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 μm 2 s-1) is only 0.07 of the in vitro value (87 μm 2 s-1); the relative dispersion among cells, σD/〈D〉 (standard deviation, σD, relative to the mean), is 0.39. Both 〈D〉 and σD/ 〈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 σD/〈D〉. Individual values of D vary by a factor of 9 × 104 but correlate well with fVPS, 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 fVPS, Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork. Copyright © 2006, American Society for Microbiology. All Rights Reserved.