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 nutrients, when they cease dividing as unbudded cells in G1 and enter stationary phase. The pathogenic budding yeast, Cryptococcus neoformans also arrests when cells grow to high density, but the cells arrest as unbudded G2 cells with fully replicated DNA. This is an unusual arrest point for stationary phase, and experiments demonstrate the C. neoformans cells arrest in response to low [O2] rather than 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, bud emergence is inhibited in early S phase and cells don’t bud until G2 phase. This arrest point is reminiscent of a checkpoint arrest observed in S. cerevisiae cells when budding is inhibited. The morphogenesis checkpoint prevents unbudded cells from undergoing mitosis and generating polyploid, 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 that arrests cells in G2. In response to various intracellular signals, some checkpoints, including the morphogenesis 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 residues on CDK, and the Cdc25 phosphatase which dephosphorylates these sites. The short-term goals of this proposal are: i) to characterize changes in cell-cycle events during transition from log-phase growth to saturation arrest to provide clues to what defects might be associated with triggering a checkpoint arrest; ii) identify changes in the cell-cycle machinery that might impact bud emergence and checkpoint arrest in G2; iii) evaluate deletion mutants of the C. neoformans orthologs, WEE1 and CDC25, as well as orthologs of other known checkpoint kinases for their role in the saturation/hypoxia arrest. These initial studies will set the stage for the long-term goal of identifying the complete molecular mechanism for the hypoxia checkpoint from the initial molecules that sense low [02] to the machinery controlling bud emergence and mitotic arrest. As checkpoints normally protect cells from dividing when cell-cycle events or the genome is perturbed, these studies are key for understanding how C. neoformans cells proliferate in low oxygen environments. Moreover, understanding the complete molecular pathways that drive cell-cycle arrest may provide ideas for novel antifungal strategies that target checkpoint components.
