p53 protects against genome instability following centriole duplication failure.
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.
Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.Show MeSH
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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).
Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.