TRANSLATIONAL DIFFUSION OF MACROMOLECULE-SIZED SOLUTES IN CYTOPLASM AND NUCLEUS

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficoll s in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aque ous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 x 10(-7) cm(2)/s with increasing dextran size (4-2,000 kD). D i n cytoplasm relative to that in water (D/D-o) was 0.26 +/- 0.01 (MDCK) and 0.27 +/- 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [R-G] 40-300 Angstrom). The fraction of mo bile FITC-dextran molecules (f(mob)), determined by the extent of fluo rescence recovery after spot photobleaching, was > 0.75 for R-G < 200 Angstrom, but decreased to < 0.5 for R-G > 300 Angstrom. The independe nce of D/D-o on FITC-dextran and Ficoll size does not support the conc ept of solute ''sieving'' (size-dependent diffusion) in cytoplasm. Pho tobleaching measurements using different spot diameters (1.5-4 mu m) g ave similar D/D-o, indicating that microcompartments, if present, are of submicron size. Measurements of D/D-o and f(mob) in concentrated de xtran solutions, as well as in swollen and shrunken cells, suggested t hat the low f(mob) for very large macromolecules might be related to r estrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/D-o was 0.25 +/- 0.02 (MDCK) and 0.27 +/- 0.03 (fibroblasts), and independent of solute size (R-G 40-300 Angstrom). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approxi mately 500 kD in cytoplasm and nucleus.