3301
Journal of Cell Science 107, 3301-3313 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
ATP depletion: a novel method to study junctional properties in epithelial
tissues
I. Rearrangement of the actin cytoskeleton
Robert Bacallao1,*, Alan Garfinkel2, Steven Monke2, Guido Zampighi3 and Lazaro J. Mandel4
1Division of Nephrology and Hypertension, Department of Medicine S-208, Northwestern University, 303 E Chicago Avenue,
Chicago, IL 60611, USA
2Department of Physiological Science and 3Department of Anatomy and Cell Biology, University of California, Los Angeles, CA,
USA
4Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
*Author for correspondence
SUMMARY
The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine
kidney (MDCK) and JTC cell lines. In prior studies, it was
shown that ATP depletion uncouples the gate and fence
functions of the tight junction. This paper extends these
observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and
confocal fluorescence microscopy in combination with
computer-aided three-dimensional reconstruction. Marked
regional differences in the sensitivity to the effects of ATP
depletion were observed in the actin cytoskeleton. Actin
depolymerization appears to first affect the cortical actin
network running along the apical basal axis of the cell. The
next actin network that is disrupted is the stress fibers
found at the basal surface of the cell. Finally, the actin ring
at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with
ultrastructural changes in tight junction strands and the
loss of the tight junction’s role as a molecular fence. During
the process of actin network dissolution, polymerized actin
aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP
depletion suggest that this may be a useful method to study
junctional complex formation and its relationship to the
actin cytoskeletal network.
INTRODUCTION
microvilli, a cortical network and the stress fibers at the base
of the cells.
Several studies in a number of epithelia have suggested that
the actin cytoskeleton interacts specifically with the tight
junction and the zonula adherens (Hirokawa and Tilney, 1982;
Madara et al., 1986; Madara and Dharmsathaphorn, 1985;
Madara, 1989; Meza et al., 1980, 1982). The junctions and the
apical ring are located at the same level. Drugs, such as
cytochalasin B and D, that depolymerize fibrillar actin (Factin) into globular actin (G-actin) increase the diffusion of
ions and small molecules through the tight junction (Rassat et
al., 1982; Madara, 1988; Madara et al., 1986, Kellerman et al.,
1990). Prior work has shown that ATP depletion can be used
to separate the gate and fence functions of the tight junctions
(Mandel et al., 1993). Ultrastructural analysis showing the
close apposition between the tight junction strands and the
actin cytoskeleton further support the idea that the actin
cytoskeleton modulates tight junction permeability (Madara,
1987). This study explores the modification of the actin
cytoskeleton after ATP depletion and attempts to correlate
these changes to both the gate and fence function of the tight
Many of the morphological features that characterize epithelial tissues result from the specialized arrangement of both the
actin cytoskeleton and intercellular junctions connecting the
cells. In the case of MDCK cells, some steps involved in the
formation of the cytoskeleton have been elucidated (R.
Bacallao et al., unpublished data; Mills and Lubin, 1986). As
early as 6 hours in culture, the actin fibers form a ring that circumscribes the plasma membrane and defines their apical pole.
This apical ring appears to precede and to form independently
of the intercellular junctions such as tight junctions and zonula
adherens. However, when intercellular junctions form, the
apical ring and the junctions are located at the same level in
the cells. The establishment of the actin cytoskeleton of
MDCK cells is completed with the formation of stress fibers
located at the surface that attaches to the substratum, followed
by the formation of a cortical network extending along the
entire apico-basal axis of the cell. Thus, in confluent MDCK
epithelia, the actin cytoskeleton exhibits three distinct arrangements: an apical ring that includes and circumscribes the
Key words: actin cytoskeleton, ATP depletion, tight junction
3302 R. Bacallao and others
junction. We show that the actin cytoskeleton of MDCK
epithelia became altered after 20-30 minutes of ATP depletion.
The three actin arrangements (the cortical actin network, the
apical ring and stress fibers) show varying degrees of sensitivity to the effects of ATP depletion. The loss of the molecular
fence function correlates with disruption of the actin ring after
60 minutes of ATP depletion.
ference between the measured TERs at the beginning (prior to
inhibitor treatment) and end of each experiment (after EGTA
addition). The MDCK cells used for these experiments are a low resistance clone (Fuller and Simons, 1986). The initial TER of these cells
grown on the Nunc filters was consistently lower than reported by
other investigators using this clone (Stevenson et al., 1988). Nevertheless as previously described these cells have well-developed tight
junctions and normal gate and fence functions (Fuller and Simons,
1986; Mandel et al., 1993).
