Increased intracellular crowding during hyperosmotic stress

Abstract

Hyperosmotic stress activates in live cells numerous processes the net result of which is readily recognized at the cellular level through morphological changes. It also promotes intracellular protein/RNA aggregation and phase separation, observable through the formation of stress granules in the cytoplasm. However, the time course and extent of these changes remain largely uncharacterized. To investigate in live cells dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress, we used Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify changes in the local environment by measuring the diffusion and the fluorescence lifetime of the monomeric enhanced Green Fluorescent Protein (eGFP), respectively. In addition, Confocal Laser Scanning Microscopy (CLSM) was used to map the acceptor/donor emission ratio (Y/C ratio) in cells expressing the glycine-inserted mutant Förster Resonance Energy Transfer (FRET) sensor (GimRET) that is indicative of MMC. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the Y/C ratio using the MMC-sensitive GimRET sensor. This suggests that eGFP molecular electronic states and/or collision frequency are affected by changes in the immediate surroundings due to MMC without requiring conformational changes as is the case for the GimRET sensor. Furthermore, slower eGFP mobility and a lower anomalous diffusion parameter (α) are measured in cells exposed to hyperosmotic stress, indicating higher intracellular viscosity due to increased MMC. Our findings reveal that changes in eGFP fluorescence lifetime and diffusion are early indicators of elevated intracellular MMC. These variables can therefore be used for quantitative characterization of MMC under various stress/disease conditions in live cells.