Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521
Fluorescence recovery after photobleaching
(FRAP) was used to quantify the translational diffusion
of microinjected FITC-dextrans and Ficolls 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 aqueous media
(viscosity 1 cP), D for the FITC-dextrans decreased
from 75 to 8.4 × 107 cm2/s with increasing dextran size
(4-2,000 kD). D in cytoplasm relative to that in water
(D/Do) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll
size (gyration radii [RG] 40-300 Å). The fraction of mobile FITC-dextran molecules (fmob), determined by the
extent of fluorescence recovery after spot photobleaching, was >0.75 for RG < 200 Å, but decreased to <0.5
for RG > 300 Å. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of
solute "sieving" (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different
spot diameters (1.5-4 µm) gave similar D/Do, indicating that microcompartments, if present, are of submicron
size. Measurements of D/Do and fmob in concentrated
dextran solutions, as well as in swollen and shrunken
cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by
immobile obstacles (such as microcompartments) or to
anomalous diffusion (such as percolation). In nucleus,
D/Do was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (RG 40-300 Å).
Our results indicate relatively free and rapid diffusion
of macromolecule-sized solutes up to approximately
500 kD in cytoplasm and nucleus.
The physical structure of cell cytoplasm has been a
topic of long-standing interest (for review see
Clegg, 1984 As more advanced biophysical techniques have been applied to study the physical state of cytoplasm, the notion is
emerging that the cytoplasm is more like a bag of slightly
viscous water than a complex gelatinous mass, at least with
respect to solute diffusion. One parameter that describes
cytoplasmic rheology is "fluid-phase viscosity," defined as
the microviscosity sensed by a small solute in the absence
of interactions with macromolecules and organelles (Fushimi and Verkman, 1991 For transport of small solutes such as metabolites and
nucleic acids, a more important parameter describing cytoplasmic rheology is the translational diffusion coefficient.
Kao et al. (1993) In earlier studies (Luby-Phelps et al., 1986 The original purpose of the study here was to use the
quantitative approach developed by Kao et al. (1993) Chemicals
FITC-dextrans (average molecular sizes 4, 10, 20, 40, 70, 150, 580, and
2,000 kD) were purchased from Sigma Chemical Co. (St. Louis, MO) or
Molecular Probes (Eugene, OR). Nonfluorescent dextrans (T4, T70,
T500, and T2000) and Ficolls (T70 and T400), as well as Sepharose CL-6B,
were purchased from Pharmacia Fine Chemicals (Piscataway, NJ). All
other chemicals were from Sigma Chemical Co.
Fluorescent Labeling of Ficolls
Ficolls T70 and T400 were activated and labeled with FITC by a modification of the method of Inman (1975) Size Fractionation of FITC-dextrans and FITC-Ficolls
70 mg of FITC-Ficoll (17.5 mg/ml) was loaded on a 3 × 100 cm column of
Sepharose CL-6B equilibrated in 20 mM Tris, pH 8.0, 50 mM KCl, and
0.02% NaN3 (buffer A). The column was eluted with buffer A and 5-ml
fractions were collected. Two fractions from the T70 Ficoll (fractions a
and b) and two from the T400 Ficoll (fractions c and d) were dialyzed
three times against water and lyophilized. FITC-dextrans (17.5 mg/ml)
were loaded on a 3 × 50-cm column of Sepharose CL-6B equilibrated
with buffer A. The column was eluted with buffer A, 5-ml fractions were
collected, and the peak fractions corresponding to ~30% of the starting
material were pooled, dialyzed against water, and lyophilized. The size-fractionated FITC-dextrans and FITC-Ficolls were then subjected to a
second round of size fractionation on a 3 × 50-cm column as described above.
Cell Culture
MDCK cells (ATCC CCL 34, passages 52-58; American Type Culture
Collection, Rockville, MD) and Swiss 3T3 fibroblasts (ATCC CL-101,
passages 60-80; American Type Culture Collection) were cultured on 18-mm-diam round glass coverslips in DME-H21 medium supplemented with
5% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were
grown at 37°C in 95% air/5% CO2 and used 1-2 d after plating at which
time they were ~80% confluent. After microinjection and incubation,
coverglasses containing cell layers were mounted in a perfusion chamber
that was positioned on the microscope stage.
Cell Microinjection
Solutions for microinjection consisted of calcium-free PBS containing 20-
40 mg/ml FITC-dextran or FITC-Ficoll. Solutions were centrifuged
(10,000 g for 10 min) to remove particulate matter. Microinjection was
performed using an Eppendorf 5170 micromanipulator and 5242 microinjector (Eppendorf North America, Inc., Madison, WI). Glass needles
were drawn from thin-walled filament capillaries (FHC, Brunswick, ME)
with a vertical needle puller (Kopf, Tujunga, CA). Cells (generally ~250
cells injected on each coverglass, ~50% injections intranuclear) were microinjected with ~4 fL of solution at an injection pressure of 120 kilopascals over 0.5 s. Slight bulging of the cell membrane was generally observed during the injection by phase-contrast microscopy.