MATERIALS AND METHODS
Depolymerization of the microtubule network
In some experiments, the effects of microtubule depolymerization
were studied. For these studies, the cells were washed in the
modified Ringer’s buffer and incubated at 37°C for 2.5 hours. The
cells were then transferred to the modified Ringer’s buffer containing 30 µM nocodazole at 4°C for 30 minutes. Preliminary experiments had shown that 30 minutes of such treatment completely
depolymerized the microtubules, as determined by immunofluorescence microscopy and by western blot analysis of the pool of polymerized and depolymerized tubulin (results not shown). The
samples were then separated into two groups, one group subjected
to ATP depletion as described above and the second group served
as an ATP replete control.
Cell culture
MDCK-clone II (Fuller and Simons, 1986) and JTC cells (Fineman
et al., 1992) were grown in DMEM media supplemented with 5%
newborn calf serum (Gemini Bioproducts, CA), 2 mM glutamine
(Sigma, MO) and penicillin/streptomycin (Sigma, MO). The cells
were incubated at 37°C in an air/5% CO2 atmosphere and were
passaged twice a week. The cells were plated on Nunc filter supports
at a low density (50,000 cells/cm2) and were allowed to grow to confluence over a 5 day period. Experiments were performed on confluent
cells grown on these filter supports, as previously described (Bacallao
et al., 1989).
ATP depletion
Since cultured cells display a combination of oxidative and glycolytic
energy metabolism, ATP depletion required inhibition of both
pathways (Mandel, 1986). Inhibition of glycolysis was accomplished
by initially washing the filters in a glucose-free modified Ringer’s
buffer supplemented with 2 mM glutamine as the sole metabolic
substrate followed by a 3 hour incubation in this buffer at 37°C to
deplete the tissue of endogenous metabolic substrates. The composition of this modified Ringer’s buffer was (in mM): NaCl (115),
NaHCO3 (25), K2HPO4 (5), MgSO4 (2), CaCl2 (1), and glutamine (2).
This solution was bubbled with air/5%CO2 to obtain a pH of 7.4.
After the initial incubation, rapid ATP depletion was achieved by
transferring the filters to modified Ringer’s buffer containing the
mitochondria inhibitor antimycin A (10 µM) and the glycolytic
inhibitor 2-deoxyglucose (10 mM). At various times following the
addition of the metabolic inhibitors, the cells on the filters were precipitated with ice-cold 3% perchloric acid. After obtaining a neutralized extract, the samples were analyzed for ATP, ADP, AMP and
hypoxanthine (HX) contents by high pressure liquid chromatography, as previously described (Mandel et al., 1988), and normalized
to their protein content. The latter was measured by the Bradford
(1976) method.
Samples designated as controls were obtained from cultures
subjected to either the initial 3 hour incubation in the modified
Ringer’s buffer, or incubated for a fourth hour in this buffer. No differences were noted in any of the measured properties between these
two groups, nor was there a measurable deterioration due to the 3 hour
initial incubation.
Measurement of the transepithelial resistance (TER)
After the 3 hour preincubation in modified Ringer’s buffer, the filters
were removed from the ring support and mounted in a specially
designed Ussing chamber maintained at 37°C by a water jacket. Cell
monolayers grown on filter supports were initially equilibrated in
modified Ringer’s buffer to obtain the initial TER values. The
solutions were subsequently changed to the same buffer containing
10 mM 2-deoxyglucose and 10 µM antimycin A to achieve ATP
depletion, as before. After 20 minutes, 5 mM EGTA was added to
both solutions to eliminate any remaining tight junctional resistance.
The TER was measured by passing a constant current pulse (5 µA)
through the tissue once per minute and measuring the resulting change
in transepithelial voltage using an automatic voltage clamp (World
Precision Instruments, CN). The initial TER was calculated as the dif-
Antibodies
Antibodies that bind ZO-1 (clone R26), α- and β-tubulin (Amersham,
IL), were used for the immunofluorescence studies. R26 (anti-ZO-1)
was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore, MD, and
the Department of Biology, University of Iowa, Iowa City, IA, under
contract NO1-HD-2-3144 from the NICHD. The actin cytoskeleton
was labeled with Bodipy-phalloidin (Molecular Probes, OR). All fluorescence-conjugated secondary antibodies were obtained from
Jackson Immunoresearch, PA.
