The Medema group has been studying cell cycle checkpoints for several years, with particular emphasis on mitotic checkpoints. The main goal is to understand the mechanisms that ensure chromosome stability in healthy cells, and how this quality control mechanism is altered in cancer.
The newly acquired insights should help us understand if we can take advantage of this defect in cancer cells to develop novel anti-cancer strategies. The main lines of research in the laboratory are:
Several years ago our lab reported that Plk1 is a target of the DNA damage checkpoint and that its inhibition is essential to enforce the G2 checkpoint (Smits et al., 2000, Nat. Cell Biol. 2: 672). However, the exact role of Plk1 during the cellular response to DNA damage was unclear. Others had reported that the Plk1 homolog Cdc5 in S. cerevisiae is required for checkpoint adaptation in cases the damage can not be repaired. Therefore, we set out to investigate the requirements for Plk1 during recovery from a checkpoint arrest in mammalian cells.
We found that Plk1 is essential for checkpoint recovery, while non-damaged cells do not require Plk1 for mitotic entry (van Vugt et al., 2004, Mol. Cell 15: 799). Furthermore, we could identify Wee1 as the crucial target of Plk1 during checkpoint recovery. Subsequently, we could show that Plk1 also acts to terminate the checkpoint signal, by promoting the degradation of Claspin (Mamely et al., 2006, Curr. Biol. 16: 1950), which acts to facilitate Chk1 activation by the ATR checkpoint kinase. Most recently, using a FRET-based biosensor for Plk1, we have been able to demonstrate that Plk1 is activated several hours prior to mitotic entry, both during a normal cell cycle, as well as during checkpoint recovery (Macůrek et al., 2008, Nature, 455: 119). We could show that this activation occurs via phosphorylation of Threonine-210 in the T-loop of Plk1. This initial activation of Plk1 is mediated by Aurora A, in combination with Bora, a previously identified cofactor for Aurora A. Both Bora and Aurora A are required for checkpoint recovery. Currently we are trying to understand how Aurora A is activated during recovery, and how exactly Aurora A/Bora and Plk1 interact.
In addition, we are currently carrying out reverse genetic screens to identify additional components of this recovery pathway, and use live cell microscopy to determine the sequential events that lead to checkpoint recovery. Using this approach, we recently identified the phosphatase Wip1 (PPM1D) as a factor that maintains a cell competent for cell-cycle re-entry during an ongoing DNA damage response in G2 (Lindqvist et al., 2009, EMBO J. 28: 3196). We could show that Wip1 function is required throughout the arrest, and that Wip1 acts by antagonizing p53-dependent repression of crucial mitotic inducers, such as Cyclin B and Plk1. These findings uncover Wip1 as a first in class recovery competence gene, and suggest that the principal function of Wip1 in cellular transformation is to retain proliferative capacity in the face of oncogene-induced stress.
At the onset of mitosis, duplicated centrosomes migrate around the nucleus, so that the DNA is positioned between the two centrosomes at the time of nuclear envelope breakdown (NEB). At NEB, the two centrosomes nucleate a large array of microtubules that together with microtubules nucleated around the chromosomes form a bipolar spindle. During spindle assembly, microtubules interact with chromosomes and align the chromosomes to the metaphase plate. Once all chromosomes are correctly positioned, the mitotic spindle segregates the two sets of sister chromosomes to the two daughter cells. Although these events were already described over 100 years ago, the molecular mechanisms controlling spindle assembly and chromosome movement are still largely unknown.
To understand how chromosomes are correctly segregated during mitosis, we are studying the molecular mechanisms that control the mitotic spindle. We are focusing on two types of proteins known to be involved in spindle assembly; microtubule-associated proteins, which control many different aspects of the spindle, including microtubule stability and attachment to chromosomes and microtubule-dependent motor proteins, which can generate forces required for spindle assembly and chromosome movement. Using a high-throughput automated RNAi screening platform, we identified RAMA1 as a novel regulator of spindle assembly (Raaijmakers et al., 2009, J.Cell Sci. 122: 2436). Depletion of RAMA1 results in severe chromosome alignment defects and a checkpoint-dependent mitotic arrest. We could show that this was due to reduced kinetochore-microtubule attachments, indicating that RAMA1 may have a direct role in mediating kinetochore-microtubule interactions.
In addition, we have shown that dynein/CLIP-170 and Lis1 produce an inward force that counteracts the outward force produced by the microtubule-dependent motor protein Eg5 (Tanenbaum et al., 2008. EMBO J. 27: 3235). More recently, we could show that kinesin12 or Kif15, another microtubule-dependent motor can take over all of the essential functions of Eg5 during spindle assembly (Tanenbaum et al., 2009, Curr. Biol. 19: 1703). We are currently trying to resolve how the function of these different kinesins is regulated in space and time.
We have found that the forkhead transcription factor FoxM1 regulates expression of a large group of G2-specific genes (Laoukili et al., 2005). We could show that depletion of FoxM1 leads to frequent errors in chromosome segregation and failed cytokinesis. We could show that FoxM1 transcriptional activity is kept inactive during the G(1)/S transition through the action of the N-terminal autorepressor domain, while phosphorylation by cyclin A/cdk complexes during G(2) results in relief of inhibition by the N terminus, allowing activation of FoxM1-mediated gene transcription. We are currently addressing the role of FoxM1-mediated transcription during the DNA damage response in G2.