Cellular stress characterisation in S. cerevisiae budding yeast using optical microscopy

The term "cellular stress" covers various environmental and metabolic events that threaten cell survival. In response, eukaryotic cells can adapt their metabolism to acute changes in their surrounding environment to maintain cellular homeostasis and ensure survival. The cell is an enclosed system with macromolecules and functional compartments diffusing in a liquid-like environment, the cytoplasm. The position in time and space of these elements influences every aspect of cell biology, from molecular interactions and enzymatic activities to the process of cell division itself. Cellular stress episodes can interfere with the timing of these physiological processes: perturbations modify the cytoplasm volume and composition (e.g., accumulation of damaged proteins), including changes of intracellular physical properties such as macromolecular crowding influencing diffusion and spatio-temporal dynamics of the whole system. Consequently, cellular stress dynamics have been of strong interest to physicists and biologists. In continuity with previous research in the field, this project aimed to explore these aspects of cellular physiology and gain new insight into cellular stress responses and crowding dynamics in the budding yeast Saccharomyces cerevisiae, a eukaryotic model sensitive to environmental stresses. This thesis sets out to investigate the influence of hyperosmotic shock, glucose availability, and cell growth on macromolecular crowding. I present a methodology developed to identify local regions of crowding in yeast cells using a previously generated Förster Resonance Energy Transfer Technology (FRET) crowding biosensor called CrGE. I describe experimental and analysis procedures to quantify crowding at subcellular levels and have developed new strains controlling the expression of fluorescently tagged cytoplasmic aggregates. To identify fluorescent clusters on cellular models, single molecule characterisations for stoichiometry and diffusion tracks in vivo were performed using bespoke Slimfield microscopy. Cellular sub-compartments were visualised and tracked over time using confocal microscopy, giving insight into polarised inheritance events in the budding yeast.