Detecting reactive oxygen species in yeast at early growth points

Abstract

Reactive Oxygen Species (ROS) at low levels can act as important signaling molecules; however, when there is a surplus of ROS this can lead to harmful and or lethal results for the cell. ROS are primarily generated in the mitochondria of cells. This development occurs in the electron transport chain (ETC) which is embedded in the inner membrane of the mitochondria. Production of the mitochondrial superoxide anion (a type of ROS), occurs at redox active prosthetic groups, or electron carriers, bound to ETC proteins where kinetic factors favor O2 to becoming the superoxide anion. These kinetic factors can include the inhibition of ETC by small molecules. If an electron interacts “early” with O2 then superoxide will form, which could ultimately result in peroxides or more toxic hydroxyl radicals (HO•). Both proteins and small molecules are used within cells to mediate ROS levels and maintain redox states within these mechanisms to ensure ROS levels do not reach damaging levels. The accumulation of too much ROS can prove to be harmful to the organism. Mutations and dysfunctional molecules can result from ROS reacting with both nuclear and mitochondrial DNA, proteins, and lipids; however, more recent research shows that low levels of ROS provide important signaling mechanisms. Our interest is in how metal cofactors are incorporated into the ETC protein complexes of yeast and how misincorporation or modulation of available metals, such as copper and iron, in yeast mitochondria leads to the production of ROS and how under these conditions ROS changes during yeast lifespan. This project is particularly interested in how ROS changes “early” in yeast lifespan, meaning timepoints before yeast cells complete stationary phase and begin to enter the death phase. To detect the ROS superoxide, yeast cultures were grown in rich media for one to three days and then stained with dyhydroethidum (DHE). DHE is a fluorescent indicator for cytosolic superoxide, and cells were assayed utilizing both a fluorescence plate reader and FACS. To enhance superoxide production cells were also treated with the ETC inhibitor Antimycin A (a Complex II inhibitor), and the control inhibitor Oligomycin A (an ATPase inhibitor). Our results indicate that we are able to detect cellular superoxide using the fluorescent dye DHE and that cells cultured in the presence of the inhibitor Antimycin A have higher DHE fluorescence values compared to cells cultured in the presence of Oligomycin A. Since Antimycin A inhibits Complex III in the ETC, these results are consistent with ROS being generated from this complex. Our current work is to culture cells in supplemental copper ranging from no treatment to 0.5 mM CuSO4, or bathocuproine disulphonate (BCS) a copper chelator. Ultimately, we intend to use DHE fluorescence to assess superoxide levels in multiple yeast strains to provide insight into how ROS changes during yeast lifespan.