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Analysis and re-wiring of cell cycle circuitry by genome engineering, modelling and quantitative imaging

Principal Supervisor: Prof. Jonathan Millar - Warwick Medical School

Co-supervisor: Prof. Bela Novak - University of Oxford

PhD project title: Analysis and re-wiring of cell cycle circuitry by genome engineering, modelling and quantitative imaging

University of Registration: University of Warwick

Project outline:

The faithful duplication and segregation of genetic material during the cell cycle is essential for life in all eukaryotes. Cell cycle progression is monitored by a series of surveillance mechanisms, called cell cycle checkpoints, that halt the cell cycle at specific points if errors in chromosome replication, repair of bi-orientation of sister chromatids is detected. Failure to arrest the cell cycle under these conditions causes a multitude of human pathologies including miscarriages, birth defects and proliferative diseases, such as cancer. Much of our understanding of the core machinery that drives the eukaryotic cell cycle has come from genetic analysis in the fission yeast Schizosaccharomyces pombe. Most of the components that have been identified are highly evolutionarily conserved. Indeed we now know three of the four key cell cycle transitions in all eukaryotes are caused by the rapid and irreversible activation of a heterodimeric cyclin dependent kinase (Cdk) complex, whereas the fourth is accompanied by rapid and irreversible Cdk inactivation. However, it remains unclear how these transitions are achieved at the cellular level. Sophistocated mathematical models to describe the fission yeast cell cycle have been developed by Novak and colleagues. However, such models can only approximate to the true regulatory mechanisms controlling cell cycle transitions in vivo as we lack detailed information as to cellular concentration and dynamic spatial re-arrangement of key cell cycle regulators and the full set of target proteins of cell cycle regulated kinases is still incomplete. More accurate models would, nevertheless, be of immense value for enabling the rapid prediction of the therapeutic potential of new drug treatments in silico so further work is merited.

In unpublished work Millar and colleagues have identified several new genetic elements that control the G1/S (START), G2/M (entry into mitosis) and M/A (metaphase to anaphase) transitions in fission yeast. The successful PhD student will identify and characterise these three new genetic elements using molecular genetics, genome sequencing, genome engineering, biochemistry and live cell fluorescence imaging. The student will use Stable Isotope labelling with amino-acids in cell culture (SILAC), phosphopeptide enrichment and liquid coupled-mass spectrometry (LC-MS/MS) and bioinformatics analysis of phosphopeptide datasets to understand how these new elements influence cell cycle regulated phosphorylation events in vivo. This information will be incorporated into existing or new algorithms to develop more sophisticated and accurate mathematical models of cell cycle behaviour in silico. The successful student will join two well-funded, enthusiastic and productive laboratories which will provide provided hands on training in all of the techniques required for the work. Additional funding is available to the student for travel and presentation of work at international conferences, for attending specialist advanced research and bioinformatics training workshops and participation in cross-departmental, regional and national cohort events with other doctoral training centre students.

References:

  1. Mora Santos, M., Hervas-Aguilar, A., Sewart, K., Lancaster, T.C., Hauf, S., Meadows, J.C. and Millar, JB (2016) Bub3-Bub1 binding to Spc7 (KNL1) toggles the spindle checkpoint switch by licensing interaction of Bub1 with Mad1-Mad2. Current Biology (in press)
  2. Silió V, McAinsh AD, Millar JB. (2015) KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores. Developmental Cell 35, 600-613
  3. Shepperd, L.A., Meadows, J.C., Sochaj, A.M., Lancaster, T.C., Rappsilber, J., Buttrick, G., Hardwick, K.G. and Millar, J.B.A. (2012) Phospho-dependent recruitment of Bub1 and Bub3 to Spc7 (KNL1) by Mph1 kinase maintains the spindle checkpoint. Current Biology 22, 891-899.
  4. Buttrick, G.J., Meadows, J.C., Lancaster, T.C., Vanoosthuyse, V., Shepperd, L.A., Hoe, K-L., Kim, D-U., Park, H-O., Hardwick, K.G. and Millar, J.B.A. (2011) Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B. Mol Biol Cell. 22, 4486-4502
  5. Meadows, J.C., Shepperd, L.A., Vanoosthuyse, V., Lancaster, T.C., Sochaj, A.M., Buttrick, G.J., Hardwick, K.G. and Millar, J.B. (2011) Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors. Developmental Cell 20, 739-750.
  6. Cundell MJ, Hutter LH, Nunes Bastos R, Poser E, Holder J, Mohammed S, Novak B, Barr FA. (2016) A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit. J Cell Biol. 29;214(5):539-54.
  7. Chica N, Rozalén AE, Pérez-Hidalgo L, Rubio A, Novak B, Moreno S. (2016) Nutritional Control of Cell Size by the Greatwall-Endosulfine-PP2A·B55 Pathway. Curr Biol. 8;26(3):319-30.
  8. Mirkovic M, Hutter LH, Novák B, Oliveira RA. (2015) Premature Sister Chromatid Separation Is Poorly Detected by the Spindle Assembly Checkpoint as a Result of System-Level Feedback. Cell Rep. 20;13(3):470-8.
  9. Kalantzaki M, Kitamura E, Zhang T, Mino A, Novák B, Tanaka TU. (2015) Kinetochore-microtubule error correction is driven by differentially regulated interaction modes. Nat Cell Biol. 17(4):421-33. Gérard C, Tyson JJ, Coudreuse D, Novák B. (2015) Cell cycle control by a minimal Cdk network. PLoS Comput Biol. 11(2):e1004056.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Molecular genetics and genome engineering Molecular biology and genome sequencing Biochemistry (for western blotting and immunoprecipitation) Stable Isotope labelling with amino-acids in cell culture (SILAC) Phosphopeptide enrichment and liquid coupled-mass spectrometry (LC-MS/MS) Bioinformatics of phosphopeptide datasets using novel algorithms. Nonlinear differential-equations for temporal dynamics of the regulatory networks Stochastic Simulation Algorithm (SSA) when number of molecules are low.

Contact: Professor Jonathan Millar, University of Warwick