Fluorescence Recovery after Photobleaching
The output of an argon ion laser (488 nm, Innova 70-4; Coherent Inc.,
Palo Alto, CA) was modulated by serial acoustooptic modulators and directed by coated mirrors (99.2% reflectivity at 488 nm) onto the stage of
an inverted epifluorescence microscope (Diaphot; Nikon Inc., Garden
City, NY) (Fig. 1). The microscope was also equipped for full-field epiillumination (halogen light source, 470 ± 20-nm interference filter) to visualize all cells to target the focused laser beam. The full-field and laser beams were reflected by a dichroic mirror (510 nm) onto the sample by an objective lens (Nikon ×20 dry, NA 0.75, unless otherwise specified). Laser
beam intensity was modulated by two acoustooptic modulators (rise time
~1.5 µs) in series using 2-mm-diam pinholes to isolate first order beams
(Kao and Verkman, 1996
Sample fluorescence was filtered by serial barrier (glass OG 515; Schott
Corp., Yonkers, NY) and interference (530 ± 15 nm) filters and detected
by a photomultiplier (9828A; Thorn EMI Electron Tubes, Inc., Rockaway, NJ) contained in a cooled housing (FACT50; Thorn EMI Electron
Tubes, Inc.). Photomultiplier signals were amplified by a transimpedance
amplifier and digitized at 1 MHz using a 14-bit analog-to-digital converter.
A gating circuit that controlled the voltage of the second dynode was used
to decrease photomultiplier gain during the photobleaching pulse. The instrument response time was <50 µs. Beam modulation, photomultiplier
gating, and data collection were software controlled. Signals were sampled before the bleach (generally 103 data points in 100 ms) and over three different time intervals after the bleach: high resolution data (1 MHz sampling rate) over 10-100 ms, low resolution data (generally 104 points) over
0.1-10 s, and "final signal" data (103 points) at a specified late time. High
and low resolution data were binned into 200 points each for storage and
analysis. Generally, in solution studies, data from five individual FRAP
experiments were averaged for each stored recovery curve; in cell studies, each recovery curve was obtained from a different spot.
Photobleaching Recovery Measurements
For measurements in aqueous solutions, specified microliter solution volumes were "sandwiched" between two coverslips to produce aqueous layers of known uniform thickness. For cell measurements, the lower coverglass contained the cultured cells (facing upward). FRAP measurements
were performed 4-6 h after microinjection to permit cell recovery. Beam
intensity and attenuation ratio were adjusted to produce <30% bleaching
and to avoid photobleaching by the probe beam. For experiments in
which cell volume was changed, cell were exposed to hypoosmolar (150 mosM, 1:1 PBS/water) or hyperosmolar (450 mosM, PBS containing 150 mM sucrose) buffer for 10 min; total internal reflection fluorescence measurements (Farinas et al., 1995 Analysis of FRAP Data
As described by Kao et al. (1993) Confocal Microscopy
Microinjected cells were visualized using a Nipkow wheel confocal microscope (Leitz with coaxial-confocal attachment [Technical Instruments,
San Francisco, CA]) and cooled CCD camera detector (Photometrics Ltd.,
Tucson, AZ) as described previously (Seksek et al., 1995 Characterization of FITC-labeled Dextrans and Ficolls
Photobleaching measurements were first carried out in
aqueous solutions containing unconjugated fluorescein
and the FITC-dextrans and FITC-Ficolls used for subsequent measurements in cells. The representative photobleaching curves in Fig. 2 A show nearly complete recovery of fluorescence back to the initial signal level (range for percentage recovery 98-101%), as expected for photobleaching in homogeneous aqueous solutions. The recovery time course was slowed with increasing fluorophore molecular size. As described previously (Kao et al.,
1993
Fig. 2 C shows the t1/2 values for each of the FITC-labeled dextrans and Ficolls. Diffusion coefficients and gyration radii (righthand axis) were computed from t1/2 values
for each of the compounds as was done by Luby-Phelps et
al. (1986) (see figure legend). The gyration radius (RG)
provides a measure of effective solute size, recognizing
that complexities of nonspherical solute shape are lost
with a single parameter description. For this reason studies
were done with the two available types of noninteracting molecules: dextrans, which have some asymmetry, and the
more spherical Ficolls. Subsequent plots will use RG to denote macromolecule size, with different symbols denoting
data obtained with FITC-dextrans and FITC-Ficolls.
