Director of Research, The Wellcome Trust/CR UK Gurdon Institute
Tel.: +44 (0)1223 334096
Regulation of mitosis in mammalian cells
We are studying how cells divide and focusing on two main aspects of cell division: how the cell first initiates mitosis, and how the cell co-ordinates mitosis by ubiquitin-mediated proteolysis via the APC/C ubiquitin ligase. Because mitosis is a highly dynamic process we are studying these processes in real time by time-lapse fluorescence microscopy. We use FRAP and photo-activation to gain a better understanding of the kinetics of protein behaviour, deconvolution to improve the spatial resolution and FRET to assay protein-protein interaction and kinase activity.
To understand how cells first initiate mitosis we are analysing the behaviour of the mitotic cyclin-CDKs, cyclins A and B1, and their regulation by phosphorylation and subcellular localisation. We use GFP-fusion proteins to determine how localisation is altered depending on the stage of the cell cycle, and to define the domains of the proteins that target them to specific subcellular structures. To identify the proteins responsible for targeting we are analysing protein complexes by mass spectrometry.
To understand how proteolysis is used to regulate mitosis we assay the degradation of the GFP-fusion proteins in living cells. We are studying the destruction of key mitotic regulators at each stage of mitosis to define the events and the mechanisms that trigger the destruction of specific proteins at specific times, and how this coordinates chromosome segregation and cytokinesis. We are using mass spectroscopy to study how the APC/C ubiquitin ligase is regulated in mitosis, and are investigating whether it is spatially regulated in mitosis; in particular whether this underlies the exquisite control of protein degradation by the spindle assembly checkpoint.
- Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J. and Pines, J. (2011) 'APC15 drives the turnover of MCC-Cdc20 to make the Spindle Assembly Checkpoint responsive to kinetochore attachment' Nat. Cell Biol. 13, 1234-1244.
- Pagliuca, F., Collins, M.O., Lichawska, A., Zegerman, P., Choudhary, J.S. and Pines, J. (2011) ‘Quantitative Proteomics Reveals the Basis for the Biochemical Specificity of the Cell Cycle Machinery’ Mol Cell, 43, 406-417.
- Izawa, D. and Pines, J. (2011) ‘Evidence for how APC/C-Cdc20 changes its substrate specificity in mitosis’ Nat. Cell Biol. 13, 223-233.
- Di Fiore, B. and Pines, J. (2010) ‘How Cyclin A destruction escapes the Spindle Assembly Checkpoint’. J. Cell Biol. 190, 501-509.
- Gavet, O. and Pines, J. (2010) ‘Progressive activation of Cyclin B1-Cdk1 coordinates entry to mitosis’ Dev. Cell 18, 533-543.
- Gavet, O. and Pines, J. (2010) ‘Activation of Cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis’ J. Cell Biol. 189, 247-259.
- Nilsson, J., Yekezare, M., Minshull, J. and Pines, J. (2008) ‘The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction.’ Nat. Cell Biol. , 10, 1411-1420.
- Di Fiore B, and Pines J (2007). ‘Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C.’ J. Cell Biol. 177, 425-437.
- Acquaviva C, Herzog F, Kraft C and Pines J (2004) The Anaphase Promoting Complex/Cyclosome is recruited to centromeres by the spindle assembly checkpoint. Nature Cell Biology 6, 892-898
- Matsusaka T, and Pines J (2004) Chfr acts with the p38 stress kinase to block entry to mitosis in mammalian cells. J Cell Biol 166, 507-516
- Lindon C and Pines J (2004) Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells. J Cell Biol 164, 233-241
- Jackman M, Lindon C, Nigg EA and Pines J (2003) Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nature Cell Biol 5, 143-148