, de novo centriole assembly is initiated after the eradication of the existing centrioles by laser ablation or microsurgery (Khodjakov et al., 2002; La Terra et al., 2005; Uetake et al., 2007). In this case a variable number of de novo centrioles are spontaneously generated. We therefore examined the effect of restoring endogenous Plk4 levels in acentriolar cells. Endogenous Plk4 levels returned to normal within 12 h of IAA washout (Fig. 6 A) and promoted the penetrant formation of de novo centrioles: by 2 d after IAA washout, de novo centriole assembly occurred in 74% of cells and increased to >95% of cells by 4 d after IAA removal (Fig. 6 B and Fig. S4 H). Knockdown of the cartwheel component STIL prevented de novo centriole assembly, suggesting that, similar to canonical centriole duplication, de novo centrioles assemble through a cartwheel-dependent mechanism (Fig. 6 C). Collectively, these data show that Plk4 levels are rate limiting for both canonical and de novo centriole assembly.

Bottom Line: Depleting p53 allowed cells that fail centriole duplication to proliferate indefinitely.Washout of auxin and restoration of endogenous Plk4 levels in cells that lack centrioles led to the penetrant formation of de novo centrioles that gained the ability to organize microtubules and duplicate.In summary, we uncover a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth after centriole duplication failure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Show MeSH
Related in: MedlinePlus