Immunofluorescence
The cells were fixed and stained as previously described (Bacallao
et al., 1989). For actin and tubulin staining, the filters were dipped
in 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl 2 (PEM
buffer) and were fixed with 0.25% glutaraldehyde (Polysciences, PA)
in PEM buffer plus 0.1% Triton X-100 (Sigma, MO). The samples
were fixed for 10 minutes at room temperature. The reaction was
quenched with three successive incubations with 1 mg/ml NaBH 4 in
PBS, pH 8.0, for 10 minutes at room temperature. The samples were
washed twice in PBS and were incubated in PBS + 0.1% Triton X100 with 0.2% fish skin gelatin (Sigma, MO). All antibodies were
diluted in this buffer. Antibody incubations were performed at 37°C
for 45 minutes. A second incubation with a fresh sample of antibody
for an additional 45 minutes was performed in all the specimens to
ensure adequate labeling. The cells were washed 6 times with PBS
+ 0.1% Triton X-100 for 15 minutes at room temperature following
each antibody incubation. Following the second antibody labeling,
the cells were washed as described above with the exception that the
final wash step was replaced with three successive washes with PBS
for 5 minutes each.
Samples that were stained for ZO-1 were fixed with 2%
paraformaldehyde (Sigma, MO) prepared as previously described
(Bacallao and Stelzer, 1989). The fixation reaction was quenched as
described above.
After the immunolabeling was completed the samples were postfixed in 4% paraformaldehyde for 30 minutes at room temperature.
The reaction was quenched with 50 mM NH4Cl dissolved in PBS.
The samples were stored at 4°C in PBS. Prior to examination with a
confocal fluorescence microscope the specimen was mounted in 50%
glycerol/PBS with 100 mg/ml of diamino-bicyclo-[2.2.2] octane
(DABCO; Sigma, MO).
ATP depletion: I. Rearrangement of the actin cytoskeleton 3303
Data acquisition, processing and visualization
Data sets were gathered using a confocal scanning laser microscope
(CLSM, Leitz, USA). The optical pathway in a confocal microscope
enables these instruments to collect light from a narrow focal plane
(in the order of 0.2-1 µm), thus discriminating against out-of-focus
information (Inoué, 1990). Each ‘optical section’ is stored in digitized
form (typically 512 × 512 × 1 byte), and a data set may consist of 50
slices at 0.4 µm spacing. The digitized data sets can then be visualized by a variety of methods.
Extended focus images
In an extended focus image, all focal planes are stacked together,
resulting in an image in which every observed structure is in focus.
Figs 4 and 5 are extended focus images encompassing the entire image
from the apical portion of the cell to the filter support.
Three-dimensional image reconstruction
Data sets were transferred to a RISC-type workstation (Kubota Pacific
750), were the intermediate slices were interpolated mathematically
to produce a solid three-dimensional volume of data. The data
volumes were visualized by rendering methods available within the
visualization software AVS (Advanced Visualization Systems).
A data volume consists of a three-dimensional array of voxels
(volume elements), with each voxel having an (x,y,z) address and an
intensity value (a) ranging from 0 to 255, representing the amount of
fluorescence detected at the point (x,y,z). Each voxel is assigned a
color based on its intensity value. In all the figures, the color scale
runs monotonically from the blue end of the spectrum for the lowest
intensities of fluorescence through green and yellow for the intermediate values and to orange and red for the highest values. The color
assignment was chosen to enhance contrasts and make them visible.
This was sometimes necessary, as the intensity values that were
recorded by the microscope sometimes had narrow ranges (on the
order of 60 values out of 256).
In addition to the color assigned to a voxel (x,y,z) on the basis of
its intensity value (a), the software enabled us to independently assign
each voxel an opacity, ranging from 0 (completely transparent) to 1
(completely opaque). This feature was important, because it allowed
us to melt away uninteresting regions and background noise by
rendering them transparent. Without this feature, foreground structures would inevitably obscure objects deeper in the volume set.
Visualization schemes most suited to confocal microscopy are
based on volume rendering methods, in which the observer can see
into the volume, which has been made somewhat transparent. The
advantage of confocal microscopy lies in its ability to image through
the object and collect a three-dimensional volume of data. Creating
opaque solid structures fails to take advantage of the power of 3-D
image collection. The most popular volume rendering methods are
based on ray tracing. A ray from the observer is cast into the object,
and each voxel that it passes through contributes some of its color,
depending on its opacity. The line-of-sight through the ray is assigned
a color by its opacity, and summing the results. This method by itself
tends to produce images that are murky and clouded. To make objects
within the volume more visible, we used gradient-based shading
methods to enhance boundaries between regions with significantly
different intensity values (Toga and Payne, 1990; Bacallao and
Garfinkel, 1994). This has the net effect of enhancing the details
observable in the final images.