Macromolecule Diffusion in Cytoplasm
The procedures used to measure fluorophore diffusion in
aqueous solutions were applied to study FITC-dextran
and FITC-Ficoll diffusion in MDCK cells. Cells were microinjected with FITC-dextran solutions and incubated for
4-6 h at 37°C before photobleaching measurements. As
seen by confocal microscopy in Fig. 3 A, only the cytoplasm or nucleus was stained in cells microinjected with FITC-dextrans of >70 kD. For smaller FITC-dextrans that
pass through nuclear pores, both the cytoplasm and nucleus
were labeled (Fig. 3 B). The confocal micrographs showed
mild heterogeneity in FITC-dextran labeling throughout
the cytoplasm and nucleus at the ~0.5-µm x,y-resolution obtainable by light microscopy. Some dye-excluding compartments were observed for the larger FITC-dextrans as
found previously in fibroblasts (Luby-Phelps et al., 1986
To determine whether degradation or metabolism of
FITC-dextrans or FITC-Ficolls occurred under the conditions of our experiments, size-exclusion chromatography
was done on fresh vs cytoplasm-exposed dye. Cytoplasm
was loaded by a 5-min incubation with 10 mg/ml FITC-dextran (70 kD) or FITC-Ficoll (fraction c) while gently rocking in the presence of 425-600-nm-diam glass beads
(McNeil and Warder, 1987 Fig. 4 A shows representative photobleaching recovery
curves for FITC-dextrans and FITC-Ficolls in cytoplasm
of MDCK cells. Compared with the photobleaching data
obtained in aqueous solutions (Fig. 2 A), the recoveries
were slower and incomplete, particularly for the 2,000-kD
FITC-dextran. Fig. 4 B shows that the shape of recovery
curves (shown for small and large macromolecules) was essentially the same as that for fluorescein (same dashed
curve as in Fig. 2 B). Data from a large set of measurements are summarized in Fig. 4 C as t1/2 (D, mobile dye;
righthand axis) vs gyration radius, RG. The recovery half-times increased approximately linearly; however, the slope
of the t1/2 vs RG plot was increased about fourfold for
FITC-dextran and FITC-Ficoll diffusion in cytoplasm compared with that in aqueous solution (shown for comparison). Fig. 4 D shows the ratio of solute diffusion in
cells vs aqueous solutions (D/Do) as a function of solute
size. Although FITC-dextran and FITC-Ficoll diffusion in
MDCK cell cytoplasm was significantly slower than that in
aqueous solutions, D/Do did not depend on dextran size:
there was no evidence of size-dependent solute sieving. This result does not agree with the findings of Luby-Phelps et al. (1986, 1987) for FITC-labeled dextrans and
Ficolls in cytoplasm of Swiss 3T3 fibroblasts, where D/Do
decreased continuously by a factor of 5 for increasing molecular size. Fig. 4 E shows that fluorescence recovery was
>75% complete for dextrans and Ficolls of RG <300 Å.
The little recovery found for the 2,000-kD FITC-dextran and FITC-Ficoll fractions with RG >400 Å (not shown)
did not permit accurate determination of the diffusion coefficients of the mobile fraction. The phenomenon of incomplete photobleaching recovery is investigated further
below.
To determine whether the absence of size-dependent
solute sieving is a cell-specific finding, similar measurements were carried out in Swiss 3T3 fibroblasts, the same
cell type studied by Luby-Phelps et al. (1986, 1987). In general, we found that microinjection was technically easier
for the fibroblasts and the data had less variability. Fig. 5
A shows a nearly linear dependence of t1/2 on RG for
FITC-dextran and FITC-Ficoll diffusion in cytoplasm. The
relative FITC-dextran diffusion coefficient in cytoplasm vs aqueous solutions (D/Do) was similar to that in MDCK
cells and again independent of solute molecular size (Fig. 5
B). For comparison, the data reported by Luby-Phelps et
al. (1986) for FITC-dextran diffusion in the cytoplasm of
Swiss 3T3 fibroblasts are shown (see Discussion). Fig. 5 C
shows that the percentage fluorescence recovery is decreased for very large solutes as was found in Fig. 4 E for
MDCK cells. In the Luby-Phelps et al. (1986, 1987) studies, the reported percentage recovery values were ~100%
for FITC-dextrans and FITC-Ficolls of RG <200 Å, decreasing to 84 and 64% for FITC-Ficolls of 227 and 248 Å,
respectively. In Fig. 5 C, the decreased recovery of the
FITC-Ficoll compared with the FITC-dextran at RG ~280
Å may be related to the more spherical shape of the Ficoll.
Reversible FITC-dextran Photobleaching at 37°C
The measurements in Figs. 2, 4, and 5 were carried out at
23°C in cells incubated for 4-6 h at 37°C after microinjection. To determine whether the temperature at the time of
measurement could account for the differences between
the data here and the results of Luby-Phelps et al. (1986, 1987), experiments were done at 37°C. Fig. 6 shows recovery curves for 3T3 fibroblasts microinjected with the 580-kD FITC-dextran. The recovery curve at 23°C (top curve)
is similar to that shown for MDCK cells in Fig. 4 A. However, after warming the same cells to 37°C, the same
bleach pulse produced more photobleaching and a remarkably faster recovery (second curve), with a t1/2 many
times less than that for the same FITC-dextran in aqueous
solution. To determine whether the recovery was related to FITC-dextran translational diffusion, photobleaching
was carried out on the same cells using a ×40 objective in
place of the ×20 objective lens. It was predicted that if the
fluorescence recovery was related to translational diffusion, then t1/2 would be 3-4 smaller with the ×40 objective
because of the smaller spot size (see Fig. 8 A below). However the t1/2 was unchanged (Fig. 6, third curve from top).
The fast recovery at 37°C, the independence of t1/2 on spot
size, and the increased bleach depth suggested reversible
photobleaching, a process that we characterized recently for fluorescein photobleaching in air-saturated viscous solutions and cells (Periasamy et al., 1996
Characterization of Partial Fluorescence Recovery
The incomplete recovery in photobleaching experiments
has been attributed to a lack of mobility of a fraction of
dye molecules because of microcompartmentation or irreversible immobilization. Recently, for photobleaching studies of membrane proteins, an alternative explanation for
incomplete recovery has been proposed based on anomalous subdiffusion (Feder et al., 1996 To determine whether mobile macromolecular solutes
could affect the percentage recovery, the translational diffusion of various FITC-dextrans was measured in solutions containing (unlabeled) dextrans of different sizes
(40, 70, 500, 2,000 kD), each at a concentration of 15% vol.
