How do cells regulate entry to mitosis? And, once in mitosis, how do cells coordinate chromosome alignment and segregation with cell division itself (cytokinesis) to ensure that the two daughter cells receive an equal and identical copy of the genome? The answer is the interplay between protein kinases and phosphatases, and ubiquitin-mediated proteolysis - in particular ubiquitination by the Anaphase Promoting Complex/Cyclosome (APC/C) - and this is the focus of our research. Because mitosis is a highly dynamic process we study living cells by time-lapse fluorescence microscopy and complement this with biochemical analyses.

Plk: Deconvolved images of Hela cells progressing through mitosis stained for Polo-like kinase 1 (green), tubulin (red) and DNA (blue). (Catherine Lindon).

To understand how cells 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 reveal the dynamics of protein localisation through the cell cycle, and to define how proteins are targeted to specific subcellular structures. To identify the proteins responsible for targeting the cyclins, and to provide insights into Cdk substrates, we analyse protein complexes by mass spectrometry. Recently, we have developed a biosensor to assay Cyclin B1-Cdk1 activity in vivo.

To understand how proteolysis regulates progress through mitosis we measure the degradation of GFP-fusion proteins in living cells and complement this with biochemical analyses of protein complexes and ubiquitination activity. These studies are revealing how the APC/C is activated, how it is able to select a particular protein for destruction at a specific time in mitosis and, most importantly, how its activity is regulated by the spindle assembly checkpoint that is essential to the control of chromosome segregation and cytokinesis. We hope that these studies will increase our understanding of how cells prevent improper chromosome segregation (aneuploidy) that is the hallmark of many cancers.

Montage of cyclin B1-Cdk1 kinase activity detected in mitosis using a novel FRET biosensor. (Red, high activity, green, low activity). (Olivier Gavet)

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:

• 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

• 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

• Walker A, Acquaviva C, Matsusaka M, Koop L and Pines J (2008) UbcH10 has a rate-limiting role in G1 phase but may not act in the spindle checkpoint or as part of an autonomous oscillator. J Cell Sci 121 2319-2326

• Pines J (2006) Mitosis: a matter of getting rid of the right protein at the right time. Trends in Cell Biology16, 55-63

A prophase cell stained for MCAK (green), microtubules (red) and DNA (blue). (Catherine Lindon)