Determinants of the translational mobility of a small solute in cell cytoplasm.

Abstract

Abstract. The purposes of this study were: (a) to measure the translational mobility of a small solute in cell cytoplasm; (b) to define quantitatively the factors that determine solute translation; and (c) to compare and contrast solute rotation and translation. A small fluorescent probe, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein (BCECF), was introduced into the cytoplasm of Swiss 3T3 fibroblasts. BCECF translation was measured by fluorescence recovery after photobleaching; rotation was measured by Fourier transform polarization microscopy. Diffusion coefficients relative to those in water (D/D0) were determined by comparing mobility in cytoplasm with mobility in standard solutions of known viscosity. At isosmotic cell volume, the relative diffusion coefficients for BCECF translation and rotation in cytoplasm were 0.27 + 0.01 (SEM, n = 24, 23~ and 0.78 + 0.03 (n = 4), respectively. As cell volume increased from 0.33 to 2 times isosmotic volume, the relative translational diffusion coefficient increased from 0.047 to 0.32, while the relative rotational diffusion coefficient remained constant. The factors determining BCECF translation were evaluated by comparing rotation and translation in cytoplasm, and in artificial solutions containing dextrans (mobile barriers) and agarose gels (immobile bartiers). It was concluded that the hindrance of BCECF translation in cytoplasm could be quantitatively attributed to three independent factors: (a) fluid-phase cytoplasmic viscosity is 28 % greater than the viscosity of water (factor 1 = 0.78); (b) 19% of BCECF is transiently bound to intracellular components of low mobility (factor 2 = 0.81); and most importantly, (c) translation of unbound BCECF is hindered 2.5-fold by collisions with cell solids comprising 13% of isosmotic cell volume (factor 3 = 0.40). The product of the 3 factors is 0.25 + 0.03, in good agreement with the measured D/D0 of 0.27 + 0.01. These results provide the first measurement of the translational mobility of a small solute in cell cytoplasm and define quantitatively the factors that slow solute translation.C ELL cytoplasm is a complex non-Newtonian fluid comprising an aqueous fluid-phase filling the space within an entangled mesh of filamentous skeletal proteins (cytomatrix) and other macromolecular structures (Bridgman and Reese, 1984;Clegg, 1984;Fulton, 1982;Gershon et al., 1985;Keith, 1973;Porter, 1984). The factors that determine the rotation and translation of solute molecules within this crowded milieu have been the topic of considerable recent interest due to their probable impact on the rates of metabolic reactions. At least three cytoplasmic factors will contribute to solute mobility: (a) fluid-phase cytoplasmic viscosity, i.e., the viscosity in the aqueous space between macromolecules; (b) solute binding to macromolecular structures; and (c) collisional (direct plus hydrodynamic) interactions between the solute and macromolecular obstacles. The relative contributions of these three factors will depend on solute size and the type...

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