Photobleaching studies were conducted to measure the translational diffusion of macromolecule-sized solutes in the cytoplasm and nucleus of fibroblasts and MDCK cells. As described in the introduction, this work followed from our previous measurements of the rotational and translational mobility of small, metabolite-sized solutes in bulk and membrane-adjacent cytoplasm and nucleus. The results here indicate that the translational diffusion of FITC-dextrans and FITC-Ficolls is slowed three- to fourfold in cytoplasm and nucleus compared with water. The degree of slowing did not depend on molecular size up to at least a 300-Å gyration radius. A large macromolecule of ~500 kD size would have a diffusion coefficient of ~2.5 × 10−8 cm2/s in cytoplasm. If significant binding of the macromolecule to slowly diffusing cytoplasmic components does not occur, then the diffusive transit time to move across a 10-μm cell would be only ~7 s. Our results indicate that this transit time would be dramatically slower for larger macromolecules or in shrunken cells, such as tubular epithelial cells exposed to strong osmotic gradients in the renal medulla.
The results in Swiss 3T3 fibroblasts were different from previous work by Luby-Phelps et al. (1986). Both studies used the same cell type, and similar microinjection procedures and incubation times after microinjection. Our microinjections involved high pressure and very thin glass capillary needles as used previously to microinject 70-nm-diam liposomes into cells and to maintain cell viability (Seksek et al., 1995
). Differences in the studies involved primarily the instrumentation, analytical procedures, and measurement temperature. Our experiments were carried out with a microsecond-resolution FRAP apparatus using acoustooptic modulators that ensured precise overlap of probe and bleach beams without alignment. Each data point generally represented the average of >30 sets of measurements, each consisting of averaged recovery curves from greater than five different spots, using at least three different cell preparations studied on different days. Direct comparison of signal-to-noise ratios and recovery curve shapes of the data here with the previous studies on fibroblasts could not be made because original recovery curves were not reported in the Luby-Phelps et al. (1986, 1987) papers. The determination of the diffusion coefficient here used standards measured on the same day using the same bleach time and objective lens, as well as similar bleach depths and sample geometries. As discussed previously (Kao et al., 1993
), we believe that this empirical approach is superior to the use of analytical approximations developed for photobleaching in two dimensions (Axelrod et al., 1976
; Lopez et al., 1988
); the bleached zone in studies of aqueous-phase dyes is a complex three-dimensional profile in which recovery occurs by translational motions in three dimensions.
A reversible photobleaching process involving triplet state relaxation was identified for FITC-dextrans in cytoplasm at 37°C. Fluorescence recovery did not depend upon solute translational diffusion because the fluorescence increase results from repopulation of the So
ground state from the T1
triplet state that became populated during the bleach pulse. As was found for BCECF diffusion in cytoplasm (Swaminathan et al., 1996
), the reversible photobleaching process was eliminated by exposing the cells to oxygen-saturated solutions. The measurements in this paper were done at 23°C to avoid the complexities associated with reversible photobleaching. Because of the relatively long bleach times and limited time resolution in the Luby-Phelps studies (1986), it is possible that unrecognized reversible photobleaching (the extent of which depends on FITC-dextran size) may have influenced the data interpretation as related to size-dependent sieving.