Kao et al. (1993)
Another possible explanation for the incomplete fluorescence recovery is light-induced covalent interaction of
the FITC-dextrans with immobile intracellular components. We think that this explanation is unlikely because of
the high percentage recovery for the smaller FITC-dextrans and FITC-Ficolls, and because a covalent complex should not exclude unbleached FITC-dextran molecules
from entering the bleached zone. Photobleaching measurements were carried out on MDCK cells at 6 h after microinjection with the fraction d FITC-Ficoll. For bleach
depths of 16 and 44%, the percentage recoveries were 56 and 59% (each ±3%). The results did not depend upon
whether the bleach depth was varied by changing bleach
beam intensity or bleach time (0.1-1 ms), suggesting that
the incomplete recovery is not due to photochemical reaction(s) and/or light-induced alterations of various intracellular components.
In our prior photobleaching study of BCECF diffusion
in cell cytoplasm (Kao et al., 1993 Fig. 7 B shows representative recovery curves for the
580- and 2,000-kD FITC-dextrans in MDCK cell cytoplasm. Interestingly, cell shrinking (450 mosM) was associated with a remarkable decrease in percentage recovery
for the 580-kD dextran. Cell swelling (150 mosM) was associated with an increase in percentage recovery for the
2,000-kD dextran, where little recovery was found under
isosmolar conditions. The results of a series of measurements are summarized in Fig. 7, C and D. Averaged D/Do
values for FITC-dextran diffusion were 0.66 (150 mosM),
0.26 (300 mosM), and 0.20 (450 mosM), and independent
of FITC-dextran size. The 2.5-fold increase in D/Do for the
swollen cells and the 0.77-fold decrease in shrunken cells are similar to corresponding values of 4 and 0.6 reported
for BCECF diffusion in 3T3 fibroblasts that were swollen
and shrunken to the same extent (Kao et al., 1993 The data above suggest that the aqueous phase of cytoplasm does not sieve solutes with sizes up to at least 500 kD, but might contain microcompartments that restrict
solute diffusion. A potentially more plausible explanation
that does not require the presence of distinct microcompartments is anomalous subdiffusion (Saxton, 1994 If FITC-dextran residence in fixed microcompartments
is responsible for the incomplete recovery, and if the sizes
of the putative diffusion-restricting compartments are as
large as ~1 µm, then the percentage recovery might depend upon laser beam spot size. Spot size was varied from
~4 to ~1.5 µm by using different objective lenses from
×20 to ×60 magnification. Experiments were carried out
with MDCK cells containing the 580-kD FITC-dextran.
Fig. 8 A shows representative recovery curves. With decreasing spot size, the recovery became faster (decreased
t1/2) as expected (Fig. 8 A). However, neither D/Do nor the
percentage fluorescence recovery was affected significantly by spot size. These findings indicate that solute diffusion is relatively rapid and unrestricted over regions of
the cell at least on the order of ~4 µm. The independence of apparent diffusion on spot size is in marked contrast
with photobleaching results in biological membranes,
where microcompartmentation, corralling, and anomalous
diffusion effects have been postulated.
Macromolecule Diffusion in Nucleus
Fig. 9 summarizes spot photobleaching measurements of
3T3 fibroblasts and MDCK cells in which the nucleus was
microinjected (or cytoplasm microinjected for the smaller
dextrans that diffuse across nuclear pores). Although
more variability in the data was found than in that for cytoplasm, the results indicate that macromolecule diffusion
in the nucleus is about fourfold slower than in aqueous solutions. There was no systematic dependence of D/Do on
solute size and the percentage of fluorescence recovery
decreased slightly with increasing solute size.
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 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 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 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 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 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.
; Mastro and Keith, 1984
; Porter, 1984
;
Luby-Phelps, 1994
). The initial descriptions of cytoplasm
were in terms of a viscous gelatinous mass without internal
structure. However, it has become clear that cytoplasm contains dissolved solutes and macromolecules in a complex array of microtubules, actin, and intermediate filaments organized into a lattice-like mesh. Popular pictorial
representations of the aqueous environment within cells
(as those by Goodsell based on measured solute concentrations; Goodsell, 1991
) suggest that the crowding might
seriously hinder solute diffusion (Fulton, 1982
)
a major
determinant of metabolism (Welch and Easterby, 1994
),
transport phenomena, signaling, and cell motility.
). Although initial estimates of this
parameter suggested relatively high values (4-100 cP, 1 cP
is viscosity of water; for review see Verkman, 1991
), more recent measurements of the rotation of small solutes by
time-resolved fluorescence anisotropy (Fushimi and Verkman, 1991
) indicated that cytoplasmic fluid-phase viscosity
in a variety of mammalian cell types (1.1-1.4 cP) is similar
to that of water. The same conclusion was reached by an
independent approach involving ratio imaging of a viscosity-sensitive fluorescent probe (Luby-Phelps et al., 1993
).