Incorporation of fluorescent phosphatydyl choline into the
apical membrane
Fluorescent lipid incorporation in the plasma membrane was achieved
by a modification of the method of Pagano and Martin (1987).
Confluent MDCK cells were incubated for 1 hour at 10°C in normal
Ringer’s buffer on the basolateral side and the same buffer containing a 1:1 mixture of 25 mM defatted bovine serum albumen and
the fluorescent lipid 1-palmitoyl-2-caproyl-sn-glycero-3-phospho-
choline-n-(lissamine rhodamine b sulfonyl) (Avanti Polar LipidsAlabaster, AL) on the apical side. After washing 3 times in regular
buffer at 10°C, the filters were incubated at 37°C in the various
solutions described above and subsequently viewed in the confocal
microscope in either the x-z or the x-y mode to obtain two-dimensional optical sections through the tissues.
Freeze fracture and electron microscopy
Freeze fracture and electron microscopy were performed as described
by Zampighi et al. (1989). The cultured cells were fixed while on the
filters by immersion for 2 hours at room temperature in 3% glutaraldehyde, 4% paraformaldehyde in 0.2 M sodium cacodylate
buffer, pH 7.4. The cells were then infiltrated with 20% glycerol in
0.2 M Na cacodylate buffer for 1 hour at room temperature, followed
by scraping from the filters. Sheets of cells were deposited on Balzer’s
holders with a minimum amount of solution, and frozen by immersion
in liquid propane. Fracture was performed in the freeze-fracture
apparatus at −100°C with a liquid nitrogen-cooled knife. The
fractured surfaces were then shadowed with platinum-carbon at 45°
and carbon at 90°. The resulting replicas were released by digesting
the tissue with 4% sodium hypochloride. The replicas were deposited
on single hole copper grids coated with Formvar for electron
microscopy.
RESULTS
ATP depletion
The course of ATP depletion after metabolic inhibitor
treatment is shown in Fig. 1 for both MDCK and JTC cells. In
both cell types, ATP content fell rapidly to about 50% of initial
values within 5 minutes, to 25% after 10 minutes, and to 15%
after 60 minutes. This drop in ATP was initially mirrored by
concomitant increases in ADP and AMP content, as seen in
Tables 1 and 2. The amount of the latter two nucleotides
increased to the same extent, and the sum of the 3 adenine
nucleotides remained constant after the first 10 minutes of
inhibitor treatment for MDCK cells (Table 1) and 20 minutes
for the JTC cells (Table 2). A continual decline in ADP content
Fig. 1. Intracellular ATP after metabolic inhibition in MDCK and
JTC cells. Rapid ATP depletion was achieved with metabolic
inhibitors, as described in Materials and Methods. The
simultaneously measured contents of ADP, AMP and HX are listed
in Tables 1 and 2. The contents of all these substances were
maintained at a constant level in the absence of metabolic inhibitors
(control).
3304 R. Bacallao and others
Table 1. Adenine nucleotide and hypoxanthine (HX)
content of MDCK cells subjected to various times of
ATP depletion
ATP
ADP
AMP
HX
ΣAXP*
N
Controls
0c
30c
60c
61±9
70±9
68±12
13±4
13±2
18±2
1.4±0.8
2.0±0.8
2.2±0.4
2.8±1.2
1.4±1.0
1.6±0.9
75±9
85±9
88±12
7
6
4
ATP depletion
5
10
20
30
60
30±6†
15±2†
14±2†
10±2†
9±2†
32±5†
18±5
5±2†
20±4†
32±10†
25±5†
26±5†
26±7†
6.4±1.7
6.6±2.0
5.2±1.2
4.8±1.1
3.4±0.9
88±6
72±10
49±5†
41±5†
38±7†
7
7
4
4
4
Time (min)
All contents are in nmoles/mg protein.
*ΣAXP = sum of ATP, ADP and AMP.
†Significantly different from the 0c (control) time point P<0.01.
was observed at the later time points, causing a significant
decrease in total adenine nucleotide content. There was only a
slight accumulation of hypoxanthine (HX), a major adenine
nucleotide breakdown product, suggesting that further hydrolysis occurred (Mandel et al., 1988).
ATP depletion caused decreased transepithelial
resistance (TER)
Intracellular ATP depletion caused the decrease of TER in both
MDCK and JTC cells, as shown in Fig. 2. We have previously
shown that in MDCK cells the fall in TER was rapid and was
preceded by a dramatic decrease in the ATP/ADP ratio, which
fell by 85% in the first 2 minutes (Mandel et al., 1993).
