Patrick Meraldi

Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53–/– cells

Aberrations in centrosome numbers have long been implicated in aneuploidy and tumorigenesis, but their origins are unknown. Here we have examined how overexpression of Aurora‐A kinase causes centrosome amplification in cultured cells. We show that excess Aurora‐A does not deregulate centrosome duplication but gives rise to extra centrosomes through defects in cell division and consequent tetraploidization. Over expression of other mitotic kinases (Polo‐like kinase 1 and Aurora‐B) also causes multinucleation and concomitant increases in centrosome numbers. Absence of a p53 checkpoint exacerbates this phenotype, providing a plausible explanation for the centrosome amplification typical of p53−/− cells. We propose that errors during cell division, combined with the inability to detect the resulting hyperploidy, constitute a major cause for numerical centrosome aberrations in tumors.

Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A.

Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. Here, we have established a centrosome duplication assay using mammalian somatic cells. We show that centrosome duplication requires the activation of E2F transcription factors and Cdk2–cyclin A activity.

Human TPX2 is required for targeting Aurora-A kinase to the spindle

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Here we show that human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity was not required for this interaction, TPX2 was readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules was abolished, although its association with spindle poles was unaffected. Conversely, depletion of Aurora-A by siRNA had no detectable influence on the localization of TPX2. We propose that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly.

A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators

Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule‐nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co‐expression of a non‐destructible B‐type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA‐related kinases.

Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins

BackgroundKinetochores are large multi-protein structures that assemble on centromeric DNA (CEN DNA) and mediate the binding of chromosomes to microtubules. Comprising 125 base-pairs of CEN DNA and 70 or more protein components, Saccharomyces cerevisiae kinetochores are among the best understood. In contrast, most fungal, plant and animal cells assemble kinetochores on CENs that are longer and more complex, raising the question of whether kinetochore architecture has been conserved through evolution, despite considerable divergence in CEN sequence.ResultsUsing computational approaches, ranging from sequence similarity searches to hidden Markov model-based modeling, we show that organisms with CENs resembling those in S. cerevisiae (point CENs) are very closely related and that all contain a set of 11 kinetochore proteins not found in organisms with complex CENs. Conversely, organisms with complex CENs (regional CENs) contain proteins seemingly absent from point-CEN organisms. However, at least three quarters of known kinetochore proteins are present in all fungi regardless of CEN organization. At least six of these proteins have previously unidentified human orthologs. When fungi and metazoa are compared, almost all have kinetochores constructed around Spc105 and three conserved multi-protein linker complexes (MIND, COMA, and the NDC80 complex).ConclusionOur data suggest that critical structural features of kinetochores have been well conserved from yeast to man. Surprisingly, phylogenetic analysis reveals that human kinetochore proteins are as similar in sequence to their yeast counterparts as to presumptive Drosophila melanogaster or Caenorhabditis elegans orthologs. This finding is consistent with evidence that kinetochore proteins have evolved very rapidly relative to components of other complex cellular structures.

Polo-like Kinase 1 Regulates Nlp, a Centrosome Protein Involved in Microtubule Nucleation

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

A dual role for Bub1 in the spindle checkpoint and chromosome congression

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore–microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live‐cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore–microtubule attachments.

The centrosome cycle

The centrosome is the major microtubule‐organizing center of animal cells. It influences cell shape and polarity and directs the formation of the bipolar mitotic spindle. Numerical and structural centrosome aberrations have been implicated in disease, notably cancer. In dividing cells, centrosomes need to be duplicated and segregated in synchrony with chromosomes. This centrosome cycle requires a series of structural and functional transitions that are regulated by both phosphorylation and proteolysis. Here we summarize recent information on the regulation of the centrosome cycle and its coordination with the chromosomal cell cycle.

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.

VHL loss causes spindle misorientation and chromosome instability

Error-free mitosis depends on fidelity-monitoring checkpoint systems that ensure correct temporal and spatial coordination of chromosome segregation by the microtubule spindle apparatus. Defects in these checkpoint systems can lead to genomic instability, an important aspect of tumorigenesis. Here we show that the von Hippel-Lindau (VHL) tumour suppressor protein, pVHL, which is inactivated in hereditary and sporadic forms of renal cell carcinoma, localizes to the mitotic spindle in mammalian cells and its functional inactivation provokes spindle misorientation, spindle checkpoint weakening and chromosomal instability. Spindle misorientation is linked to unstable astral microtubules and is supressed by the restoration of wild-type pVHL in pVHL-deficient cells, but not in naturally-occurring VHL disease mutants that are defective in microtubule stabilization. Impaired spindle checkpoint function and chromosomal instability are the result of reduced Mad2 (mitotic arrest deficient 2) levels actuated by pVHL-inactivation and are rescued by re-expression of either Mad2 or pVHL in VHL-defective cells. An association between VHL inactivation, reduced Mad2 levels and increased aneuploidy was also found in human renal cancer, implying that the newly identified functions of pVHL in promoting proper spindle orientation and chromosomal stability probably contribute to tumour suppression.