Low fluid-phase viscosity was also found in the aqueous
compartment of the nucleus (Fushimi and Verkman, 1991
) and in the thin layer of cytoplasm adjacent to the cell plasma membrane (Bicknese et al., 1993
).
measured the translational mobility of a
small fluorescent probe (BCECF) in the cytoplasm of
Swiss 3T3 fibroblasts using fluorescence recovery after
photobleaching (FRAP).1 Instrumentation and calibration
procedures were developed for accurate measurement of
intracellular solute diffusion coefficient. BCECF translational diffusion in cytoplasm was approximately four times
slower than in water. Three independently acting factors were identified that accounted quantitatively for the fourfold slowed diffusion: (a) slowed diffusion in fluid-phase
cytoplasm, (b) probe binding to intracellular components,
and (c) probe collisions with intracellular components.
The latter factor, probe collisions, was determined to be
the principal diffusive barrier that slowed the translational
diffusion of small solutes. The resistance to diffusion because of cytoplasmic solids was determined from BCECF diffusion coefficients measured as a function of cell volume. The exponentially increasing resistance to BCECF
diffusion with decreasing cell volume was modeled to give
the cytoplasmic content of cell solids and the resistance to
diffusion imposed by the solids. Recently, BCECF translational diffusion in membrane-adjacent cytoplasm was
measured by total internal reflection-FRAP to be about
twofold lower than that in bulk cytoplasm (Swaminathan
et al., 1996
), suggesting slowed diffusion resulting from the
high density of proteins near the cell membrane.
, 1987
), spot
photobleaching was used to measure the translational diffusion of larger solutes: microinjected, fluorescently labeled
dextrans and Ficolls. As dextran or Ficoll molecular size
was increased, diffusion in cytoplasm progressively decreased relative to that in water, suggesting a cytoplasmic
"sieving" mechanism that was proposed to involve the
skeletal mesh. The possibility of solute sieving was supported by diffusion measurements in concentrated artificial solutions containing F-actin and albumin (Hou et al., 1990
). If correct, such a sieving mechanism would have important implications concerning the restricted movement
of macromolecule-sized solutes in the cytoplasm.
to
define the determinants of mobility of macromolecule-sized
solutes in cell cytoplasm. Based on the prior work by
Luby-Phelps and colleagues mentioned above, it was anticipated that size-dependent sieving of FITC-dextrans would be found. Our plan was to confirm and extend these
observations by: (a) comparing the sieving properties of
epithelial cells vs fibroblasts, (b) determining whether cytoplasmic sieving required an intact actin cytoskeleton, (c)
testing whether solute sieving occurred in nucleoplasm,
and (d) developing a quantitative model of sieving from
data on the cell volume dependence of solute diffusion. Contrary to expectations, sieving was not found in fibroblasts or epithelial cells for solutes of apparent molecular
size to at least 500 kD. An important result was that the
translational diffusion of large solutes in cytoplasm and
nucleus was slowed three- to fourfold relative to their diffusion in water, similar to results for diffusion of a small
(~0.5 kD), metabolite-sized solute (Kao et al., 1993
). Taken
together with our previous studies of the mobility of small
solutes (Fushimi and Verkman, 1991
; Kao et al., 1993
;
Bicknese et al., 1993
; Swaminathan et al., 1996
), the results
here do not support the view that cytoplasm and nucleus are so crowded that solute motion is seriously impeded.
Materials and Methods
. 650 mg of Ficoll was dissolved in 9 ml
of freshly prepared 1.35 M sodium chloroacetate (pH 7). After addition of
2.5 ml of 10 N NaOH, the reaction mixture was incubated at 25°C for 3 h.
The reaction was terminated by addition of 0.1 ml of 2 M NaH2PO4 and titration to pH 7 with 6 N HCl. The product was dialyzed against water, lyophilized, and resuspended in 25 ml water. 3.3 g of ethylenediamine dihydrochloride followed by 650 mg of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride was added and the mixture was stirred at
25°C while maintaining a pH of 4.7. The resultant amino-Ficoll was dialyzed extensively and lyophilized. 500 mg of amino-Ficoll was dissolved in
10 ml of cold 100 mM NaHCO3 buffer, pH 8.2, 78 mg of FITC was added,
and the mixture was warmed to 20°C and stirred for 3 h. The FITC-Ficoll
was dialyzed extensively and lyophilized.
). For most experiments, the laser beam power
was set to 50-100 mW (488 nm) and the attenuation ratio (the ratio of
bleach to probe beam intensity) was set to 5,000-15,000.
Fig. 1.
Schematic of the photobleaching apparatus. The laser
beam is modulated by acoustooptic modulators (AOM 1 and
AOM 2) and fluorescence is recorded from a focused spot using a
photomultiplier that is gated off during the photobleaching pulse.
See Materials and Methods for details.
[View Larger Version of this Image (21K GIF file)]
) showed sustained cell swelling (150 mosM) and shrinking (450 mosM) under these conditions. In some experiments, the cell bathing solution was saturated with oxygen by bubbling with 100% oxygen for 15 min and maintaining the cell chamber in an oxygen atmosphere (Swaminathan et al., 1996
). Unless otherwise specified,
measurements were done at 23°C in a temperature-controlled darkroom;
studies at 37°C were done with a heated stage with feedback temperature
control.
