Dissecting the roles of Polo and Aurora kinases during cytokinesis. Animal growth, development and reproduction depend on the accurate process of cell division, which faithfully partitions the genomic information between the two daughter cells. The overarching aim of this project is to dissect the functions of two mitotic kinases – Polo-like kinase 1 (Plk1) and Aurora-B – during the last phase of cell division, cytokinesis. Plk1 and Aurora B play multiple roles during mitosis, including spindle assembly, centrosome maturation, chromosome condensation, alignment, and segregation (1, 2). Because of these early mitotic functions, it has so far been difficult to dissect the roles of these kinases during cytokinesis. To overcome this limitation, the student will employ a combination of chemical genetics, mass spectrometry, time-lapse imaging and drug treatments to investigate the possible cross talk between these two kinases and identify their targets during cytokinesis.
The specific aims of this project are:
- Characterise the localisation patterns of Plk1 and Aurora B during cytokinesis, using both immuno-fluorescence and time-lapse imaging. Plk1 and Aurora B specific inhibitors will also be used to investigate the localisation dependency of these two kinases, including their active forms, and their effects on the distribution of other cytokinesis proteins.
- Initial studies have indicated that Plk1 and Aurora B localisations are slightly different, leading to the hypothesis that their distribution might be functionally relevant. To address this, we will engineer a Plk1 mutant able to mimic Aurora B localisation and vice versa and then study the effects of these Plk1 and Aurora B variants on cytokinesis.
- To identify substrates phosphorylated by Plk1 and Aurora B specifically during cytokinesis in vivo, we propose to employ a chemical genetic screen that has already been successfully used to identify AMPK targets (3). We will engineer the Plk1 and Aurora B kinase domains in order to enable these mutants to accept ‘bulky’ ATP analogues. We will then express these mutants in HeLa cells and after synchronisation in telophase we will incubate them with ‘bulky’ ATPS. Thio-phosphorylated substrates will be immuno-precipitated using a thiophosphate ester-specific antibody and identified by mass spectrometry.
- V. Archambault, D. M. Glover. Nat Rev Mol Cell Biol 10, 265-275 (2009)
- S. Ruchaud, et al. Nat Rev Mol Cell Biol 8, 798-812 (2007)
- M. R. Banko, et al. Mol Cell 44, 878-892 (2011)