Abstract. During mitosis in Ptkl cells anaphase is not initiated until, on average, 23 +_

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Abstract. To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK t cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r 2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle.We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment-well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.T HE transition from metaphase to anaphase is a key cell cycle event that commits the cell to exit mitosis and enter a new interphase (reviewed in Murray, 1992;Sluder and Rieder, 1993). Since the equal segregation of chromosomes at mitosis is predicated on each acquiring a bipolar attachment to the spindle before anaphase onset, the metaphase-anaphase transition must be tightly coordinated with proper completion of chromosome attachment. Ensuring the essential coordination of these events would be relatively straightforward if the mitotic portion of the cell cycle was fixed in duration, but sufficiently long so that all chromosomes had time to acquire a proper attachment before disjoining. However, the stochastic nature of spindle formation makes chromosome attachment an unpredictable and errorprone process. In vertebrate cells a chromosome first attaches when one of its kinetochores, usually the one closest to and facing a spindle pole at nuclear envelope breakdown

Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing gamma-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures-distinct from elements of the microtubule-organizing center-that is required for the somatic cell cycle to progress through G1 into S phase. Once the cell is in S phase, these core structures are not needed for the G2-M phase transition.

Lambrus et al. show that centrosome loss or a prolonged mitosis activates a USP28–53BP1–p53–p21 signaling axis that prevents the growth of cells with an increased propensity for mitotic errors.

Summary The mitotic checkpoint maintains genomic stability by blocking the metaphase-anaphase transition until all kinetochores attach to spindle microtubules [1, 2]. However, some defects are not detected by this checkpoint. With low concentrations of microtubule targeting agents the checkpoint eventually becomes satisfied though the spindles may be short and/or multipolar[3, 4] and the fidelity of chromosome distribution and cleavage completion are compromised. In real life, environmental toxins, radiation, or chemotherapeutic agents may lead to completed but inaccurate mitoses. Once the checkpoint is satisfied and cells divide it has been assumed that the daughter cells would proliferate regardless of prometaphase duration. However, when continuously exposed to microtubule inhibitors, untransformed cells eventually slip out of mitosis after 12-48 hours and arrest in G1[5-8] (also see [9]). Interestingly, transient but prolonged treatments with nocodazole allow completion of mitosis but the daughter cells arrest in interphase [10, 11](also see [9, 12]). We characterize the relationship between prometaphase duration and the proliferative capacity of daughter cells. Our results reveal the existence of a mechanism that senses prometaphase duration; if prometaphase lasts >1.5 hours, this mechanism triggers a durable p38 -p53 dependent G1 arrest of the daughter cells despite normal division of their mothers.

No abstract

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting “maternal” centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: ∼5–8 h after ablation, clouds of pericentriolar material (PCM) containing γ-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2–14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.