Imaging of nucleocytoplasmic transcription factor dynamics in yeast

Licentiate thesis, 2010

Fluorescence microscopy has proven to be an invaluable tool in identifying and understanding cell signalling, intracellular spatial and temporal dynamics, and cell-to-cell variability. This thesis focuses on imaging of nucleocytoplasmic transcription factor dynamics at the single cell level in yeast. This area is interesting because it is still unclear in most cases how stress is introduced and transduced throughout signalling pathways and correlated to transcription factor dynamics. The thesis also deals with different aspects of single cell microscopy experiments, including cell recognition, image analysis, and quantitative data analysis of dynamic intracellular events. The general stress response factor Msn2p was studied in budding yeast, Saccharomyces cerevisiae. Msn2p enters a nucleocytoplasmic shuttling behaviour or completely translocates from the cytoplasm to the nucleus as a response to light exposure. The phenomenon is dependent on the light intensity and is induced at light doses commonly used in fluorescence microscopy experiments. Therefore, the measurement technique itself could alter the biological conditions and introduce multiple variables that make data interpretation difficult. In order to provide guidelines for non-invasive imaging, the localization of Msn2p tagged with GFP was used as a reporter for the stress level at commonly used exposure settings. The results showed that for a constant light dose a combination of low intensities and longer exposure times induced a lower stress level, compared to the opposite. From a microscopy point of view, high fluorescence S/N ratio should be gained by e.g. using high copy plasmids, since the abundance of GFP, did not seem to be correlated to the stress response. In order to understand how light exposure affects the oscillatory behaviour of Msn2p and how light induced stress can be used to evaluate transcription factor dynamics, I used automated epi-fluorescence microscopy and continuously exposed yeast cells at various constant light intensities. Evaluation of the nucleocytoplasmic dynamics showed that at high light intensities Msn2p exhibited both an oscillatory phase followed by permanent nuclear localization. A decrease in light intensity resulted in a prolonged oscillatory phase and some cells did not even exhibit the permanent nuclear localization. The data indicate that illumination with a constant light intensity results in continuously increasing stress levels, presumably due to an accumulating concentration of toxic photoproducts. The oscillatory behaviour was found to be affected and negatively controlled by protein kinase A activity. A lower activity resulted in an increasing stress response, mainly due to regulation by more frequent nuclear localizations, and not by a significant increase in the average time spent for each nuclear localization period.