Primary Supervisor: Dr Masanori Mishima, Warwick Medical School
Secondary supervisor: Jonathan Millar
PhD project title: A synthetic biology approach to dissect the post-anaphase functions of mitotic kinases
University of Registration: University of Warwick
Cell division is essential for life. The complex mechanochemical processes of chromosome segregation and cytokinesis are coordinated by the group of multifunctional protein kinases (mitotic kinases) such as Aurora kinases and Polo kinases under the master regulation of the cyclin-dependent kinases. They dynamically change their localization to different parts of cell division machinery depending on the stage of cell division and regulate various subprocesses of cell division by phosphorylating various target proteins. For example, Aurora B kinase controls the chromosome-kinetochore attachment at the centromere before anaphase onset, initiation of cytokinesis at the spindle midzone during anaphase, and final separation of the daughter cells (abscission) at the midbody. The defects of mitotic kinases can cause abnormal cell division, which leads to aneuploidy, chromosomal instability, and cancer. Since mitotic kinases work primarily in the proliferating cells, their chemical inhibitors have been under clinical trial for cancer chemotherapy.
We have been studying the regulation of cytokinesis by mitotic kinases in mammalian cells and C. elegans embryos, focusing on the phosphoregulation of centralspindlin, a key regulator of cytokinesis (refs. 1-3). However, the multi-functionality of the mitotic kinases makes this problematic. In the case of Aurora B, its full depletion/inhibition results in the prometaphase arrest and, eventually, the exit from mitosis without proper chromosome segregation and cytokinesis. Although we can still assess its function in cytokinesis by observing the phenotypes of the partial depletion or weak mutants, interpretation of such experiments can't be very clearcut. A possible approach to overcome this difficulty is to treat cells with specific chemical inhibitors after the chromosome segregation starts at the onset of anaphase. However, this still suffers from a limitation that synchronization of the cell division, which itself can interfere with the normal progression of cell division, is necessary for the analysis of a large population of the cells (eg. biochemistry).
Recently, our lab has been investigating synthetic biology approaches to render cells sensitive to a kinase inhibitor in a manner specific to the stages of cell division, especially post-anaphase stages. A strategy that seems to be promising for Aurora B is to combine a chemical inhibitor, a known drug-resistant mutation of Aurora B, and a peptide motif that induces protein degradation (degron) during anaphase. The degron-tagged, drug-resistant version of Aurora B can make the cell insensitive to the chemical inhibitor only before anaphase onset, but not after this as it disappears due to the degradation.
The first aim of the project is to optimize the design of the synthetic construct and the method of its expression in mammalian cultured cells. The goal is to establish a combination of a chemical inhibitor and a cell line suitable for a CRISPR-based genome-wide knock-out screen for genetic modifiers of Aurora B important for cytokinesis. The second aim is to extend this approach to other mitotic kinases such as Polo kinases. These will provide us with effective platforms for a better understanding of the regulatory mechanisms of the later cell division events by mitotic kinases.
- Douglas, M.E., Davies, T., Joseph, N., and Mishima, M. (2010). Aurora B and 14-3-3 coordinately regulate clustering of centralspindlin during cytokinesis. Curr. Biol. 20, 927–933. PMID: 20451386
- Joseph, N., Hutterer, A., Poser, I., and Mishima, M. (2012). ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration. Embo J 31, 2604–2614. PMID: 22580824
- Mishima, M. (2016). Centralspindlin in Rappaport's cleavage signaling. Semin. Cell Dev. Biol. 53, 45–56. PMID:26964770
BBSRC Strategic Research Priority: Understanding the Rules of Life: Systems Biology & Integrated Understanding of Health: Pharmaceuticals
Techniques that will be undertaken during the project:
- Molecular cell biology
- Live microscopy including super-resolution techniques
- Image analysis
- Structural bioinformatics
Contact: Dr Masanori Mishima, University of Warwick