, fluorophore diffusion coefficients (D)
were determined from recovery t1/2 using standards consisting of thin
(2-10 µm) layers of 1 mM fluorescein in water-glycerol solutions of specified viscosity (glycerol concentration measured by refractometry). The t1/2
value was determined as the time after the bleach pulse when the fluorescence was equal to the mean of the fluorescence just after the bleach and
when essentially all recovery occurred (postbleach). The fluorescence just
after the bleach was determined operationally as the average fluorescence at 50-75 µs after the end of the bleach. The postbleach fluorescence was
generally determined as the average fluorescence at a time equal to 25-
100 t1/2 intervals; as discussed below, the postbleach signal remained essentially constant beyond ~25 t1/2 intervals. The percentage recovery was
computed from pre- and postbleach fluorescence and the percentage
bleaching. t1/2 was computed using a quadratic polynomial fitted to a small
interval of the recovery curve surrounding an estimated half-time that was
computed from an initial exponential regression of the full recovery curve.
). Cells were
viewed using a ×60 oil immersion objective (NA 1.4) with a z-resolution
of ~1 µm.
Results
), the recovery t1/2 was taken as a quantitative measure of fluorophore translational diffusion. The use of a
single parameter t1/2 to describe the recovery curve formally requires that the curve shape be the same for each
compound. Fig. 2 B shows that, after scaling each curve in
the time direction (to account for different diffusion
rates), recovery curve shapes were essentially the same as
that for fluorescein. These results also indicate that the
size-fractionated FITC-dextrans and FITC-Ficolls are fairly
homogeneous in size. Experiments done with singly size-fractionated FITC-dextrans and FITC-Ficolls also showed
essentially identical curve shapes and t1/2 values; however,
curve shapes were different for large unfractionated FITC-dextrans (not shown) because of contamination by smaller
dextrans.
Fig. 2.
Photobleaching recovery measurements on aqueous
solutions of FITC-dextrans and
Ficolls. (A) Recovery curves for
fluorescein (0.1 mM) and indicated FITC-dextrans and Ficolls
(4 mg/ml) in PBS at 23°C.
Bleach time was 0.5 ms, solution
layer thickness was 5 µm, and
the ×20 objective lens was used.
Final fluorescence (at 10 s) was
98-101% of initial (prebleach)
fluorescence. (B) Curves shown
in A were scaled in time and amplitude to compare shape. Identical dashed curves are shown
overlying each data curve to facilitate visual comparison. (C)
Averaged recovery half-times (t1/2, mean ± SEM, n = 15-25)
for FITC-dextrans and Ficolls.
Purchased FITC-dextrans (molecular sizes indicated) and synthesized FITC-Ficolls were size
fractionated twice (see Materials
and Methods). Gyration radii
(RG) were computed according
to Luby-Phelps et al. (1986) and
a measured fluorescein diffusion
coefficient of 2.6 × 106 cm2/s
from the relation, RG (in Å) = 2.74 t1/2 (in ms).
[View Larger Version of this Image (41K GIF file)]
;
Provance et al., 1993
), but no vesicular staining was seen.
The inset in Fig. 3 A shows a wide-field epifluorescence
micrograph of a cell at lower magnification as visualized in
the photobleaching measurements. The laser probe beam
(bright dot indicated by arrow) was directed to a spot in the cytoplasm. For these photobleaching studies, the
bleached volume was generally <2% of the cytoplasmic or
nuclear volume, and little fluorophore diffusion occurred
during the brief bleach pulse.
Fig. 3.
Micrographs of labeled MDCK cells. (A) Cell cytoplasm or nucleus was microinjected with 580-kD FITC-dextran as
described in Materials and Methods. Confocal micrographs
(z-resolution ~1 µm) were obtained using a ×60 oil objective
(N.A. 1.4) and cooled CCD camera detector. (Inset) Low magnification (×20) wide-field micrograph showing laser spot (arrow).
(B) Confocal micrograph of MDCK cells microinjected with 20-kD FITC-dextran. (C) Size-exclusion chromatograms of FITC-dextran (70 kD) and FITC-Ficoll (fraction c) before (filled circles) vs after (open circles, dashed lines) remaining in cytoplasm
for 6 h. No change in size distribution was found. Bars, 20 µm.
[View Larger Versions of these Images (19 + 65K GIF file)]
). Cells were homogenized after
6 h and the soluble fraction was subject to size-exclusion
chromatography on Sepharose CL-6B. Representative
chromatograms in Fig. 3 C show no change in FITC-dextran or FITC-Ficoll size after a 6-h exposure to cytoplasm.
Fig. 4.
Photobleaching recovery measurements of
FITC-dextran and Ficoll diffusion in MDCK cell cytoplasm. (A) Representative
spot photobleaching recovery data (0.5 ms bleach time,
×20 objective) for cells microinjected with indicated
FITC-dextrans and Ficolls. Cells were incubated for 4-6 h
at 37°C before measurements
done at 23°C. (B) Curves
shown in A were scaled in
time and amplitude to compare shape. Identical dashed
curves over each experimental curve are the same as in
Fig. 2 B. (C) Dependence of
recovery half-time (t1/2) and
deduced diffusion coefficient (D) on gyration radius.
