Illumination in single cell optical microscopy studies - friend and foe

Doktorsavhandling, 2012

This thesis focuses on how to use light as a biological stress factor in optical microscopy experiments and simultaneously use the illumination to image the spatial and temporal protein dynamics in single yeast cells. The stress responses to environmental changes other than light have been extensively investigated in budding yeast, Saccharomyces cerevisiae, often using techniques based on population averages. Fluorescence microscopy has evolved to a powerful technique to study proteins in vivo in individual cells. However, little attention has been brought to the effects of light exposure during long-term fluorescence microscopy experiments, even though it is well known that visible light can cause cellular dysfunction. This thesis shows that light-induced stress can be used as a powerful tool to exert stress during in vivo optical microscopy studies of transcription factor dynamics. In addition, mechanisms responsible for the light-induced stress response are investigated and guidelines for non-invasive imaging are suggested. Msn2p is one of the most important transcription factors involved in the environmental stress response in yeast. The amount of nuclear localization of Msn2p-GFP was therefore used as a reporter to quantitatively characterize the level of blue light-induced stress. Importantly, it was found that it was more stressful for a cell to be exposed to high intensity and short exposure times compared to low intensity and long exposure times, even though the light dose was the same. Fluorescent proteins themselves could potentially induce the cellular toxicity by acting as photosensitizers, but the data presented here point towards endogenous compounds with absorption bands similar to GFP, possibly flavin containing molecules that could cause H2O2 production. Based on the study above, continuous blue light exposure with relatively low intensity was used to stress cells and to study the spatial and temporal responses from the transcription factors Msn2p, Msn4p and Crz1p. All three transcription factors responded to light induced-stress through translocation from the cytoplasm to the nucleus, but they displayed distinctly different nucleocytoplasmic response patterns. Msn2p and Msn4p exhibited nucleocytoplasmic oscillations in response to stress, whereas Crz1p located permanently to the nucleus after a short delay time. Analysis of the localization patterns of the transcription factors revealed different responses compared to other stresses and showed that adaptation to small environmental changes can have drastic effects on the sensitivity and response for each transcription factor. Interestingly, peroxiredoxins were found to be necessary for the nucleocytoplasmic response of Msn2p to light-induced stress. Peroxiredoxins were initially characterized as H2O2-scavenging enzymes, implicating H2O2 as a potential reactive oxygen species formed upon light exposure. In addition, Msn2p hyper-responded in cells lacking the possibility to reduce thioredoxins. In a final study of light stress, the colony forming ability of the gene knockout collection was studied under white light illumination. This genome-wide screen implicated previously unknown systems in the stress response to illumination. In particular, several components of the HOG MAPK cascade were picked up as sensitive. Deletion mutants resistant to illumination displayed enrichment of mitochondrial components.