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. 2012 Dec 15;26(24):2684-9.
doi: 10.1101/gad.207027.112.

The Autoregulated Instability of Polo-like Kinase 4 Limits Centrosome Duplication to Once Per Cell Cycle

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The Autoregulated Instability of Polo-like Kinase 4 Limits Centrosome Duplication to Once Per Cell Cycle

Andrew J Holland et al. Genes Dev. .
Free PMC article

Abstract

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centriole duplication occurs once per cell cycle and is controlled by Polo-like kinase 4 (Plk4). We showed previously that Plk4 phosphorylates itself to promote its degradation by the proteasome. Here we demonstrate that this autoregulated instability controls the abundance of endogenous Plk4. Preventing Plk4 autoregulation causes centrosome amplification, stabilization of p53, and loss of cell proliferation; moreover, suppression of p53 allows growth of cells carrying amplified centrosomes. Plk4 autoregulation thus guards against genome instability by limiting centrosome duplication to once per cell cycle.

Figures

Figure 1.
Figure 1.
The autoregulated destruction of Plk4 limits centrosome duplication to once per cell cycle. To allow for continued cell growth, cells analyzed in A–C express the SV40 large T-antigen; cells in D–H did not. (A) Images show the level of Plk4 at the centrosome. (Red) γ-Tubulin; (green) Plk4; (blue) DNA. Bar, 5 μm. (B) Quantitative immunofluorescence analysis of Plk4 levels at the centrosome. Points show the fluorescence intensity of individual cells from at least two independent experiments. The horizontal line represents the mean. (C) The number of Plk4 molecules per cell was determined using AQUA mass spectrometry. Bars represent the mean of at least two replicas. Error bars represent the standard deviation (SD). (D) Thin-section transmission electron micrographs of cells 2 d after infection with adenoviral Cre (Ad-Cre). Bar, 0.5 μm. (E) Fraction of cells with more than two centrosomes at various times after infection with Ad-Cre. Points show the mean of at least three independent experiments. Error bars represent the standard error of the mean (SEM). (F) Fold increase in cell number at various times starting 2 d after infection with Ad-Cre. Points show the mean of at least three independent experiments. Error bars represent the SEM. (G) Images of crystal violet-stained colonies. (H) Percent congenic survival of the indicated cells. Bars represent the mean of at least three independent experiments carried out in triplicate. Error bars represent the SEM.
Figure 2.
Figure 2.
Loss of Plk4 autoregulation leads to p53/p21 stabilization and cell cycle arrest. (A) Time-lapse images of cells stably expressing histone H2B-mRFP (red) and EYFP-α-Tubulin (green). Filming began 24 h after infection with Ad-Cre. Numbers in the top left refer to the time in minutes after nuclear envelope breakdown. (B) Quantification of the proportion of cells with normal, multipolar, or clustered spindle poles at the time of division. Cells were filmed for 16 h beginning 1 d after infection with Ad-Cre. Bars show the mean of >116 cells per condition from at least two independent experiments. Error bars represent the SEM. (C) Duration of mitosis in normal or abnormally dividing cells. Cells were filmed for 16 h beginning 1 d after infection with Ad-Cre. The line shows the mean of >116 cells per condition from at least two independent experiments. (D) Fraction of cells dividing in a 12-h period beginning 1 or 2 d after Ad-Cre infection. (E) Immunoblots of protein harvested 48 h after Ad-Cre infection or 12 h after UV irradiation.
Figure 3.
Figure 3.
Centrosome amplification inhibits proliferation of RPE1 cells. (A) Schematic of the strategy for doxycycline (Dox)-inducible expression of Plk4AA in RPE1 cells. (B) Immunoblots showing the levels of protein in Plk4AA cells at various times after doxycycline addition. Cells were left untreated, grown in the continuous presence of doxycycline, or transiently exposed to doxycycline for 48 h. (C) Fraction of RPE1 and Plk4AA cells with more than two centrosomes at various times after doxycycline addition. Points show the mean of two independent experiments, and error bars represent the SEM. (D) Percent congenic survival for the indicated cells. Immunoblots of p53 protein in UV-irradiated cells stably expressing a p53 shRNA. Bars represent the mean of two independent experiments carried out in triplicate. Error bars represent the SEM. (E) Fraction of cells with more than two centrosomes at various times after doxycycline addition. Points show the mean of two independent experiments, and error bars represent the SEM. (F) Percent congenic survival for the indicated cells. Bars represent the mean of two independent experiments carried out in triplicate. Error bars represent the SEM.
Figure 4.
Figure 4.
p53 inhibits the proliferation of cells defective in Plk4 autoregulation. (A) Crystal violet-stained colonies of the indicated cell lines. (B) Percent congenic survival for indicated cells. Bars represent the mean of at least two independent experiments carried out in triplicate. Error bars represent the SEM. (C) Images show the level of Plk4 at the centrosome of SV40-RPE1 clones that were isolated after infection with Ad-Cre. (Red) hSPD-2; (green) Centrin; (blue) DNA. Bar, 5 μm. (D) Fraction of SV40-RPE1 clones with more than two centrosomes. Bars represent the mean of at least two independent experiments, and error bars represent the SEM. (E) Thin-section transmission electron micrographs of SV40-RPE1 clones. Bar, 0.5 μm. (F) Immunoblots of p53 protein levels in UV-irradiated cells stably expressing a p53 shRNA. (G) Crystal violet-stained colonies of the indicated cell lines. (H) Percent clonogenic survival for the indicated cells. Bars represent the mean of at least two independent experiments carried out in triplicate. Error bars represent the SEM.

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