Molecular Dissection of the Centrosome Overduplication Pathway in S-Phase Arrested Cells

The formation of a bipolar mitotic spindle is crucial for the even segregation of genetic material into two daughter cells during cell division. Each pole of the spindle is organised by a centrosome, therefore it is paramount that two, and only two, centrosomes are present in the cell at the time that it enters mitosis. The presence of too many centrosomes can lead to the formation of multipolar spindles and the uneven segregation of chromosomes. Indeed, cancer cells frequently display supernumerary centrosomes which may contribute to chromosome missegregation and aneuploidy. Normally, following the completion of mitosis, each cell contains a single centrosome. This then duplicates in a semiconservative, templated manner that is tightly linked to the cell cycle. In some instances supernumerary centrosomes arise through uncoupling of this event from the cell cycle such that centrosomes overduplicate within a single cell cycle. Experimentally, centrosome overduplication can be induced in certain cell types by treatment with drugs, such as hydroxyurea, that inhibit DNA synthesis and thereby provoke an S-phase arrest. This assay has been exploited in CHO, U2OS and p53-/- MEF cells to decipher a molecular pathway for centrosome overduplication using a range of pharmacological inhibitors. Distinct granules containing the protein centrin were identified by fluorescence microscopy as early intermediates in this process and shown to form within the nucleus in a Cdk-dependent manner. These foci are then trafficked from the nucleus to the cytoplasm dependent upon the nuclear export machinery. Here, they recruit modified tubulin, PCM-1 and pericentrin, and resemble centriolar satellites. Microtubules and dynein are required to focus these satellites around the centrosome and the formation of centrioles as recognised by electron microscopy. Finally, Hsp90 is required for the recruitment of γ-tubulin to the newlyformed centrioles to construct functional microtubule organising centres. Significantly, intermediate steps in this pathway show similarities to events associated with the de novo centriole formation pathway and the centriolar and acentriolar pathways of ciliogenesis. Together, this work substantially increases our understanding of how supernumerary centrosomes are generated in cells and identifies key events that may be targeted to prevent centrosome overduplication in cancer cells.