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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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Depletion of p53 allows continued growth in the absence of Plk4. (A) Graph showing the fold increase in cell number after IAA addition. Points show the mean of at least two independent experiments performed in triplicate. (B) Quantification of the number of Centrin foci per cell in interphase at indicated times after IAA addition. Bars represent the mean of >150 cells from two independent experiments. (C) Selected images of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells immunostained with α-tubulin, γ-tubulin, and Centrin. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the duration of mitosis, the frequency of divisions resulting in the formation of misshapen nuclei, and the frequency of cytokinesis failure and chromosome missegregation in acentriolar Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >100 cells from two independent experiments. All error bars in the figure represent the SEM. (E) Selected images from a time-lapse series of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. The acentriolar cell spends longer in mitosis and fails cytokinesis. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Bars: (main) 5 µm; (inset) 1 µm.
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fig5: Depletion of p53 allows continued growth in the absence of Plk4. (A) Graph showing the fold increase in cell number after IAA addition. Points show the mean of at least two independent experiments performed in triplicate. (B) Quantification of the number of Centrin foci per cell in interphase at indicated times after IAA addition. Bars represent the mean of >150 cells from two independent experiments. (C) Selected images of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells immunostained with α-tubulin, γ-tubulin, and Centrin. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the duration of mitosis, the frequency of divisions resulting in the formation of misshapen nuclei, and the frequency of cytokinesis failure and chromosome missegregation in acentriolar Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >100 cells from two independent experiments. All error bars in the figure represent the SEM. (E) Selected images from a time-lapse series of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. The acentriolar cell spends longer in mitosis and fails cytokinesis. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Bars: (main) 5 µm; (inset) 1 µm.

Mentions: RPE1 cells maintain a normal p53 response. Plk4AID/AID cells but not Plk4+/+ cells showed stabilization of p53 at 2 and 3 d after IAA addition (Fig. S4 A). We therefore tested whether stabilization of p53 contributes to the irreversible cell cycle arrest that occurs after Plk4 depletion and centriole duplication failure. Preventing p53 accumulation using a stably expressed p53 shRNA (Fig. S4 B) allowed the continued growth of cells lacking Plk4 (Fig. 5 A) and led to a partial recovery of the clonogenic survival of this population (Fig. S4, C and D). Our findings indicate that a failure of centriole duplication increases p53 levels, eliciting a p53-dependent cell cycle arrest. This is consistent with prior work showing that knockout cells lacking proteins essential for centriole duplication also fail to proliferate in the presence of p53 (Bazzi and Anderson, 2014; Izquierdo et al., 2014).


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)

Depletion of p53 allows continued growth in the absence of Plk4. (A) Graph showing the fold increase in cell number after IAA addition. Points show the mean of at least two independent experiments performed in triplicate. (B) Quantification of the number of Centrin foci per cell in interphase at indicated times after IAA addition. Bars represent the mean of >150 cells from two independent experiments. (C) Selected images of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells immunostained with α-tubulin, γ-tubulin, and Centrin. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the duration of mitosis, the frequency of divisions resulting in the formation of misshapen nuclei, and the frequency of cytokinesis failure and chromosome missegregation in acentriolar Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >100 cells from two independent experiments. All error bars in the figure represent the SEM. (E) Selected images from a time-lapse series of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. The acentriolar cell spends longer in mitosis and fails cytokinesis. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Bars: (main) 5 µm; (inset) 1 µm.
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Related In: Results  -  Collection

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fig5: Depletion of p53 allows continued growth in the absence of Plk4. (A) Graph showing the fold increase in cell number after IAA addition. Points show the mean of at least two independent experiments performed in triplicate. (B) Quantification of the number of Centrin foci per cell in interphase at indicated times after IAA addition. Bars represent the mean of >150 cells from two independent experiments. (C) Selected images of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells immunostained with α-tubulin, γ-tubulin, and Centrin. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the duration of mitosis, the frequency of divisions resulting in the formation of misshapen nuclei, and the frequency of cytokinesis failure and chromosome missegregation in acentriolar Plk4AID/AID;p53 shRNA cells. Bars represent the mean of >100 cells from two independent experiments. All error bars in the figure represent the SEM. (E) Selected images from a time-lapse series of untreated control or acentriolar Plk4AID/AID;p53 shRNA cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. The acentriolar cell spends longer in mitosis and fails cytokinesis. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Bars: (main) 5 µm; (inset) 1 µm.
Mentions: RPE1 cells maintain a normal p53 response. Plk4AID/AID cells but not Plk4+/+ cells showed stabilization of p53 at 2 and 3 d after IAA addition (Fig. S4 A). We therefore tested whether stabilization of p53 contributes to the irreversible cell cycle arrest that occurs after Plk4 depletion and centriole duplication failure. Preventing p53 accumulation using a stably expressed p53 shRNA (Fig. S4 B) allowed the continued growth of cells lacking Plk4 (Fig. 5 A) and led to a partial recovery of the clonogenic survival of this population (Fig. S4, C and D). Our findings indicate that a failure of centriole duplication increases p53 levels, eliciting a p53-dependent cell cycle arrest. This is consistent with prior work showing that knockout cells lacking proteins essential for centriole duplication also fail to proliferate in the presence of p53 (Bazzi and Anderson, 2014; Izquierdo et al., 2014).

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