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. 2006 Sep;188(17):6115-23.
doi: 10.1128/JB.01982-05.

Crowding and confinement effects on protein diffusion in vivo

Michael C Konopka  1 , Irina A Shkel, Scott Cayley, M Thomas Record, James C Weisshaar
Affiliations

Crowding and confinement effects on protein diffusion in vivo

Michael C Konopka et al. J Bacteriol. 2006 Sep.

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-mum-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, <phi>, and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, <D>, in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/<D> (standard deviation, sigma(D), relative to the mean), is 0.39. Both <D> and sigmaD/<D> remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =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 Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/<D>. Individual values of D vary by a factor of 9 x 10(4) 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 Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta 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.

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Figures

FIG. 1.
FIG. 1.
(a) Optical layout showing laser paths, beam splitters (a), mirrors (e), electronic shutters (c and d), collimating telescope (f), and lenses (b and g). (b) Schematic of camera region of interest, including one cell, bleach beam area, and probe beam area.
FIG. 2.
FIG. 2.
Sequences of FRAP images for B-strain cells grown in 0.24 osmolal (Osm) LB and upshifted as shown. The intensity scale is renormalized to 100 for each image. The prebleach (Pre) image and subsequent times are indicated. Bar, 2 μm. The range of x of 0 to L over which the diffusion analysis was carried out is shown in the first image of each sequence. Rel, relative.
FIG. 3.
FIG. 3.
(a) Mean intensity in each pixel column (each value of x) versus distance along the cell axis for the Δ = 0 recovery sequence shown in Fig. 1 at different times as shown; the upper trace is the prebleach image. (b) Least-squares single exponential fits of equation 4 to the decay of n = 1 cosine mode intensity for cells shown in Fig. 1 at the osmotic upshifts shown.
FIG. 4.
FIG. 4.
(Top) Images of B-strain cells grown in 0.24 osmolal (Osm) LB in isosmotic resuspension buffer (a) or with an upshift of 0.44 osmolal (b). Two VPSs are marked by arrows. Bar, 1 μm. (Bottom) Distribution of D and log D for single cells grown in 0.24 osmolal LB and upshifted as shown. Table 1 contains additional quantitative details.
FIG. 5.
FIG. 5.
Semilog plot of <D> versus Δ for cells grown in 0.24 osmolal LB. Horizontal bars are individual measurements. Dashed lines are merely guides to the eye. The GFP diffusion coefficient in buffer is indicated. mOsm, milliosmolal.
FIG. 6.
FIG. 6.
Correlation of log D versus fVPS = − ΔVVPS/Vpre-VPS for cells exhibiting VPSs at Δ values of 0.28 osmolal (Osm), 0.39 osmolal, and 0.44 osmolal. Horizontal error bars indicate ±20% uncertainty in fVPS. Gray swath, smooth curve drawn by hand through the data; blue horizontal lines, log D for 14 cells at a Δ of 0.28 osmolal exhibiting no VPSs; black horizontal lines, log D for 39 cells at a Δ of 0.
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References

    1. Amsden, B. 1998. Solute diffusion within hydrogels. Mechanisms and models. Macromolecules 31:8382-8395.
    1. Banks, D. S., and C. Fradin. 2005. Anomalous diffusion of proteins due to molecular crowding. Biophys. J. 89:2960-2971. - PMC - PubMed
    1. Bremer, H., and P. P. Dennis. 1996. Modulation of the chemical composition and other parameters of the cell by growth rate, p. 1553-1569. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, vol. 2. ASM Press, Washington, D.C.
    1. Cayley, D. S., H. J. Guttman, and M. T. Record, Jr. 2000. Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress. Biophys. J. 78:1748-1764. - PMC - PubMed
    1. Cayley, S., and M. T. Record, Jr. 2003. Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine. Biochemistry 42:12596-12609. - PubMed

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