Although the declines in ATP and the ATP/ADP ratio occurred
with a similar time course in the JTC cells, the TER fell much
more slowly in these cells, and was only inhibited by 50% after
the 20 minute experimental protocol (Fig. 2).
Reorganization of the actin cytoskeleton after ATP
depletion
The normal organizational pattern of the F-actin cytoskeleton
in MDCK cells is shown in a three-dimensional reconstruction
in Fig. 3A. The similar F-actin pattern displayed by JTC cells
Table 2. Adenine nucleotide and hypoxanthine (HX)
content of JTC cells subjected to various times of ATP
depletion
Time (min)
ATP
ADP
AMP
HX
ΣAXP*
N
Controls
0c
30c
60c
49±12
52±7
49±9
11±2
12±3
7±3
2.4±0.8
2.0±0.8
0.4±0.8
1.2±0.4
1.2±0.4
1.6±0.8
62±12
67±7
56±10
6
6
3
ATP Depletion
5
10
20
30
60
26±7†
13±2†
12±2†
9±3†
7±4†
24±4†
13±3
10±4
21±7†
22±8†
40±14†
24±3†
20±4†
3.6±0.8†
4.8±2.0†
4.4±1.2†
3.2±2.0†
2.0±0.4
71±7
49±8
62±14
33±5†
27±6†
6
6
4
3
3
All contents are in nmoles/mg protein.
*ΣAXP= sum of ATP, ADP and AMP.
†Significantly different from the 0c (control) time point P<0.01.
Fig. 2. Fractional change in transepithelial resistance (TER) as a
function of time after inhibitor treatment. The average initial TER
was 25±4 and 20±2 ohm/cm2, respectively, for MDCK and JTC
cells. Although the time course of ATP depletion was similar in the
two cell types, the decline in TER was much slower in the JTC cells.
is seen in Fig. 4A in an extended focus image. The three characteristic cytoskeletal patterns described in the Introduction are
seen in both cell lines. On the apical side of the cell, the actin
cytoskeleton forms the microvilli and in addition a ring of actin
staining is present at the level of the tight junction and the
zonula adherens. Second, along the lateral membrane there are
cortical actin microfilaments that run parallel to the apicalbasal axis of the cells along the cytoplasmic face of the lateral
membrane. Third, at the base of the cells there are stress fibers
that stabilize the interactions with the substratum (Burridge et
al., 1988).
Alterations in this pattern are first observable after 20
minutes of ATP depletion, becoming more discernible after 30
minutes of ATP depletion. Since all the samples were treated
and processed identically, these images are semi-quantitative.
At this time, the actin cytoskeleton forms aggregates that are
found in the cytoplasm, typically around the nucleus (Fig. 3B;
data not shown for JTC cells), while the cortical actin staining
at the cell borders is lost and there is an apparent thinning of
the stress fibers in the MDCK cells and in the JTC cells (Fig.
3B, data not shown for JTC cells). The total cellular F-actin
content seems to decline at this time. After 60 minutes of ATP
depletion, these structural changes become more striking in
both cell types (Figs 3C, 4E and 4F), including the disappearance of the actin ring and stress fiber staining. However, the
total F-actin content in the perinuclear region appears to
increase, as compared to the 30 minute time point. The changes
in the actin cytoskeleton are diagramed in the schematic
presented in Fig. 9.
ATP depletion does not affect the organization of
the microtubule network
The possibility of an interaction between microtubules and the
actin cytoskeleton has been recently suggested (Buendia et al.,
1990). Several types of experiments were performed to
determine whether such an interaction played a role in the
actin alterations just described. First, we observed the microtubule network during ATP depletion. As seen in Fig. 5, the
microtubule network showed no evidence of depolymerization
or reorganization even with 60 minutes of ATP depletion.
Second, depolymerization of the microtubule network by
nocodazole, prior to the onset of ATP depletion, did not affect
ATP depletion: I. Rearrangement of the actin cytoskeleton 3305
Fig. 3. Stereo pairs
images of F-actin
distribution in MDCK
cells after various times
of ATP depletion. The
cells were fixed,
permeabilized, and
stained with Bodipyphalloidin after
metabolic inhibitor
treatment. All samples
were treated identically
and the threedimensional
reconstruction of the
confocal images utilized
the same color scale
throughout: from blue
for low intensity
staining to red for the
highest intensity
staining. (A) Control
conditions with no
inhibitors. The apical
ring structures are
demarcated by arrows
and the cortical actin
network, which runs
along the apical-basal
axis of the cell, by
arrowheads. Stress fibers
can be seen at the base
of the cells (open
arrows). This image is a
segment of a larger field
of cells; 16 complete
MDCK cells are
presented. The
dimensions along the x
and y axes are
approximately 40 µm
and 35 µm, respectively.
