Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells.

Abstract

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.The non-Newtonian properties of cytoplasm have been well documented during more than a century of study, but the physical chemical basis for the non-Newtonian properties of cytoplasm is not understood (for reviews, see refs. 1-12). While such macroscopic non-Newtonian phenomena as viscoelasticity and thixotropy imply that cytoplasm possesses some sort of submicroscopic intermolecular organization not found in a dilute, aqueous solution, the possible forms of this organization range from a liquid crystal structure due to the high concentration of protein in cytoplasm, to a meshwork of entangled filamentous proteins, to a crosslinked gel network. A fundamental problem in approaching this question has been the difficulty of studying living cells with high enough resolution. Until recently there has been no method of obtaining data on a molecular level without the necessity of first fixing the cells for electron microscopy or fractionating the cells for subsequent biochemical analysis. Each of these approaches contains the potential for artifacts that make it uncertain how far the results of such experiments can be extended to the structure and function of living cells. Two relatively new techniques have made it possible to study the behavior of specific molecules in living cells while keeping perturbation of the cells' normal structure and function to a minimum. Fluorescent analog cytochemistry (FAC) can be used to study the subcellular distribution of fluorescent derivatives (analogs) of specific molecules (13), and fluorescence recovery after photobleaching (FRAP) can be used to study quantitatively the mobility of these analogs within living cells (14-21). By com...

Implications of a poroelastic cytoplasm for the dynamics of animal cell shape

“…We estimate pore diameters of 30-60nm are required to account for data on blebbing and local sucrose perfusion using a poroelastic model ( [18] and GC, TM and LM, manuscript in preparation). This value is consistent with that obtained from analyzing diffusion of different sized probes in some [19], but not all experiments [20]. 30-60nm is smaller than the pore size we expect from cytoskeletal networks alone [21,22], with the possible exception of the extreme leading edge [23], where actin filaments are highly concentrated.…”

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

“…The data from these experiments are presented in Table III. In the perinuclear region, aldolase had a Dcyto of 1.1 x 10 -7 cm2/s, a value 4.5-fold lower than its aqueous diffusion coefficient. This reduction may be explained simply by the higher viscosity of the aqueous phase of the cytoplasm, which has been reported to be between 3 and 6 centipoise (40,42,51,55). However, the immobile fraction of 23 % is not explained by cytoplasmic viscosity, and may reflect a bound fraction of aldolase in the perinuclear region.…”

When is a carrier not a membrane carrier? The cytoplasmic transport of amphipathic molecules

“…4,70 This requirement greatly limits the molecules that may be studied. Moreover, the addition of a fluorescent side…”