How do cells regulate entry to mitosis? And, once in mitosis, how do cells coordinate chromosome segregation with cell division (cytokinesis) to ensure that the two daughter cells receive an equal and identical copy of the genome? The answer lies in the interplay between protein kinases, protein phosphatases, and APC/C-mediated proteolysis, and this is the focus of our research. Because mitosis is a highly dynamic process we study living cells by time-lapse fluorescence microscopy but to complement this with biochemical analyses we are using somatic cell recombination to knock-out and mutate specific mitotic regulators.This has given us remarkably accurate and precise in vivo kinetics for protein degradation.

To understand how cells initiate mitosis we are analysing the behaviour of the key mitotic kinases, the Cyclin A- and B-dependent kinases, and their regulation by phosphorylation and dephosphorylation.We recently developed a FRET biosensor to assay Cyclin B1-Cdk1 activity in vivo and are using this to define the pathways that regulate the timing of mitosis.To identify the proteins responsible for regulating the Cyclin-Cdks, and provide insights into Cyclin-Cdk substrates, we have analysed protein complexes through the cell cycle by SILAC mass spectrometry and are following up some of the exciting results from this screen.

To understand how proteolysis regulates progress through mitosis we complement the analysis of APC/C-dependent degradation in living cells with biochemical analyses of protein complexes and ubiquitination activity.These studies are revealing how the APC/C is activated and how it is able to select a particular protein for destruction at a specific time. Our research is also providing insights into how APC/C activity is regulated by the spindle assembly checkpoint that is essential to the control of chromosome segregation and cytokinesis. In particular, we are beginning to elucidate the key events in the checkpoint pathway and their antagonism by the APC/C itself.

Mass spectroscopy analysis reveals the dynamic interactions of the different cyclins through the cell cycle. Credit Felicia Walton-Pagiluca & Mark Collins (Sanger Institute)

Plain English:
We are trying to understand how one cell divides into two identical cells. It is particularly important that these two new 'daughter' cells receive the same set of genes because when this goes wrong, and one cell receives an extra or a damaged chromosome, this can lead to cancer. We now know that cells guard against problems in their division using particular proteins to block the next stage in the process, and it is only when the cell senses that everything is correct that these proteins are destroyed. This is the process that we are trying to understand, in essence: how does the cell destroy the right protein at the right time?

Selected publications:

• Mansfeld J, Collin P, Collins MO, 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 MO, Lichawska A, Zegerman P, Choudhary JS and Pines J (2011) Quantitative proteomics reveals the basis for the biochemical specificity of the cell cycle machinery. Mol Cell 43, 406-417.

• Gavet O and Pines J (2010) Progressive activation of Cyclin B1-Cdk1 coordinates entry to mitosis. Dev Cell 18, 533-543.

• 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

• Wolthuis R, Clay-Farrace L, van Zon W,Yekezare M, Ogink J, Medema R and Pines J (2008) Cdc20 and Cks direct the spindle checkpoint-independent destruction of Cyclin A. Mol Cell 30, 290-302