Characterizing a novel hypoxia cell-cycle checkpoint in Cryptococcus

Like many microbes S. cerevisiae cells will grow in liquid culture until they utilize all the nutrientswhen they cease dividing as unbudded cells in G1 and enter stationary phase. The pathogenicbudding yeast Cryptococcus neoformans also arrests when cells grow to high density but the cellsarrest as unbudded G2 cells with fully replicated DNA. This is an unusual arrest point for stationaryphase and experiments demonstrate the C. neoformans cells arrest in response to low [O2] ratherthan depletion of nutrients. Cells reproducibly arrest at the same [O2] levels (approximately 6%)regardless of the starting conditions. As cells approach the saturation arrest and deplete O2 budemergence is inhibited in early S phase and cells dont bud until G2 phase. This arrest point isreminiscent of a checkpoint arrest observed in S. cerevisiae cells when budding is inhibited. Themorphogenesis checkpoint prevents unbudded cells from undergoing mitosis and generatingpolyploid multinucleate cells. We hypothesize that in saturated cultures of C. neoformans low [O2]inhibits budding and triggers a morphogenesis checkpoint or directly activates a checkpoint thatarrests cells in G2. In response to various intracellular signals some checkpoints including themorphogenesis checkpoint act to inhibit mitotic cyclin/CDK complexes to prevent entry into mitosis.Inhibition of these complexes is regulated by the Wee1 kinase that phosphorylates inhibitory residueson CDK and the Cdc25 phosphatase which dephosphorylates these sites. The short-term goals ofthis proposal are: i) to characterize changes in cell-cycle events during transition from log-phasegrowth to saturation arrest to provide clues to what defects might be associated with triggering acheckpoint arrest; ii) identify changes in the cell-cycle machinery that might impact bud emergenceand checkpoint arrest in G2; iii) evaluate deletion mutants of the C. neoformans orthologs WEE1 andCDC25 as well as orthologs of other known checkpoint kinases for their role in the saturation/hypoxiaarrest. These initial studies will set the stage for the long-term goal of identifying the completemolecular mechanism for the hypoxia checkpoint from the initial molecules that sense low [02] to themachinery controlling bud emergence and mitotic arrest. As checkpoints normally protect cells fromdividing when cell-cycle events or the genome is perturbed these studies are key for understandinghow C. neoformans cells proliferate in low oxygen environments. Moreover understanding thecomplete molecular pathways that drive cell-cycle arrest may provide ideas for novel antifungalstrategies that target checkpoint components.