RT Journal Article SR Electronic T1 Increased intracellular crowding during hyperosmotic stress JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.12.30.522363 DO 10.1101/2022.12.30.522363 A1 Kitamura, Akira A1 Oasa, Sho A1 Kawaguchi, Haruka A1 Osaka, Misato A1 Vukojević, Vladana A1 Kinjo, Masataka YR 2023 UL http://biorxiv.org/content/early/2023/05/01/2022.12.30.522363.abstract AB Hyperosmotic stress activates in live cells numerous processes and also promotes intracellular protein/RNA aggregation and phase separation. However, the time course and the extent of these changes remain largely uncharacterized. To investigate dynamic changes in intracellular macromolecular crowding (MMC) induced by hyperosmotic stress in live cells, we used Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Correlation Spectroscopy (FCS) to quantify changes in the local environment by measuring the fluorescence lifetime and the diffusion of the monomeric enhanced Green Fluorescent Protein (eGFP), respectively. Real-time monitoring of eGFP fluorescence lifetime showed that a faster response to environmental changes due to MMC is observed than when measuring the acceptor/donor emission ratio using the MMC-sensitive Förster Resonance Energy Transfer sensor (GimRET). 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, eGFP diffusion assessed by FCS indicated higher intracellular viscosity due to increased MMC during hyperosmotic stress. 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 in live cells.Competing Interest StatementThe authors have declared no competing interest.