The cells are 18 µm
high. (B) After 30
minutes of ATP
depletion. The cortical
actin network is no
longer present and the
height of the cells has
decreased. Fewer stress
fibers are seen compared
to the control cells. The
apical ring of actin is
intact. The total content of F-actin appears diminished. There are 14 cells visible in this section. The dimensions along the x and y axis are the
same as in A. The cells are 10-11 µm high. (C) After 60 minutes of ATP depletion. Most of the F-actin is in a perinuclear location, showing
little structure. The apical actin ring and the stress fibers have disappeared. The total F-actin content appears larger than observed after 30
minutes, but it is difficult to compare to the control condition. The dimensions of the x and y axes are the same as in A. The cells are 12-15 µm
high.
the aforementioned alterations of the actin cytoskeleton (data
not shown). These results suggest that the perinuclear aggregates of actin are not drawn in from the cell periphery by a
microtubule-dependent mechanism, but may rather be the
result of re-polymerization of G-actin at a perinuclear
location.
Structural alterations in the tight junctions (TJ)
occur only after prolonged ATP depletion
Previous results demonstrated only minimal alterations in the
TJ after 10 minutes of ATP depletion (Mandel et al., 1993).
Since the present results demonstrate that the time frame for
the actin alterations after ATP depletion was 20-60 minutes,
3306 R. Bacallao and others
Fig. 4. Extended focus
image of F-actin
cytoskeleton in JTC
cells after various times
of ATP depletion.
(A,B,C) Control
conditions. (A) An
extended focus image
that spans 5.6 µm of the
apical side of the cells.
The punctate staining
corresponds to
microvilli, while the
apical ring can be seen
at the periphery of the
cells (arrows).
(B) Extended focus
image spanning 5.6 µm
in the mid-region of the
cell. Punctate staining is
still observed and
corresponds to
microvilli. Cortical actin
staining is observed
near the cell border
(arrowheads). (C) Basal
5.6 µm extended focus
image. The linear
streaks of actin staining
are stress fibers
(arrowheads) (D,E,F)
After 60 minutes of
ATP depletion. The
image intensity and
photographic settings
are equivalent to the
settings used to obtain
the control images.
(D) An extended focus
image that spans 5.6 µm
of the apical side of the
cell. Note the decrease
in microvilli staining
that is absent in some
cells. The ring of actin
is disrupted.
(E) Extended focus
image spanning 5.6 µm
in the mid-region of the
cell. Aggregates of
cytoplasmic actin are
found, mostly in a
perinuclear location
(arrowheads). Note the
absence of the actin ring
or cortical actin
network. (F) Basal 5.6
µm extended focus image. There is a marked decrease in the number of stress fibers as compared to control. The cortical actin network is
disrupted and some aggregates of actin are observed (arrowhead). Bar, 10 µm.
structural changes in the TJ and their effect on the molecular
fence function were examined before and at 60 minutes of ATP
depletion.
Untreated control MDCK epithelia exhibited tight junctions
composed of, on average, three tight junctional strands. These
strands fractured, leaving rows of particles on the P fracture
ATP depletion: I. Rearrangement of the actin cytoskeleton 3307
Fig. 5. Extended focus image of microtubule network in MDCK cells. No differences were observed between control conditions (A,B) and 60
minutes after ATP depletion (C,D). (A,C) Extended focus image of the apical side of the cells 4.6 µm deep. (B,D.) Extended focus image of the
basal side of the cells 6.0 µm deep. Both control and ATP-depleted samples were imaged and photographed using the same settings.
Bar, 10 µm.
face (the protoplasmic leaflet of the membrane) and complementary furrows on the E fracture face (the external leaflet of
the membrane), as previously shown by Mandel et al. (1993).
Thus, each tight junctional strand formed a continuous ring
around the entire cell perimeter. Only the P fracture face is
shown in Fig. 6A.
Epithelia that were ATP depleted for 60 minutes showed
dramatic alterations of this fracture pattern. The overall
appearance of the tight junction was still recognizable but
the P face strands contained extensive gaps or vacancies
(Fig. 6B). The E face furrows become filled with particles
that, in places, appeared almost as a regular P face strand.
