Understanding how cells trigger mitosis

We hope to discover exactly how the cell makes the decision to divide into two. This is a crucial decision because when it goes wrong the two daughter cells that are formed can go on to cause cancer, or become resistant to current anti-cancer drugs. In the course of this research we hope to uncover new targets for anti-cancer drugs and to identify ways to make current anti-cancer drugs much more effective.

We aim to understand what controls the entry to mitosis in human cells. Although many important regulators have been identified it is not known whether we have identified all the necessary factors, nor how they act on each other to make the final decision to enter mitosis. This question is important to the understanding and treatment of cancer because when cells enter mitosis with damaged DNA this can lead to chromosome breakage or loss and consequent aneuploidy, which can clearly contribute to the development of cancer. To provide a definitive answer to this problem we will combine single cell imaging with cell engineering and biochemistry to determine: how Cyclin A promotes mitosis; and how the decision to activate Cyclin B1-Cdk1 is finally made; and how the cell cycle machinery is coordinated in throughout the cell. By mass-spectrometry we have already identified the proteins specifically associated with Cyclin A and Cyclin B1 at each stage of the cell cycle, which has revealed a number of candidates for proteins that could be involved in the control of mitosis and will be pursued. Single cell imaging will provide the necessary temporal resolution to determine when and how cells enter mitosis and we will use FRET probes for CyclinA-Cdk and for Cyclin B1-Cdk1 activity to assay both the exact time when each kinase is turned on, and the kinetics with which it is activated in living cells. In addition to transgenic mouse embryos, recent advances in gene targeting in human cells now allow us to combine the resolution of single cell imaging with biochemical analysis in defined genetic backgrounds. Using gene targeting we will introduce epitope tags and point mutations into specific genes to generate cell lines containing analogue-sensitive kinases, conditionally unstable proteins, and epitope-tagged proteins that will enable us both to assay their level and localisation in living cells and to identify their interaction partners and post-translational modifications by mass spectroscopy. The analogue-sensitive kinases and conditionally unstable proteins will enable us specifically to inactivate a chosen protein at a defined point in the cell cycle, using an ATP-analogues for kinases, and either Cre-mediated excision or an inducible-degron for other proteins, such as phosphatases. With this multi-pronged approach we will be able to identify the molecular mechanisms that control entry to mitosis and how this is coordinated with the unreplicated DNA and damaged DNA checkpoint pathways.