Each point is the mean ± SEM for 30-45 independent
measurements done with
FITC-dextrans (open circles)
and FITC-Ficolls (filled circles). For comparison, the t1/2
vs RG data are shown for diffusion in aqueous solutions
(from Fig. 2 C). (D) Ratio of
the relative FITC-dextran
diffusion coefficient in cells to
that in aqueous solution (D/
Do) as a function of RG. (E)
Percentage fluorescence recovery as a function of RG.
[View Larger Version of this Image (28K GIF file)]
Fig. 5.
Photobleaching recovery measurements of FITC-dextran and Ficoll diffusion in cytoplasm of Swiss 3T3 fibroblasts.
Measurements were carried out as in Fig. 4. (A) Dependence of
recovery half time (t1/2) and deduced diffusion coefficient (D) on
RG. Each point is the mean ± SEM for 30-45 independent measurements done with FITC-dextrans (open circles) and FITC-Ficolls (filled circles). For comparison, the t1/2 vs RG line data are
shown for diffusion in aqueous solutions. (B) Ratio of the relative
FITC-dextran diffusion coefficient in cells to that in aqueous solution (D/Do) as a function of RG. For comparison, data from
Luby-Phelps et al. (1986) are plotted on the same scale. (C) Percentage fluorescence recovery as a function of RG.
[View Larger Version of this Image (13K GIF file)]
; Swaminathan et
al., 1996
) and for photobleaching of the green fluorescent
protein (Swaminathan et al., 1997
). If triplet state relaxation is responsible for the fast recovery at 37°C, then it
was predicted that 100% oxygen should accelerate triplet
state relaxation so that it would be unobservably fast and
only the irreversible bleach process would be observable
(Periasamy et al., 1996
). Fig. 6 (bottom curve) shows that
100% oxygen remarkably slowed recovery, giving a t1/2 only ~30% less than that at 23°C. The recovery after
100% oxygen was strongly dependent on spot size (not
shown). A reversible photobleaching process was also observed for the 20-kD FITC-dextran in cytoplasm at 37°C.
There was no effect of 100% oxygen on the fluorescence
recovery kinetics for the 20- and 580-kD FITC-dextrans in
cytoplasm at 23°C. These results indicate the existence of a
reversible photobleaching process for FITC-dextrans in
cytoplasm at 37°C in which fluorescence recovery is rapid
and unrelated to solute translational diffusion; an unrecognized reversible photobleaching process could have influenced the interpretation of previous similar studies (see
Discussion). The reversible bleach process was not observed at 23°C so that subsequent measurements were
done only at 23°C.
Fig. 6.
Reversible photobleaching of FITC-dextran in cytoplasm of Swiss 3T3 fibroblasts at 37°C. Spot photobleaching experiments were done in cells microinjected with 580-kD FITC-dextran (RG 291 nm). Measurements were made as indicated at
23°C vs 37°C, in solutions equilibrated with air vs 100% O2, and
with the ×20 vs ×40 objectives.
[View Larger Version of this Image (20K GIF file)]
Fig. 8.
Effect of spot size on apparent solute diffusion. (A)
Representative photobleaching recovery curves for MDCK cells
microinjected in the cytoplasm with 580-kD FITC-dextran. (B)
Relative diffusion in cytoplasm vs water (D/Do) and percentage
recoveries shown (n = 4). Objective lenses were: ×20 (dry, N.A.
0.75), ×40 (dry, N.A. 0.55), and ×60 (oil, N.A. 1.4). Bleach times
were 1 (×20), 0.5 (×40), and 0.2 ms (×60).
[View Larger Version of this Image (24K GIF file)]
). We conducted several types of experiments to investigate the low percentage recovery for the largest solutes, including measurements in
concentrated dextran solutions, in swollen and shrunken
cells, and in cells using different bleach beam intensities,
beam spot sizes, and recovery time acquisitions.
reported that diffusion of fluorescein in
15% vol dextran was slowed about threefold compared with that in water, and that the magnitude of slowing did
not depend on dextran size. The percentage recovery for
photobleaching of FITC-dextrans in the dextran solutions
was consistently >97%, suggesting that simple mobile obstacles in the concentration range studied here cannot account for the incomplete fluorescence recovery in cells.
Fig. 7 A shows the dependence of FITC-dextran diffusion coefficient on the size of unlabeled dextran. Diffusion of
the smallest FITC-dextran (4 kD) did not depend on the
size of the larger unlabeled dextrans, in agreement with
the paradigm that it is the concentration but not the size of
large obstacles that determines the diffusion coefficient of
a smaller fluorescent probe (Phillies, 1987
, 1989
; Furakawa
et al., 1991
). Diffusion of the larger FITC-dextrans was
slowed with increased size of the unlabeled dextrans; the
dependence showed a saturation phenomenon in which
diffusion became independent of dextran size when the size of the unlabeled dextran was large relative to the
FITC-dextran. In cells (Figs. 4 D and 5 B), it was found
that D/Do for the FITC-dextrans was ~0.26 and independent of dextran size. From the results in artificial dextran
solutions in Fig. 7 A, it is concluded that the effective concentration of "obstacles" in cytoplasm is just under 15%,
and that the effective size of the obstacles is >~70 kD.
