The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B–Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B–Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B–Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B–Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B–Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.

BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1. Bidirectional microtubule-based transport is responsible for the positioning of a large variety of cellular organelles, but the molecular mechanisms underlying the recruitment of microtubule-based motors to their cargoes and their activation remain poorly understood. In particular, the molecular players involved in the important processes of nuclear and centrosomal positioning prior to the onset of cell division are not known. In this study we focus on the function of one of the mammalian homologues of Drosophila Bicaudal D, an adaptor for the microtubule minus-end-directed dynein-dynactin motor complex. Previously, Drosophila Bicaudal D and its mammalian homologues were shown to act as linkers between the dynein motor and mRNP complexes or secretory vesicles. Here, we identify a new cargo for mammalian Bicaudal D2 (BICD2)–the nucleus. We show that BICD2 specifically binds to nuclear pore complexes in cells in G2 phase of the cell division cycle. We also show that this interaction is required for G2-specific recruitment of dynein to the nuclear envelope and thus for proper positioning of the nucleus relative to centrosomes prior to the onset of mitosis. Further, our findings demonstrate that the motor protein kinesin-1 opposes dynein's activity during this process and requires BICD2 for its activity. Our study therefore reveals BICD2 as the critical molecular adaptor that allows molecular motors to regulate nuclear and centrosomal positioning before cell division.

Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression. When active, the enzyme cyclin B1–cyclin-dependent kinase 1 (Cdk1) commits a growing cell to the process of mitotic cell division and chromosome separation. Cyclin B1–Cdk1 activation is controlled in many ways, but once its activity rises above a certain level, further activation of cyclin B1–Cdk1 is catalyzed by a positive-feedback loop. This generates highly active cyclin B1–Cdk1 and triggers the start of mitosis, which can only be completed when cyclin B1–Cdk1 activity is properly shut off again. However, it is not clear how cyclin B1–Cdk1 activity develops in human cells or how the switch between its inactive and active states is controlled. Our work combines activation measurements with a kinetic model to study how cyclin B1–Cdk1 activity accumulates just before and during mitosis. We show that cyclin B1–Cdk1 activity develops gradually in early mitosis and that different activity levels are required for initiation of, and progression through, mitosis. We also demonstrate that once cyclin B1–Cdk1 activation is truly launched, it is bound to continue and will not lightly drop back again. We propose that the successive cyclin B1–Cdk1 activity levels by themselves may coordinate the progression through the distinct phases of mitosis.

Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.