A Possible Conserved Role for SR Protein Kinases in the Regulation of Cell Death (thesis)
McGhee, Virginia Elizabeth
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Apoptosis, programmed cell death, is a process that mammalian cells frequently undergo to suppress tumors and regulate cell growth. It is unclear whether or not apoptosis is conserved across both multicellular and unicellular eukaryotes. In baker's yeast, Saccharomyces cerevisiae, specific stress conditions can cause nuclear DNA fragmentation and cytoplasmic release of reactive oxygen species (ROS), the molecular markers of apoptosis in mammalian cells [1,2]. However, it is unclear whether these markers are part of a fungal specific stress response pathway or indicate that the machinery controlling cell death is conserved. One complication to resolving this question has been that the probes used to quantify DNA fragmentation and ROS levels are usually used to stain fixed cells. This means cells are only observed at a fixed point in time and the fate of a cell following appearance of apoptotic markers cannot be tracked. We are unable to confirm yeast cell death without observing the cells after the markers of cell death appear because we do not know if the presence of ROS is indicative of a stress response or cell death pathway. To address this we developed a novel technique using live-cell confocal microscopy to quantify the levels of ROS in yeast grown in different stress conditions. Preliminary data show that once ROS appear in a cell it never buds again over a 10-hour time course. This correlation between expression of the mammalian apoptotic markers and loss of cell viability in yeast suggest that this is a cell death pathway. Here, we focus on a potentially conserved role for SR protein kinases in the regulation of apoptosis in yeast and mammals using this system. Previous work has demonstrated a role for the mammalian SRPK2 in apoptosis. Specifically, SRPK2 appears to be cleaved in a caspase dependent system; the N-terminus then translocates to the nucleus where it may be involved in a nuclear apoptotic pathway . Yeast encode for a single SR protein, Sky1 . In response to cation toxicity, such as LiCl, we see induction of apoptosis markers in yeast. Interestingly, the deletion of SKY1 reduces the presence of the molecular markers of apoptosis compared to wild-type (WT) in both standard (1% and 2% respectively) and stress conditions (3% and 14% respectively) . Consistent with this, cells that carry a sky1[delta] grow better than WT under stress conditions; moreover, cells that have constructs that overexpress SKY1 grow much worse under stress conditions. Using our live-cell microscopy assay, we quantify and compare release of ROS in WT to sky1[delta] constructs and WT to sky1-overexpression constructs. Furthermore, we are investigating whether the cleavage of Sky1 is conserved by using yeast with N- and C-terminal fluorescent tags on Sky1. Determining that these results are representative of an apoptotic cell death pathway in yeast opens the door to using this genetically facile model organism to further probe the molecular mechanics of apoptosis.