Crowding and confinement effects on protein diffusion in vivo

Area of Science:

• Cellular biology
• Biophysics
• Molecular dynamics

Background:

• Cellular environments are crowded with macromolecules, impacting molecular transport.
• Understanding protein diffusion is crucial for cellular function and processes.
• Previous studies lacked in vivo measurements of diffusion versus cytoplasmic volume fraction.

Purpose of the Study:

• To measure protein diffusion coefficients in vivo across varying cytoplasmic biopolymer concentrations.
• To investigate the impact of osmotic stress and plasmolysis on protein diffusion in E. coli.
• To differentiate between crowding, binding, and confinement effects on diffusion.

Main Methods:

• Utilized fluorescence recovery after photobleaching (FRAP) to measure green fluorescent protein diffusion.
• Employed osmotic upshifts using hyperosmotic buffers to alter cytoplasmic volume fraction.
• Quantified diffusion coefficients and their dispersion in Escherichia coli cytoplasm.

Main Results:

• In vivo protein diffusion was significantly slower (0.07x) than in vitro, even at low osmotic stress.
• Diffusion coefficients and dispersion remained constant up to an osmotic upshift of 0.28 osmolal.
• At higher osmotic stress (>0.28 osmolal), diffusion decreased dramatically (380-fold) with plasmolysis space formation, increasing dispersion.

Conclusions:

• Cytoplasmic crowding and confinement significantly impede protein diffusion in vivo.
• The formation of visible plasmolysis spaces dramatically alters protein diffusion dynamics.
• Crowding alone cannot fully explain the observed diffusion behavior; binding and confinement likely play roles.