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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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Plk4 depletion leads to a failure of centriole duplication followed by a cell cycle arrest. (A) Graph showing the fold increase in cell number after IAA addition. Plk4 destruction leads to a cell cycle arrest in Plk4AID/AID cells. 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 >100 cells from two independent experiments. (C) Graph showing the fold increase in cell number after IAA washout (WO) and restoration of Plk4 levels. Centriole loss leads to an irreversible cell cycle arrest. Points show the mean of at least two independent experiments performed in triplicate. (D) Quantification of the duration of mitosis. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >60 cells from two independent experiments. (E) Quantification of relative cell proliferation and fraction of cells undergoing chromosome missegregation or cytokinesis failure. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. (F) Quantification of the frequency of divisions resulting in the formation of misshapen nuclei. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. All error bars in the figure represent the SEM. (G) Selected images from a time-lapse series of untreated or IAA-treated Plk4AID/AID cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Note that the cell treated with IAA for 2 d exhibits an asymmetric spindle with one acentriolar pole. The interphase nucleus (asterisk) at 189 min later left the field of view. Bars: (main) 5 µm; (inset) 1 µm.
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fig3: Plk4 depletion leads to a failure of centriole duplication followed by a cell cycle arrest. (A) Graph showing the fold increase in cell number after IAA addition. Plk4 destruction leads to a cell cycle arrest in Plk4AID/AID cells. 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 >100 cells from two independent experiments. (C) Graph showing the fold increase in cell number after IAA washout (WO) and restoration of Plk4 levels. Centriole loss leads to an irreversible cell cycle arrest. Points show the mean of at least two independent experiments performed in triplicate. (D) Quantification of the duration of mitosis. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >60 cells from two independent experiments. (E) Quantification of relative cell proliferation and fraction of cells undergoing chromosome missegregation or cytokinesis failure. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. (F) Quantification of the frequency of divisions resulting in the formation of misshapen nuclei. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. All error bars in the figure represent the SEM. (G) Selected images from a time-lapse series of untreated or IAA-treated Plk4AID/AID cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Note that the cell treated with IAA for 2 d exhibits an asymmetric spindle with one acentriolar pole. The interphase nucleus (asterisk) at 189 min later left the field of view. Bars: (main) 5 µm; (inset) 1 µm.

Mentions: We next examined the chronic effect of Plk4 depletion in RPE1 cells. Although Plk4+/+ cells proliferated normally in the presence of IAA, addition of IAA to Plk4AID/AID cells resulted in a cell cycle arrest 48 h after treatment began (Fig. 3 A). Interphase Plk4AID/AID cells exhibited a dramatic reduction in centriole number by 24 h after IAA addition (Fig. 3 B). Centriole number decreased further by 48 h after Plk4 degradation, giving rise to 22% of cells that lack centrioles. After this time, centriole content changed only slowly, consistent with the fact that the vast majority of cells cease proliferating by 48 h after IAA addition.


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)

Plk4 depletion leads to a failure of centriole duplication followed by a cell cycle arrest. (A) Graph showing the fold increase in cell number after IAA addition. Plk4 destruction leads to a cell cycle arrest in Plk4AID/AID cells. 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 >100 cells from two independent experiments. (C) Graph showing the fold increase in cell number after IAA washout (WO) and restoration of Plk4 levels. Centriole loss leads to an irreversible cell cycle arrest. Points show the mean of at least two independent experiments performed in triplicate. (D) Quantification of the duration of mitosis. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >60 cells from two independent experiments. (E) Quantification of relative cell proliferation and fraction of cells undergoing chromosome missegregation or cytokinesis failure. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. (F) Quantification of the frequency of divisions resulting in the formation of misshapen nuclei. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. All error bars in the figure represent the SEM. (G) Selected images from a time-lapse series of untreated or IAA-treated Plk4AID/AID cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Note that the cell treated with IAA for 2 d exhibits an asymmetric spindle with one acentriolar pole. The interphase nucleus (asterisk) at 189 min later left the field of view. Bars: (main) 5 µm; (inset) 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Plk4 depletion leads to a failure of centriole duplication followed by a cell cycle arrest. (A) Graph showing the fold increase in cell number after IAA addition. Plk4 destruction leads to a cell cycle arrest in Plk4AID/AID cells. 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 >100 cells from two independent experiments. (C) Graph showing the fold increase in cell number after IAA washout (WO) and restoration of Plk4 levels. Centriole loss leads to an irreversible cell cycle arrest. Points show the mean of at least two independent experiments performed in triplicate. (D) Quantification of the duration of mitosis. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >60 cells from two independent experiments. (E) Quantification of relative cell proliferation and fraction of cells undergoing chromosome missegregation or cytokinesis failure. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. (F) Quantification of the frequency of divisions resulting in the formation of misshapen nuclei. Measurements were taken over a 24-h period at indicated times after IAA addition. Bars represent the mean of >40 cells from two independent experiments. All error bars in the figure represent the SEM. (G) Selected images from a time-lapse series of untreated or IAA-treated Plk4AID/AID cells coexpressing histone TagRFP-tubulin, EGFP-Histone H2B, and EGFP-CEP63. Insets show EGFP-CEP63 at the centrosome. Time is indicated in minutes relative to nuclear envelope breakdown (time point 0). Note that the cell treated with IAA for 2 d exhibits an asymmetric spindle with one acentriolar pole. The interphase nucleus (asterisk) at 189 min later left the field of view. Bars: (main) 5 µm; (inset) 1 µm.
Mentions: We next examined the chronic effect of Plk4 depletion in RPE1 cells. Although Plk4+/+ cells proliferated normally in the presence of IAA, addition of IAA to Plk4AID/AID cells resulted in a cell cycle arrest 48 h after treatment began (Fig. 3 A). Interphase Plk4AID/AID cells exhibited a dramatic reduction in centriole number by 24 h after IAA addition (Fig. 3 B). Centriole number decreased further by 48 h after Plk4 degradation, giving rise to 22% of cells that lack centrioles. After this time, centriole content changed only slowly, consistent with the fact that the vast majority of cells cease proliferating by 48 h after IAA addition.

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