We study how the cell biology of mitosis is co-ordinated with cell cycle progression. The spindle checkpoint normally prevents cells with spindle or kinetochore defects from initiating chromosome segregation. Mutations in the MAD (mitotic arrest defective) or BUB (budding uninhibited by benomyl) genes inactivate the checkpoint and allow cells with defective spindles to proceed through mitosis. Such cell divisions lead to inaccurate chromosome segregation, aneuploidy and
death.
A single unattached kinetochore is sufficient to activate the spindle checkpoint, and we are particularly interested in how such kinetochores generate signals that delay
anaphase onset throughout the mitotic apparatus.Mad3 and Mad2 interact with Cdc20 to form the mitotic checkpoint complex (MCC) which is crucial for inhibition of the anaphase-promoting complex (APC), and thereby delaying the metaphase to anaphase transition. There appear to be several distinct modes of Cdc20-APC inhibition by checkpoint proteins, including direct Cdc20- APC binding and proteolytic turnover of Cdc20.
A major focus of our current research is to identify important substrates of the Mps1, Bub1 and Aurora (Ark1) kinases. These protein kinases localise to kinetochores where they act in concert to ensure efficient chromosome bi-orientation. Mph1 kinase activity is critical for spindle checkpoint arrest (Fig. 1A) and we have found that it phosphorylates both Mad2 and Mad3. In certain situations the chromosomal passenger proteins, including Aurora B kinase (Ipl1 in budding yeast and Ark1 in fission yeast), are also required for spindle checkpoint arrest. Ipl1-dependent phosphorylation of Mad3 is necessary to delay anaphase onset in response to lack of tension at budding yeast kinetochores. Ark1 activity is required to maintain spindle checkpoint arrests in fission yeast, even in situations where there are many unattached kinetochores. Inhibition of Ark1 leads to APC activation and degradation of cyclin B within a few minutes (see Fig. 1B).
We developed a novel spindle checkpoint silencing assay, and screened for downstream factors that are necessary for cyclin B degradation when Ark1 kinase is inhibited. Upon testing fission yeast protein phosphatases we found that the kinetochore-localised PP1 (Dis2) is critical for cyclin destruction (see Fig. 1C). We recently identified two key recruitment factors for PP1-mediated checkpoint silencing, the kinetochore protein Spc7 (KNL1/Blinkin/Spc105) and the Kinesin-8 family members Klp5 and 6.
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Selected publications:
Meadows, J.C., Shepperd, L.A., Vanoosthuyse, V., Lancaster, T.C., Sochaj, A.M., Buttrick, G.J., Hardwick, K.G., and Millar, J.B.A. (2011) Spindle checkpoint silencing requires association of PP1 to both Spc7 and Kinesin-8 motors. Developmental Cell,
20, 739-750.
Vanoosthuyse, V. and Hardwick, K.G. (2009). A novel Protein Phosphatase 1-dependent spindle checkpoint silencing mechanism. Current Biology, 19, 1176-81.
King, E.M.J., Rachidi, N., Morrice, N., Hardwick, K.G., and Stark, M.J.R. (2007). Ipl1p-dependent phosphorylation of Mad3p is required for the spindle checkpoint response to lack of tension at kinetochores. Genes and Development, 21, 1163-8.
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