Investigating molecular crowding during cell division in budding yeast with FRET

TL;DR

This study employs FRET-based sensors and advanced microscopy to quantify molecular crowding during yeast cell division, revealing spatial heterogeneity and its potential regulatory roles in cellular processes.

Contribution

It introduces optimized FRET measurement techniques combined with multi-color imaging to analyze molecular crowding at high resolution during yeast cell division.

Findings

Crowding hotspots are observed at the bud neck during division.

Molecular crowding varies between mother and daughter cells.

Inherited organelles like vacuoles influence local crowding levels.

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 F\"{o}rster 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.