Fig. 7.
Influence of cell
volume on diffusion of FITC-dextrans in MDCK cell cytoplasm. (A) Photobleaching of
FITC-dextrans in 5-µm solution films containing 15% vol
unlabeled dextran (10 mg/
ml). Recovery t1/2 as a function of the size of the nonfluorescent dextran. (B) MDCK
cells were microinjected
with FITC-dextrans, incubated for 4-6 h, and then
subjected to photobleaching
measurements after a 5-20-min incubation in PBS (300 mosM), PBS diluted 1:1 with
water (150 mosM), and PBS
containing 150 mM sucrose
(450 mosM). Bleach time was
1 ms and the ×20 objective
lens was used. Representative recovery curves for 580- and 2,000-kD FITC-dextran and indicated solution osmolalities. (C) Dependence of
percentage recovery on RG.
(D) Dependence of D/Do
on RG.
[View Larger Version of this Image (28K GIF file)]
), analysis of recovery kinetics as a function of cell volume provided quantitative
information about the characteristics of the diffusive barrier imposed by cell solids. Similar experiments were carried out for diffusion of FITC-dextran in the cytoplasm of
MDCK cells. After microinjection and incubation for 6 h,
photobleaching measurements were made on cells bathed in PBS (300 mosM), PBS diluted 1:1 with water (150 mosM), and PBS containing excess 150 mM sucrose (450 mosM). If incomplete fluorescence recovery is due to a
percolation phenomena (Almeida and Vaz, 1995
) or to the
presence of apparent "submicroscopic compartments" in
which solute diffusion is restricted, then it is predicted that
the percentage recovery would increase with cell swelling
and decrease with cell shrinkage. If diffusion of the mobile
fraction of FITC-dextran is restricted by cell solids (as was
the case for BCECF), then it is predicted that FITC-dextran diffusion (D/Do) would increase with cell swelling because of the decreased concentration of solids, and would
correspondingly decrease with cell shrinkage. Quantitative
values for percentage recovery and D/Do permit the testing of various models as described below.
). The
dependence of D/Do on cell volume supports the view that
the change in FITC-dextran diffusion results from a
change in the concentration of dissolved cytoplasmic solids.
), where
the diffusion coefficient of a particular solute molecule is
time dependent because of diffusion into regions of differing viscous properties. Anomalous subdiffusion has been proposed to account for some photobleaching results for
membrane components (Feder et al., 1996
), where lateral
diffusion is orders of magnitude slower than that predicted
for simple lipid membranes. Because anomalous diffusion
models do not involve distinct subcompartments that restrict solute diffusion, it is predicted that fluorescence recovery will continue to occur, albeit slowly, even over a
time scale of 10-10,000 (or more) t1/2. For MDCK cells
containing fraction d FITC-Ficoll in the cytosol, t1/2 was
~450 ms and the fraction recovery (at 10 s, ~22 t1/2 intervals) was 0.58 (Fig. 4 E). Measurements were done to determine whether the percentage recovery changed over
the interval 10-240 after bleaching. Averaged percentage recovery values were: 0.59 (10 s), 0.56 (60 s), and 0.56 (240 s)
(n = 3). Similar studies for the 2,000-kD FITC-dextran
also showed no significant fluorescence recovery between
10 and 240 s. In these studies the probe beam was turned
off after the bleach pulse until the late measurement time
to ensure no photobleaching by the probe beam. The absence of further recovery at late times does not provide evidence for an anomalous subdiffusion model for solute
translational movement in cells; however, recovery over
very long times (up to 105 t1/2) could not be studied because of mechanical instrument drift and laser beam drift/
fluctuations. Although extremely slow recovery could formally be described in terms of anomalous subdiffusion or
percolation, such recovery has also been classified as "long-tail" kinetics (Nagle, 1992
).
Fig. 9.
Photobleaching recovery measurements of FITC-dextran diffusion in nuclei of MDCK cells and Swiss 3T3 fibroblasts.
Experiments were done as in Figs. 4 and 5 after intranuclear injection of FITC-dextrans. (A) Dependence of t1/2 on FITC-dextran RG. For comparison, the t1/2 vs RG data are shown for diffusion in aqueous solutions. (B) Dependence of D/Do on RG. (C)
Dependence of percentage recovery on RG.
[View Larger Version of this Image (17K GIF file)]
Discussion
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.
). 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.
), 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.
), 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.
; 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
).
Received for publication 18 March 1997 and in revised form 12 May 1997.
Please address all correspondence to Alan S. Verkman, Departments of Medicine and Physiology, 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA 94143-0521. Tel.: (415) 476-8530. Fax: (415) 665-3847. e-mail: verkman{at}itsa.ucsf.eduWe thank Drs. H. Pin Kao, N. Periasamy, and M. Saxton for helpful discussions; Dr. R. Swaminathan for the analysis software; and Katherine Chen for cell culture.
This work was supported by grants DK43840, DK35124, and HL42368 from the National Institutes of Health. O. Seksek was supported in part by a fellowship from the California Lung Association.
D, diffusion coefficient; FRAP, fluorescence recovery after photobleaching; RG, gyration radius.
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