Complementary matching of the particles and pits of the P
and E faces appeared lost (data not shown). Another
important difference between control and ATP-depleted cells
was detected in the P face strands. In untreated controls, the
3308 R. Bacallao and others
C
Fig. 6. Freeze-fracture electron microscopy of tight
junctions in MDCK cells. (A) Control conditions. The P
fracture face shows three parallel strands of particles on
average, and they contain a fair number of interruptions.
×125,000. (B) P fracture face after 60 minutes of ATP
depletion. Three strands can still be identified, however,
the number and average length of the interruptions are
greatly increased. ×125,000. (C) P fracture face after 60 minutes of ATP depletion. The higher magnification (×400,000) shows folding of the
tight junction strands (arrow) and aggregation of the tight junction particles.
particles were of uniform diameter and associated in well
defined, extensive rows. After 60 minutes of ATP depletion,
the P face strands were shorter. At places, the tight junction
strands appeared to be composed of particles of larger
diameter and irregular morphology. Careful inspection of
these images, however, suggested that the strands broke down
into short rows formed from 5-6 particles. These short rows
curved and in some cases the strands appeared to fold
together (Fig. 6C, arrow).
Additional information regarding the integrity of the tight
junction was obtained by examining the distribution of the tight
junction-associated protein, ZO-1 (Stevenson et al., 1986).
Both cell lines showed the characteristic unbroken pattern of
ZO-1 staining delineating the TJ that was observed under
ATP depletion: I. Rearrangement of the actin cytoskeleton 3309
Fig. 7. The effect of ATP
depletion on ZO-1 staining in
MDCK cells. Extended focus
images obtained by confocal
fluorescence microscopy.
(A) Control MDCK cells. ZO-1
forms a linear pattern at the cell
contact sites. (B) After 60
minutes of ATP depletion.
Breaks in the linear pattern are
readily observed. Bar, 10 µm.
control conditions (Fig. 7A). This pattern was intact in both
cell lines until the cells were ATP depleted for 60 minutes. At
this time, discontinuities in ZO-1 staining were observed, as
illustrated in Fig. 7B. Similar results were observed in the JTC
cell line (data not shown). The continuity of the molecular
fence was also tested at this time through the use of apical fluorescent phosphatydyl choline (PC). Previous work had shown
that fluorescent PC incorporated into the apical membrane of
MDCK cells did not diffuse to the basolateral side under
control conditions (Fig. 8A) (Dragsten et al., 1981; van Meer
and Simons, 1986; Mandel et al., 1993). After 60 minutes of
ATP depletion (Fig. 8B), fluorescent PC diffused to the basolateral side, suggesting that the TJ molecular fence had been
compromised. Therefore the time required to disrupt the fence
function of the tight junction corresponds to the time at which
the actin ring is compromised.
DISCUSSION
The results establish the temporal sequence of changes in tight
junctional properties and cytoskeleton organization upon
depletion of intracellular stores of ATP. These experiments
expand a previous communication, in which we reported on
the ability to separate the paracellular gate and molecular fence
functions of the tight junctions (Mandel et al., 1993). Two
different renal cell lines originating from the proximal (JTC)
and the distal tubule (MDCK) were used to test the generality
of the present findings. ATP depletion was achieved rapidly in
both cell types through use of simultaneous glycolytic and
oxidative inhibitors. The pattern of ATP hydrolysis was similar
to that found in freshly isolated renal tubules subjected to
anoxia. The initial breakdown of ATP into ADP and AMP was
followed by continued adenine nucleotide breakdown over
time (Mandel et al., 1988). The hypoxanthine content did
increase slightly, but little accumulation of this ATP
breakdown product occurred, suggesting that further hydrolysis took place. It should be noted that total adenine nucleotide
content decreased with time. This information on the metabolic
profile of the cells was recently utilized to formulate a protocol
that enabled a rapid reversal of ATP depletion (Doctor et al.,
Fig. 8. Confocal optical sections of MDCK cells, showing the
fluorescence of lissamine-rhodamine phosphatydylcholine
incorporated into the apical membrane. (A) x-y section obtained 2
mm below the apical surface under control conditions, showing
mainly endocytic vesicles. No evidence of lateral membrane staining
is seen. (B) x-y section 2 mm below the apical surface delineating the
lateral membranes and showing fewer endocytic vesicles.
Bar, 10 µm.
3310 R. Bacallao and others
Fig. 9. Diagram of the changes in the actin cytoskeleton following ATP depletion. (A) Actin organization in control cells. The actin
cytoskeleton is composed of an apical ring and longitudinal fibers running along the apical basal axis of the cells. Actin structures not shown
are the microvilli and the stress fibers. These were omitted to improve the clarity of the diagram. The dotted lines at the base of the cells reflect
cell boundaries rather than an actin structure. (B) Actin organization after 30 minutes of ATP depletion. The longitudinal actin network has
been lost. The cells decrease in height but the apical ring remains intact. The dotted lines at the base of the cells delineate cell boundaries.
