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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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De novo centrioles recruit PCM and act as MTOCs. (A) Selected images from a time-lapse series showing de novo centriole formation in cells expressing histone EGFP-Centrin. De novo centrioles duplicate in the second cycle after they are born. Time is indicated in hours relative to the time of IAA washout (time point 0). (B) Quantification of the time of de novo centriole formation relative to the time of cell division. Measurements were made using time-lapse movies of EGFP-Centrin expressing cells from two independent experiments. Line represents the mean of >50 cells. (C) Selected images illustrating the different configurations adopted by de novo centrioles. Cells were costained with Centrin and CEP192 to identify centrioles. (D) Selected images of de novo centrioles in interphase and mitotic cells at 1 and 2 d after IAA washout. The mitotic spindle segregates freestanding de novo centrioles in the first division and pairs of replicated centrioles in the second cell division. Cells were costained with α-tubulin, Centrin, and γ-tubulin or Pericentrin. (E) Selected images of pairs of de novo centrioles in interphase cells at 2 d after IAA washout. Cells were costained with Centrin, CNAP1, and CEP164. Bars: (main) 5 µm; (inset) 1 µm.
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fig8: De novo centrioles recruit PCM and act as MTOCs. (A) Selected images from a time-lapse series showing de novo centriole formation in cells expressing histone EGFP-Centrin. De novo centrioles duplicate in the second cycle after they are born. Time is indicated in hours relative to the time of IAA washout (time point 0). (B) Quantification of the time of de novo centriole formation relative to the time of cell division. Measurements were made using time-lapse movies of EGFP-Centrin expressing cells from two independent experiments. Line represents the mean of >50 cells. (C) Selected images illustrating the different configurations adopted by de novo centrioles. Cells were costained with Centrin and CEP192 to identify centrioles. (D) Selected images of de novo centrioles in interphase and mitotic cells at 1 and 2 d after IAA washout. The mitotic spindle segregates freestanding de novo centrioles in the first division and pairs of replicated centrioles in the second cell division. Cells were costained with α-tubulin, Centrin, and γ-tubulin or Pericentrin. (E) Selected images of pairs of de novo centrioles in interphase cells at 2 d after IAA washout. Cells were costained with Centrin, CNAP1, and CEP164. Bars: (main) 5 µm; (inset) 1 µm.

Mentions: To examine the kinetics of de novo centriole assembly, we generated acentriolar cells stably expressing EGFP-Centrin and performed time-lapse imaging after IAA washout. Approximately 9 h before mitosis, a variable number of EGFP-Centrin foci formed dispersed throughout the cytosol (Fig. 8, A and B). These foci increased in size and intensity until they were indistinguishable from EGFP-Centrin foci observed at normal centrioles in control cells. Immunofluorescence staining showed that the newly formed Centrin foci colocalized with CEP192 and are thus likely to represent de novo centrioles (Fig. 8 C). Once de novo centrioles had formed, no additional Centrin foci were generated, consistent with the observation that de novo centriole assembly is inhibited by the presence of centrioles (Fig. 8 A and Video 5; Marshall et al., 2001; La Terra et al., 2005). To test if a single centriole is capable of suppressing de novo centriole formation, we transiently treated EGFP-Centrin–expressing Plk4AID/AID;p53 shRNA cells with IAA to generate a fraction of cells with a single centriole, and then removed IAA and monitored de novo centriole formation by time-lapse microscopy. Strikingly, whereas 80% of acentriolar cells underwent de novo centriole formation within 36 h of IAA washout, only 15% of cells with a single centriole underwent de novo centriole assembly in the same period (Fig. S5 E).


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)

De novo centrioles recruit PCM and act as MTOCs. (A) Selected images from a time-lapse series showing de novo centriole formation in cells expressing histone EGFP-Centrin. De novo centrioles duplicate in the second cycle after they are born. Time is indicated in hours relative to the time of IAA washout (time point 0). (B) Quantification of the time of de novo centriole formation relative to the time of cell division. Measurements were made using time-lapse movies of EGFP-Centrin expressing cells from two independent experiments. Line represents the mean of >50 cells. (C) Selected images illustrating the different configurations adopted by de novo centrioles. Cells were costained with Centrin and CEP192 to identify centrioles. (D) Selected images of de novo centrioles in interphase and mitotic cells at 1 and 2 d after IAA washout. The mitotic spindle segregates freestanding de novo centrioles in the first division and pairs of replicated centrioles in the second cell division. Cells were costained with α-tubulin, Centrin, and γ-tubulin or Pericentrin. (E) Selected images of pairs of de novo centrioles in interphase cells at 2 d after IAA washout. Cells were costained with Centrin, CNAP1, and CEP164. Bars: (main) 5 µm; (inset) 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: De novo centrioles recruit PCM and act as MTOCs. (A) Selected images from a time-lapse series showing de novo centriole formation in cells expressing histone EGFP-Centrin. De novo centrioles duplicate in the second cycle after they are born. Time is indicated in hours relative to the time of IAA washout (time point 0). (B) Quantification of the time of de novo centriole formation relative to the time of cell division. Measurements were made using time-lapse movies of EGFP-Centrin expressing cells from two independent experiments. Line represents the mean of >50 cells. (C) Selected images illustrating the different configurations adopted by de novo centrioles. Cells were costained with Centrin and CEP192 to identify centrioles. (D) Selected images of de novo centrioles in interphase and mitotic cells at 1 and 2 d after IAA washout. The mitotic spindle segregates freestanding de novo centrioles in the first division and pairs of replicated centrioles in the second cell division. Cells were costained with α-tubulin, Centrin, and γ-tubulin or Pericentrin. (E) Selected images of pairs of de novo centrioles in interphase cells at 2 d after IAA washout. Cells were costained with Centrin, CNAP1, and CEP164. Bars: (main) 5 µm; (inset) 1 µm.
Mentions: To examine the kinetics of de novo centriole assembly, we generated acentriolar cells stably expressing EGFP-Centrin and performed time-lapse imaging after IAA washout. Approximately 9 h before mitosis, a variable number of EGFP-Centrin foci formed dispersed throughout the cytosol (Fig. 8, A and B). These foci increased in size and intensity until they were indistinguishable from EGFP-Centrin foci observed at normal centrioles in control cells. Immunofluorescence staining showed that the newly formed Centrin foci colocalized with CEP192 and are thus likely to represent de novo centrioles (Fig. 8 C). Once de novo centrioles had formed, no additional Centrin foci were generated, consistent with the observation that de novo centriole assembly is inhibited by the presence of centrioles (Fig. 8 A and Video 5; Marshall et al., 2001; La Terra et al., 2005). To test if a single centriole is capable of suppressing de novo centriole formation, we transiently treated EGFP-Centrin–expressing Plk4AID/AID;p53 shRNA cells with IAA to generate a fraction of cells with a single centriole, and then removed IAA and monitored de novo centriole formation by time-lapse microscopy. Strikingly, whereas 80% of acentriolar cells underwent de novo centriole formation within 36 h of IAA washout, only 15% of cells with a single centriole underwent de novo centriole assembly in the same period (Fig. S5 E).

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