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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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Inducible destruction of endogenous Plk4. (A) Schematic outlining the strategy for the auxin-inducible destruction of Plk4. (B) Western blot showing the levels of immunoprecipitated Plk4-AID-FLAG at the indicated times after IAA addition. (C) Plk4AID/AID cells immunostained for CEP192, Plk4, and FLAG after treatment with IAA for the indicated time. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated times after IAA addition. Each condition represents the mean of >70 cells from at least two independent experiments. (E) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated cell cycle stages. Horizontal line represents the mean of >70 cells from two independent experiments. (F) Quantification of Plk4 protein levels at the centrosome at the indicated time after IAA washout. Plk4 levels return to normal within 3 h of auxin removal. Each bar represents the mean of >70 cells from at least two independent experiments. All error bars in the figure represent the SEM.
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fig1: Inducible destruction of endogenous Plk4. (A) Schematic outlining the strategy for the auxin-inducible destruction of Plk4. (B) Western blot showing the levels of immunoprecipitated Plk4-AID-FLAG at the indicated times after IAA addition. (C) Plk4AID/AID cells immunostained for CEP192, Plk4, and FLAG after treatment with IAA for the indicated time. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated times after IAA addition. Each condition represents the mean of >70 cells from at least two independent experiments. (E) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated cell cycle stages. Horizontal line represents the mean of >70 cells from two independent experiments. (F) Quantification of Plk4 protein levels at the centrosome at the indicated time after IAA washout. Plk4 levels return to normal within 3 h of auxin removal. Each bar represents the mean of >70 cells from at least two independent experiments. All error bars in the figure represent the SEM.

Mentions: We stably expressed osTIR1-9xMyc in Plk4AID/AID cells to place the stability of endogenous Plk4AID under the control of exogenous auxin (Fig. 1 A). To analyze Plk4 protein levels, we immunoprecipitated Plk4AID-3xFLAG from cell lysates using a FLAG antibody and determined the abundance of Plk4 by immunoblot. Addition of the auxin indole-3-acetic acid (IAA) to Plk4AID/AID cells led to rapid Plk4 degradation, with Plk4 falling below the limit of detection within 10 min of IAA addition (Fig. 1 B). The level of Plk4 at the centrosome was measured at various times after IAA addition using antibodies raised to the FLAG tag or to the C terminus of Plk4. Staining with the monoclonal FLAG antibody revealed that the level of Plk4 at the centrosomes of interphase cells declined by >95% within 30 min of IAA addition (Fig. 1, C and D), whereas staining with a polyclonal antibody to the C terminus of Plk4 revealed a >80% reduction during the same time period (Fig. S1 F). Plk4 destruction occurred in all cell cycle phases (Fig. 1 E) and required the presence of the osTIR1 F-box protein (Fig. S1 G). Importantly, the degradation of Plk4 was fully reversible, with the level of Plk4 at the centriole recovering to original levels within 3 h of IAA removal (Fig. 1 F).


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)

Inducible destruction of endogenous Plk4. (A) Schematic outlining the strategy for the auxin-inducible destruction of Plk4. (B) Western blot showing the levels of immunoprecipitated Plk4-AID-FLAG at the indicated times after IAA addition. (C) Plk4AID/AID cells immunostained for CEP192, Plk4, and FLAG after treatment with IAA for the indicated time. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated times after IAA addition. Each condition represents the mean of >70 cells from at least two independent experiments. (E) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated cell cycle stages. Horizontal line represents the mean of >70 cells from two independent experiments. (F) Quantification of Plk4 protein levels at the centrosome at the indicated time after IAA washout. Plk4 levels return to normal within 3 h of auxin removal. Each bar represents the mean of >70 cells from at least two independent experiments. All error bars in the figure represent the SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Inducible destruction of endogenous Plk4. (A) Schematic outlining the strategy for the auxin-inducible destruction of Plk4. (B) Western blot showing the levels of immunoprecipitated Plk4-AID-FLAG at the indicated times after IAA addition. (C) Plk4AID/AID cells immunostained for CEP192, Plk4, and FLAG after treatment with IAA for the indicated time. Bars: (main) 5 µm; (inset) 1 µm. (D) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated times after IAA addition. Each condition represents the mean of >70 cells from at least two independent experiments. (E) Quantification of the level of FLAG-tagged Plk4-AID at the centrosome at the indicated cell cycle stages. Horizontal line represents the mean of >70 cells from two independent experiments. (F) Quantification of Plk4 protein levels at the centrosome at the indicated time after IAA washout. Plk4 levels return to normal within 3 h of auxin removal. Each bar represents the mean of >70 cells from at least two independent experiments. All error bars in the figure represent the SEM.
Mentions: We stably expressed osTIR1-9xMyc in Plk4AID/AID cells to place the stability of endogenous Plk4AID under the control of exogenous auxin (Fig. 1 A). To analyze Plk4 protein levels, we immunoprecipitated Plk4AID-3xFLAG from cell lysates using a FLAG antibody and determined the abundance of Plk4 by immunoblot. Addition of the auxin indole-3-acetic acid (IAA) to Plk4AID/AID cells led to rapid Plk4 degradation, with Plk4 falling below the limit of detection within 10 min of IAA addition (Fig. 1 B). The level of Plk4 at the centrosome was measured at various times after IAA addition using antibodies raised to the FLAG tag or to the C terminus of Plk4. Staining with the monoclonal FLAG antibody revealed that the level of Plk4 at the centrosomes of interphase cells declined by >95% within 30 min of IAA addition (Fig. 1, C and D), whereas staining with a polyclonal antibody to the C terminus of Plk4 revealed a >80% reduction during the same time period (Fig. S1 F). Plk4 destruction occurred in all cell cycle phases (Fig. 1 E) and required the presence of the osTIR1 F-box protein (Fig. S1 G). Importantly, the degradation of Plk4 was fully reversible, with the level of Plk4 at the centriole recovering to original levels within 3 h of IAA removal (Fig. 1 F).

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