Global Cellular Controls in Fission Yeast

The major focus of the proposal is how CDKs ensure orderly progression through the cell cycle. The levels of potential CDK regulators through the cell cycle and their association with chromatin and the SPB will be determined to investigate cell size and ploidy monitoring mechanisms. The principle that cell cycle temporal ordering is established by differential CDK substrate sensitivity will be tested through multiplexed in vitro CDK and in vivo CDK assays, and by investigating different CDKs and CDK accessory proteins. The influence of cellular localisation on CDK activity will be examined by altering CDK location and through activity localisation assays. Using an in vivo single cell assay, the dynamics of CDK activity and its consequences on orderly cell cycle progression will be assessed and potential positive feedback mechanisms investigated. The phosphorylation changes that occur after TOR pathway inhibition will be measured at fine temporal resolution and correlated with changes in rates of RNA and protein synthesis, to investigate how overall cellular rates of synthesis are regulated. The mechanisms underpinning nuclear size homeostasis will be explored by testing the hypothesis that a major role is played by barrier membrane proteins that regulate membrane flow between organelles.