Investigating molecular crowding during cell division in budding yeast with FRET

Cell division, aging, and stress recovery triggers the spatial reorganization of cellular components in the cytoplasm, but also of membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model eukaryotic unicellular organism to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a F&oumlrster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to growth and osmotic stress, and its dependence on mother and daughter cells during division, and find unexpectedly the presence of hot spots of crowding across the bud neck in the burgeoning daughter cell, which might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges, and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third colour, which can be used as an organelle marker and to readout membrane morphology. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.