UC San Diego

Oxidative stress has been a widely studied topic owing to its hazardous characteristic to biological functioning and involvement in the pathogenesis of multiple diseases. It is crucial to understand the regulation of oxidative stress as well as how homeostasis is maintained. To further realize cell responses to oxidative stress, we used microfluidic technology to investigate heterogeneity in yeast cells when affected by oxidative stressors hydrogen peroxide and diamide. We have identified that under both H2O2 and diamide stress, a constitutive GFP in yeast cells will respond with three types of intensity fluctuations: one with continuous maintenance of the GFP intensity, another with a drastic drop in intensity when the stress is added but followed with intensity recovery after removal of the stress, and lastly the type that also has a drastic drop in intensity but no recovery is seen even after the stress is removed. This heterogeneity in the yeast cells can be seen while under 0.5mM to 1mM H2O2 stress and 4mM to 7mM diamide stress; however, in comparison to diamide, the cells under H2O2 stress respond with a more defined distinction among the three cell types that better corresponds with the stress concentration. Building off these results, we integrated a superfolder pHluorin gene into the yeast genome and were able to conclude that the GFP intensity fluctuations are indeed correlated to intracellular pH alterations upon H2O2 addition but not diamide addition. Alternative possibilities of the heterogeneity under H2O2 and diamide stress are discussed in this paper.