Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

TL;DR

This study employs advanced biosensing and microscopy techniques to map and analyze molecular crowding within single yeast cells, revealing how environmental factors like glucose and ionic strength influence cellular microenvironments.

Contribution

It introduces a combined approach of FRET biosensing and single-molecule microscopy to quantify and visualize subcellular crowding in live cells, providing new insights into cellular responses to environmental changes.

Findings

Crowding is reduced in yeast grown with higher glucose levels.

Cytosolic crowding is broadly uniform across cells over seconds.

Cell membrane is largely inaccessible to crowding sensors.

Abstract

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.