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Increased intracellular crowding during hyperosmotic stress.

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Hyperosmotic stress rapidly alters intracellular macromolecular crowding (MMC). Measuring eGFP fluorescence lifetime and diffusion offers early detection of these dynamic cellular changes.

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Area of Science:

  • Cellular Biology
  • Biophysics

Background:

  • Hyperosmotic stress induces cellular processes, including protein/RNA aggregation and phase separation.
  • The dynamics and extent of these stress-induced intracellular changes are not well understood.

Purpose of the Study:

  • To investigate dynamic changes in intracellular macromolecular crowding (MMC) in live cells under hyperosmotic stress.
  • To compare the response time of eGFP fluorescence lifetime and diffusion with a Förster resonance energy transfer (FRET) sensor.

Main Methods:

  • Utilized fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy (FCS).
  • Quantified MMC changes by measuring the fluorescence lifetime and diffusion of monomeric enhanced green fluorescent protein (eGFP).

Main Results:

  • eGFP fluorescence lifetime responded faster to MMC changes than the GimRET FRET sensor.
  • FCS revealed increased intracellular viscosity due to elevated MMC during hyperosmotic stress.
  • eGFP lifetime and diffusion serve as early indicators of increased intracellular MMC.

Conclusions:

  • Changes in eGFP fluorescence lifetime and diffusion dynamics are rapid indicators of hyperosmotic stress-induced MMC.
  • This approach allows real-time observation of transient states in MMC dynamics within live cells.