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...

Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After labeling, a laser was used to bleach small (∼0.4 μm radius) spots in the heterochromatin and euchromatin of cells of both types. These spots were observed to persist for >1 h, implying that interphase chromatin is immobile over distance scales ⩾0.4 μm. Over very short times (<1 s), a partial fluorescence recovery within the spots was observed. This partial recovery is attributed to independent dye motion, based on comparison with results obtained using ethidium homodimer-1, which binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear lamina, or the nuclear matrix.

The coupling between molecular diffusion and the structure and function of the rat liver mitochondrial matrix was explored using fluorescence anisotropy techniques and electron microscopy. The results confirm that matrix ultrastructure and the concentration of matrix protein are influenced by the respiratory state of mitochondria and the osmolarity of the external medium. At physiological osmolarity, a fluorescent metabolite-sized probe was found to diffuse slowly in the mitochondrial matrix but not to be completely immobile. In addition, significant differences in diffusion rates were found to exist between different mitochondrial respiratory states, with the slowest diffusion occurring in states with the highest matrix protein concentration. These data support the concept of a matrix structure in which diffusion is considerably hindered due to limited probe-accessible water and further suggest that volume-dependent regulation of matrix protein packing may modulate metabolite diffusion and, in turn, mitochondrial metabolism.

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.