The PIDDosome activates p53 in response to supernumerary centrosomes

Abstract

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

Keywords: cell division; centrosome; cytokinesis failure; p53.

Figure 1.

Caspase-2 constrains polyploidization after cytokinesis failure by MDM2 processing, leading to p53 stabilization. (A) A549 cells transfected with the indicated siRNAs targeting either luciferase (Gl2) or Caspase-2 (C2) were treated for 24 h with reversine (Rev), nocodazole (Noc), or taxol (Tax) alone or in combination and processed for immunoblotting. (B–D) Following transfection with siRNAs, cells were treated with the indicated cytokinesis inhibitors for different times and processed for DNA content analysis in a flow cytometer (B) or, in parallel, for immunoblotting (C,D).

Figure 2.

The PIDDosome is required to activate p53 after cytokinesis failure. A549 CASP2, PIDD1, or RAIDD knockout cells obtained using two different small guide RNAs (sgRNAs) were treated with ZM447439 for either 24 h and processed for immunoblotting (A) or up to 72 h and processed for DNA content analysis (B). hTERT-RPE1 cells were transduced with the indicated constructs targeting either mouse CD8 (mCD8) as a negative control or RAIDD and then treated with ZM447439 for either 24 h and processed for immunoblotting (C) or up to 72 h and processed for DNA content analysis (D).

Figure 3.

PIDDosome-activated p53 shares features with nongenotoxic activation via Nutlin-3 and functions via p21. (A) A549 knockout cells obtained using the indicated sgRNAs were treated with doxorubicin (DXR), nocodazole, or ZM447439 for 24 h and processed for immunoblotting. (B,C) A549 knockout cells obtained using the indicated sgRNAs were treated for the indicated times with Nutlin-3, ZM447439, or ZM447439 followed by the addition of Nutlin-3 and processed for DNA content analysis (B) or immunoblotting (C). (D,E) Following transfection with siRNAs, cells were treated with ZM447439 for 48 h and processed for immunoblotting (D) or DNA content analysis (E).

Figure 4.

The PIDDosome constrains hepatocyte polyploidization in vivo via p53. (A) Hepatocytes were isolated by collagen perfusion of 7-wk-old mice of the indicated genotypes and subjected to DNA content analysis. (B) Quantification of the DNA content distribution for each genotype displayed in A. Bars represent mean percentages ± SEM. n = 3–5. (C) DNA content analysis and immunoblotting for hepatocytes obtained from animals of the indicated genotypes either unweaned (−) or weaned (+) at the age of 20 and 25 d, respectively. (D) Quantification of the DNA content distribution for each genotype displayed in C. n = 3. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 using unpaired Student's t-test.

Figure 5.

Extra centrosomes are necessary and sufficient for PIDDosome activation. (A) Scheme of the protocol used to generate polyploid cells with or without extra centrosomes. A549 cells were treated with either solvent control (DMSO), ZM447439 (ZM), or nocodazole plus reversine (NocRev) to promote cytokinesis failure in the absence or presence of 24 or 48 h of centrinone treatment. The combination of nocodazole plus reversine appeared best suited for faithful enumeration of centrioles by immunofluorescence (see below). Cells treated as in A were subjected to DNA content analysis (B), immunofluorescence (C,D), and immunoblotting with the indicated antibodies (F). (D) Scatter plot of cells stained as in C subjected to counting of C-Nap1-positive centrioles per cell. Fifty cells per condition were analyzed. Black dashed lines represent the median. (E) Summary of the features measured across the indicated treatments concerning ploidy and centrosomal content. The cartoon summarizes the typical centrosomal arrangement, with blue cylinders representing centrioles, ±green triangles representing the appendage structures decorating them, and markers (as Cep164) for mother centrioles. (G,H) U2OS-Trex-MYC-PLK4 cells were left untreated or treated with doxycycline (DOX) for 72 h and subjected to immunoblotting with the indicated antibodies (G) or DNA content analysis (H).

Figure 6.

Extra mother centrioles activate the PIDDosome. (A) Scheme of the protocol used to time-resolve the appearance of extra centrioles at different maturation stages and PIDDosome activation. U2OS-Trex-MYC-PLK4 cells were arrested in S phase with thymidine. After 14 h, they were either left untreated or induced for an additional 10 h with doxycyline (Dox). Cells were either processed for immunofluorescence (Supplemental Fig. S10A) or immunoblot directly during S arrest or released for 24 and 48 h. (B) Scatter plot for the abundance of C-Nap1-positive and Cep164-positive centrioles assessed in 50 individual cells. (C) PIDDosome activation was followed by immunoblot analysis using the indicated antibodies. (D) Localization of PIDD1 at extra mother centrioles generated by PLK4 overexpression. (E–G) A549 cells transfected with the indicated siRNAs were subjected to immunofluorescence with the indicated antibodies (E) or treated with ZM447439 for the indicated times and subjected to either immunoblotting (F) or DNA content analysis (G).

Figure 7.

Proposed model of PIDDosome-mediated cell cycle arrest in response to supernumerary centrosomes. Cycling somatic cells carry one centrosome in G1, composed of one mother centriole (1)—decorated by appendages (triangles) and positive for PIDD1 (red)—and one daughter centriole (2). Both centrioles are connected by a flexible linker. During S phase, each centriole nucleates one procentriole (3 and 4). Upon mitotic traverse, the daughter centriole matures to mother and acquires appendages and PIDD1 positivity (2). In the absence of cytokinesis, the two mother centrioles (1 and 2) that would normally be inherited by the two daughter cells undergo clustering and promote PIDDosome assembly. This promotes Caspase-2 activation, which results in MDM2 cleavage at D367. The N-terminal fragment of MDM2 binds p53, leading to its stabilization and p21 transcription-dependent cell cycle arrest.