Temporal and spatial control of cyclin B1 destruction in metaphase.

The proteolysis of key regulatory proteins is thought to control progress through mitosis. Here we analyse cyclin B1 degradation in real time and find that it begins as soon as the last chromosome aligns on the metaphase plate, just after the spindle-assembly checkpoint is inactivated. At this point, cyclin B1 staining disappears from the spindle poles and from the chromosomes.

MPF localization is controlled by nuclear export.

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells.

Microtubule dependence of chromosome cycles in Xenopus laevis blastomeres under the influence of a DNA synthesis inhibitor, aphidicolin.

The spindle-assembly checkpoint of the cell cycle develops in Xenopus laevis embryos at the midblastula transition (MBT). Our previous experiments using animal-cap blastomeres indicate that the checkpoint is regulated by a mechanism that depends on age, but not on the nucleocytoplasmic (N/C) ratio (Clute and Masui, 1995). In the present study, the time of appearance of the spindle-assembly checkpoint is examined in animal-cap blastomeres whose N/C ratio is reduced by treatment with aphidicolin.

Regulation of the appearance of division asynchrony and microtubule-dependent chromosome cycles in Xenopus laevis embryos.

Divisions of animal-cap blastomeres dissociated from Xenopus laevis embryos are synchronous mostly up to 12th cleavage or the 13th cell cycle, but become asynchronous afterward, during the midblastula transition (MBT), and at the same time, chromosome cycles become microtubule-dependent and are arrested in mitosis if treated with nocodazole. To investigate causes for these changes in cell-cycle control, we observed division synchrony in animal-cap blastomeres dissociated from embryos whose nucleocytoplasmic ratio (N/C) had been altered by constriction of zygotes or by delaying nucleation into zygote halves and compared their mitotic indices in the presence and absence of nocodazole. Thus, we found that asynchronous divisions always commenced when N/C reached the value of 128 to 256 times that of an animal blastomere of the 32-cell embryo, corresponding to the 12th and 13th cycles of a normal embryo, while the number of synchronous cycles became variable, ranging from 9 to 14, depending on the initial N/C.

Development of Microtubule-Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos: (Xenopus/cell cycle/chromosomes/microtubutes/midblastula transition).

Animal-cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M-) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%.