Centriole overduplication through the concurrent formation of multiple daughter centrioles at single maternal templates

Abstract

Abnormal centrosome numbers are detected in virtually all cancers. The molecular mechanisms that underlie centrosome amplification, however, are poorly characterized. Based on the model that each maternal centriole serves as a template for the formation of one and only one daughter centriole per cell division cycle, the prevailing view is that centriole overduplication arises from successive rounds of centriole reproduction. Here, we provide evidence that a single maternal centriole can concurrently generate multiple daughter centrioles. This mechanism was initially identified in cells treated with the peptide vinyl sulfone proteasome inhibitor Z-L(3)VS. We subsequently found that the formation of more than one daughter at maternal centrioles requires cyclin E/cyclin-dependent kinase 2 as well as Polo-like kinase 4 and that overexpression of these proteins mimics this phenotype in the absence of a proteasome inhibitor. Moreover, we show that the human papillomavirus type 16 E7 oncoprotein stimulates aberrant daughter centriole numbers in part through the formation of more than one daughter centriole at single maternal templates. These results help to explain how oncogenic stimuli can rapidly induce abnormal centriole numbers within a single cell-division cycle and provide insights into the regulation of centriole duplication.

Figure 1. Aberrant centriole configuration following treatment with the proteasome inhibitor Z-L₃VS

(A) Fluorescence microscopic analysis of U-2 OS cells stably expressing centrin-GFP to visualize centrioles (arrows; inserts) after either treatment with 0.1% DMSO (top panels) or 1 μM Z-L₃VS (bottom panels) for 48 h. Nuclei stained with DAPI. Scale bar indicates 10 μm. (B) Quantification of U-2 OS/centrin-GFP cells with more than one daughter centriole per maternal centriole after treatment with either 0.1% DMSO or 1 μM Z-L₃VS for 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.

Figure 2. Concurrent formation of multiple daughter centrioles at a single maternal centriole following Z-L₃VS

Electron microscopic analysis of U-2 OS/centrin-GFP cells either DMSO-treated with 0.1% DMSO (A) or treated with 1 μM Z-L₃VS for 48 h (B). Scale bars indicate 500 nm.

Figure 3. Z-L₃VS-induced formation of excessive daughter centrioles at maternal centrioles requires cyclin E, CDK2 and PLK4

(A) Immunoblot analyses of U-2 OS/centrin-GFP cells transfected with either control siRNA duplexes (control) or siRNAs targeting cyclin E1, cyclin E2, cyclin A2, CDK2 or PLK4 for the indicated time intervals. Immunoblots for actin are shown to demonstrate loading of equal amounts of protein. (B) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transfected with control siRNA duplexes (top panels) or siRNAs targeting cyclin E1 (bottom panels) following treatment with 1 μM Z-L₃VS for 48 h. Cells were co-transfected with DsRED fluorescent protein as transfection marker. Nuclei stained with DAPI. Scale bar indicates 10 μm. (C) Quantification of U-2 OS/centrin-GFP cells transfected with the indicated siRNAs followed by either control treatment with 0.1% DMSO or 1 μM Z-L₃VS for 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.

Figure 4. Overexpression of cyclin E/CDK2 and PLK4 stimulates the concurrent formation of multiple daughter centrioles at single maternal centrioles

(A) Quantification of U-2 OS/centrin-GFP cells with more than one daughter centriole per maternal centriole at 48 h after transfection with empty vector (neo), cyclin E, CDK2 and PLK4 either alone or in combination. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment. (B) Quantification of IMR-90 normal human fibroblasts with more than one daughter centriole per maternal centriole at 48 h after transfection with empty vector (neo), cyclin E, CDK2 and PLK4 either alone or in combination. Centrioles were visualized by immunofluorescence microscopy for centrin. Each bar represents mean and standard error of at least three independent experiments with a minimum of 50 cells counted per experiment.

Figure 5. Aberrantly synthesized daughter centrioles can function as mitotic spindle poles

(A) Fluorescence microscopic analysis of mitotic U-2 OS/centrin-GFP cells. A normal bipolar metaphase is shown in comparison to a pseudobipolar metaphase cell and a multipolar (tripolar) anaphase cell. More than two images are merged for middle and right panels to show all centrioles. Chromosomes stained with DAPI. (B) Quantification of mitotic abnormalities in U-2 OS/centrin-GFP populations after treatment with 0.1% DMSO or 1 μM Z-L₃VS for 24 h and incubation in normal media for an additional 24 h. Only dividing cells were evaluated. Each bar represents mean and standard error of three independent experiments with at least 100 mitotic cells counted per experiment.

Figure 6. HPV-16 E7 rapidly stimulates the concurrent formation of more than one daughter centriole at maternal templates

(A) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transiently transfected with either empty vector (control) or HPV-16 E7 after 24 h or 48 h. Note the formation of two daughters at a single maternal centriole after 24 h in a HPV-16 E7-transfected cell (middle panels, insert). After 48 h, this pattern was less frequent and the majority of cells with abnormal centriole numbers showed a more dispersed arrangement (bottom panels, insert, arrows). Cells were co-transfected with DsRED fluorescent protein as transfection marker. Nuclei stained with DAPI. Scale bar indicates 10 μm. (B) Quantification of U-2 OS/centrin-GFP cells with more than four centrioles in a random arrangement (gray bars) in comparison to cells with more than four centrioles and a concurrent formation of more than one daughter per maternal centriole (black bars). Cells were transfected with empty vector (control) or HPV-16 E7 for 24 h or 48 h. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment. (C) Quantification of U-2 OS/centrin-GFP cells with more than four centrioles and concurrent formation of more than one daughter centriole at a maternal centriole after treatment with 1 mM HU for the indicated time intervals. dH₂O-treated cells are shown as controls. Each bar represents mean and standard error of at least three independent experiments with a minimum of 100 cells counted per experiment.