An interesting observation was that little fluorescence recovery occurred after photobleaching of the largest FITC-dextrans and FITC-Ficolls in the cytoplasm. Measurements in concentrated dextran solutions suggested that the incomplete recovery was not due to simple cytoplasmic crowding with mobile macromolecular obstacles. Measurements with different beam intensities and bleach times suggested that the photochemical reaction was not responsible for incomplete recovery. Apparent solute diffusion coefficients and recovery extents did not depend on spot size, suggesting that microcompartments, if present, were much smaller than ~1 μm. The lack of further fluorescence recovery over long periods of time did not provide evidence for anomalous diffusion or long-tail phenomena, although recoveries over very long periods of time could not be studied because of technical limitations. The experiments in swollen and shrunken cells showed a strong effect of cell volume on the extent of fluorescence recovery, such that an essentially immobile 2,000-kD FITC-dextran in normovolemic cells became mobile after twofold cell swelling. Taken together, these findings are consistent with the possibilities that the incomplete recovery is related to the presence of immobile microcompartments of submicroscopic dimensions, or to anomalous diffusion such as percolation. For several reasons we believe that percolation is most likely. First, it is unclear which cellular components could comprise the putative microcompartments. More importantly, the percolation threshold is expected to be very sensitive to the size of the diffusing particle (Saxton, 1993
), as was found here, and to the cell volume. In contrast, relatively small effects of cell volume on the percentage recovery vs solute size relation are predicted for a sieving mechanism in which solute diffusion is restricted by a meshlike skeletal network. The next important steps in the analysis will be the construction of instrumentation to measure recoveries over many minutes/ hours, as well as the development of a theoretical model of anomalous diffusion and percolation phenomena in three dimensions.
The photobleaching data indicated similar rates of FITC-dextran diffusion in the cytoplasm and nucleus. The nucleus is spatially organized into several distinct domains bounded by the nuclear envelope, which consists of two membranes bridged in places by nuclear pores. Within the nucleus are the nucleoli (for ribosome production), nuclear lamina, and possibly specialized domains for the localization of replication, transcription, and splicing. The existence of a “nuclear matrix,” consisting of a scaffolding structure seen in electron micrographs of detergent-extracted nuclei (Capco et al., 1982
; Fey et al., 1986
; He et al., 1990
; Raska et al., 1992
), has been controversial. The nuclear matrix has been proposed to act as an anchoring site for biochemical and molecular events and would provide a structural basis for nuclear organization (Berezney, 1991
; Cook, 1991
; Jacobson, 1995
). Information on diffusion in the nucleus is available only for relatively small and very large objects. Based on photobleaching experiments on fluorescently labeled dextrans (3–150 kD), Lang et al. (1986)
reported that diffusion in the nucleus is about fivefold slower than in dilute buffer, with no evidence of sieving. Although these studies were performed on large polyethylene glycol–fused, multinucleated cells, the results are in agreement with the conclusions here. In contrast, from analysis of the trajectories of much larger, naturally occurring cytoplasmic inclusions, Alexander and Rieder (1991)
reported that diffusion in the matrix was several hundredfold slower than in dilute solution. The results here indicate the absence of solute sieving of FITC-dextrans up to an apparent gyration radius of 300 Å, which does not support the existence of a scaffolding structure with characteristic dimensions of under ~250 Å. The existence of a functional nuclear substructure of greater dimensions will require the measurement of single particle trajectories in three dimensions (Kao and Verkman, 1994
Several potential concerns should be noted in evaluating the strength of our conclusions. As in previous studies of this type, the introduction of FITC-dextrans required an invasive microinjection procedure. To minimize effects of cell trauma, cells were incubated for 4–6 h after microinjections, during which time severely damaged cells would be released and minor damage to microinjected cells would likely be repaired. In control studies, rhodamine-phalloidin staining patterns of microinjected and control cells were similar at 4–6 h after FITC-dextran microinjection (not shown). Another issue is the analysis of photobleaching recovery data based on t1/2 values and comparison with solution standards of known viscosity. This approach was chosen to permit quantitative determination of diffusion coefficients without the need to develop complex models of solute diffusion in three dimensions. The use of t1/2 values was justified here on the basis of the nearly identical appearance of recovery curve shapes, which also indicated relative size homogeneity in the size-fractionated FITC-dextrans and FITC-Ficolls. Therefore, although we believe that the principal conclusions about solute diffusion are valid, diffusion coefficients must be viewed as representing averaged physical properties of the cytoplasm or nucleus. Finally, because the shapes of FITC-dextran and FITC-Ficoll molecules are nonspherical, the data must formally be interpreted in terms of effective hydrodynamic radii or, as used here, gyration radii. It is noted that this caveat does not affect the principal conclusion that macromolecule diffusion in cells relative to that in water is independent of macromolecule size.