Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

Abstract

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including 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 system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster 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 cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding 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 color, which can be used as a marker that labels organelle membranes. 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.

The uniformity and stability of cellular mass density in mammalian cell culture

“…With these considerations in mind, we binned cells at the G1, S, and G2-M stages by their total Hoechst intensity in the nucleus and found no significant difference in protein or lipid density in any of the compartments between any 2 cell cycle stages in either HeLa or NIH3T3 cells ( Figures 3B–E,G–J ). This result is consistent with the previous density measurement by refractive index ( Kim and Guck, 2020 ) but contrasts with measurements of the cell cycle-dependent molecular crowding ( Lecinski et al, 2021 ; Yamamoto et al, 2021 ) and diffusion rate ( Pradeep and Zangle, 2022 ). Although mass density, molecular crowding, and diffusion rate are related, they are evaluated by molecules of very different size, which may result in distinct behaviors.…”

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

“…The experimental state-of-the art is to use a Förster resonance energy transfer (FRET)-based probe to measure crowding. One such that we have used previously is called crGE2.3 [26][27][28], consisting of a donor fluorophore, mGFP and acceptor, mScarlet-I, linked by a flexible amino acid hairpin, forming an efficient FRET pair system as reported and characterized in the literature [29][30][31]. As molecular crowding increases, the donor and fluorophore are pushed closer together, and the FRET signal increases [26,32,33].…”

iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

“…Aggregates may be actively recognized by cells and sequestrated in the mother cell volume, additionally, physicochemical properties such as local viscosity (101) and the molecular crowding at the junction between the two cells can potentially influence aggregate localization, as suggested by the results of our previous study (72) on the investigation subcellular crowding dynamics.…”