Crowding and Confinement Effects on Protein Diffusion In Vivo

ABSTRACT

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-μm-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K⁺ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, <φ>, and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Δ, of 0), the mean diffusion coefficient, <D>, in cytoplasm (6.1 ± 2.4 μm² s⁻¹) is only 0.07 of the in vitro value (87 μm² s⁻¹); the relative dispersion among cells, σ_D/<D> (standard deviation, σ_D, relative to the mean), is 0.39. Both <D> and σ_D/<D> remain remarkably constant over the range of Δ values of 0 to 0.28 osmolal. For a Δ value of ≥0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in <D> by a factor of 380 over the range of Δ values of 0.28 to 0.70 osmolal and a substantial increase in σ_D/<D>. Individual values of D vary by a factor of 9 × 10⁴ but correlate well with f_VPS, the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Δ values for cells lacking a VPS, perhaps related to variation in φ or biopolymer organization during the cell cycle, and stronger dispersion at high Δ values related to variation in f_VPS. Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.