Abstract
Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved.
Highlights
To proliferate, mammalian cells copy their genome during S phase and divide the two copies between two daughter cells during mitosis (M phase)
Cell cycle progression is controlled by cyclin-dependent kinases (CDKs), which are activated by the appropriate cyclin proteins and by the interplay between activating and inhibitory kinases and phosphatases
While 3 h RO-3306 treatment before mitosis improved bipolar spindle maintenance in control-depleted cells, this effect was all but abolished in cells depleted of HURP (Fig 4A and 4B; control siRNA, dimethyl sulfoxide (DMSO), 69.2 ± 4.7% monopolar; control siRNA, RO3306, 42.5 ± 4.8% monopolar; HURP siRNA, DMSO, 100 ± 0% monopolar; HURP siRNA, RO-3306, 97.6 ± 1.0% monopolar; n61), suggesting that high K-MT stability is required for RO-3306-induced bipolar spindle maintenance
Summary
Mammalian cells copy their genome during S phase and divide the two copies between two daughter cells during mitosis (M phase). S and M phases of cultured mammalian cells have intervening G1 and G2 phases. Cell cycle progression is controlled by cyclin-dependent kinases (CDKs), which are activated by the appropriate cyclin proteins and by the interplay between activating and inhibitory kinases and phosphatases. CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules [1, 2]. At the G2/M transition, the activity of CDK-1 coupled with cyclin B controls mitotic entry and progression [3]. Because CDK-1 activity is thought to be switch-like and sudden at the onset of mitosis [4], CDK-1 inhibition by small molecule inhibitors is often used to synchronize cells before entry into mitosis [5]
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