We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.

Centrioles function as the major components of centrosomes, which organize microtubule (MT) arrays in proliferating cells, and as basal bodies for primary cilia formation in quiescent cells. Centrioles and basal bodies are structurally similar, barrel-shaped organelles composed of MTs. In proliferating cells, two new centrioles, termed procentrioles, form during the S phase of the cell cycle in close proximity to the proximal ends of the two preexisting parental centrioles, often at a near-orthogonal angle. Considerable progress has been made toward understanding the biogenesis of centrioles, but the mechanisms that determine their lengths remain unknown. Here we show that overexpression of the centriolar protein CPAP in human cells enhances the accumulation of centriolar tubulin, leading to centrioles of strikingly increased length. Consistent with earlier work, we also find that elongated MT structures can be induced by depletion of the distal end-capping protein CP110 from centrioles. Importantly, though, these structures differ from genuine primary cilia. We thus propose that CPAP and CP110 play antagonistic roles in determining the extent of tubulin addition during centriole elongation, thereby controlling the length of newly formed centrioles.

Faithful chromosome segregation in mitosis requires the formation of a bipolar mitotic spindle with stably attached chromosomes. Once all of the chromosomes are aligned, the connection between the sister chromatids is severed by the cysteine protease separase. Separase also promotes centriole disengagement at the end of mitosis. Temporal coordination of these two activities with the rest of the cell cycle is required for the successful completion of mitosis. In this study, we report that depletion of the microtubule and kinetochore protein astrin results in checkpoint-arrested cells with multipolar spindles and separated sister chromatids, which is consistent with untimely separase activation. Supporting this idea, astrin-depleted cells contain active separase, and separase depletion suppresses the premature sister chromatid separation and centriole disengagement in these cells. We suggest that astrin contributes to the regulatory network that controls separase activity.

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.

SummaryCentrosomes represent the major microtubule organizing centers (MTOCs) of animal somatic cells and orchestrate bipolar spindle assembly during mitotic cell division. In meiotic cells, the kinesin HSET compensates for the lack of centrosomes by focusing acentrosomal MTOCs into two spindle poles. By clustering multiple centrosomes into two spindle poles, HSET also mediates bipolar mitosis in cancer cells with supernumerary centrosomes. However, although dispensable in non-transformed human cells, the role of HSET in cancer cells with two centrosomes has remained elusive. In this study, we demonstrate that HSET is required for proper spindle assembly, stable pole-focusing and survival of cancer cells irrespective of normal or supernumerary centrosome number. Strikingly, we detected pronounced acentrosomal MTOC structures in untreated mitotic cancer cells. While in most cancer cells these acentrosomal MTOCs were rapidly incorporated into the assembling bipolar spindle, some cells eventually established bipolar spindles with acentrosomal poles and free centrosomes. These observations demonstrate that acentrosomal MTOCs were functional and that both centrosomal and acentrosomal mechanisms were required for bipolar spindle organization. Our study shows that HSET is critical for clustering acentrosomal and centrosomal MTOCs during spindle formation in human cancer cells with two bona fide centrosomes. Furthermore, we show that in checkpoint-defective cancer cells, acentrosomal spindle formation and HSET-dependence are partially mediated by a constitutive activation of the DNA damage response. In summary, we propose that acentrosomal spindle assembly mechanisms are hyperactive in cancer cells and promote HSET, a key driver of acentrosomal spindle organization, as an attractive target for cancer therapy.