(C) Actin organization After 60 minutes of ATP depletion. Actin has polymerized throughout the cytoplasm and the apical actin ring is
disrupted.
1994). Due to the decline in adenine nucleotide content by 30
minutes of energy depletion, re-establishment of energy metabolism after 30 minutes of energy depletion would not be
expected to rapidly restore ATP content, since ATP synthesis
in the short term is primarily from ADP and AMP. Such a
result was previously obtained in LLC-PK1 cells following
removal of metabolic inhibitors in which the ATP content was
only restored to 50% of control at 6 hours (Canfield et al.,
1991). More recently, ATP content was restored within 30
minutes of energy depletion by inhibiting purine breakdown
with allopurinol and exogenous addition of adenosine (Doctor
et al., 1994).
In both cell types, the fall in the ATP/ADP ratio preceded
the decrease in TER. Both of these variables were affected
more rapidly in the MDCK than the JTC cells. Furthermore,
the TER was essentially abolished in the MDCK cells whereas
it only fell by 50% during the 20 minute experimental period
in the JTC cells. The latter result is similar to that reported in
LLC-PK1 cells (Canfield et al., 1991). It is presently unclear
what could account for these differences, especially in light of
the limited information available regarding the molecular basis
of the paracellular barrier. Clearly, epithelial cells differ in
their ability to modulate paracellular permeability. It is
possible that these differences are also reflected in the reaction
to ATP depletion. These early changes in TER are unlikely to
be caused by the disruption of the actin ring, since it precedes
the disruption of the actin ring by 40 minutes.
The present results with ATP depletion indicate that the
functional alterations are quite different from those obtained
with low Ca2+-EDTA. As described earlier, ATP depletion
permits the separation between the paracellular barrier and the
molecular fence functions of the TJ, whereas Ca2+ depletion
rapidly abolishes both functions (Volberg et al., 1986; van
Meer and Simons, 1986). Structural alterations in ZO-1 did not
occur until 60 minutes after ATP depletion, whereas internalization and breaks in both ZO-1 and cingulin structure have
been reported after 15 minutes of low Ca2+ treatment (van
Meer and Simons, 1986; Citi, 1992). Recently it was shown
that inhibition of protein kinase C activity with H-7 prevented
the TJ disruption by low Ca2+, suggesting that protein kinase
activity was required for this event (Citi, 1992). This result
contrasts with the conditions of ATP depletion, where the low
ATP content would be expected to inhibit kinase activity due
to lack of substrate. Therefore, the expectation would be for
molecular events possibly involving protein phosphatases and
protein dephosphorylation. Further experiments are needed to
test the possibility that ATP depletion and low Ca2+ treatment
may act through different pathways.
Correlation between disruption of the actin ring and
the loss of molecular fence function of the tight
junction
The molecular fence function of the TJ was lost after 60 minutes
of ATP depletion, as measured by the appearance of rhodamineconjugated PC on the basolateral membrane. The loss of the
molecular fence seemed to be accompanied by profound
rearrangements in ZO-1 and freeze fracture patterns of tight
junction strands. The normal linear ridge pattern of the TJ was
transformed to one in which the TJ particles formed large aggregates and left extensive gaps. The coincidence between these
structural alterations and the loss of the fence function is consistent with previous models in which this function depends on
the contact between particles forming the strand within each cell
(Madara, 1989). In contrast, the paracellular gate (barrier)
function depends on the contact between strands located in
apposing cells. These two functions of the TJ could be separated
after 10 minutes of ATP depletion, since the gate function was
essentially abolished at this time while the fence function
appeared to be intact. No alterations in the linear structure of
the ridges and furrows were observable after 10 minutes of ATP
depletion (Mandel et al., 1993). Our data suggests that the
integrity of the paracellular barrier function is dependent upon
ATP or can be altered by the phosphorylation state of TJ
proteins. Indirect evidence in support of this idea comes from
the work by Citi and Stevenson that showed that cingulin and
ZO-1 are phosphoproteins (Stevenson et al., 1989; Nigam et al.,
1991). Additional supportive evidence comes from work by
Ojakian, which showed that activation of protein kinase C by
phorbol esters caused a drop in TER (Ojakian, 1981). The data
in this paper suggest that dephosphorylation of TJ proteins
causes a rapid loss of paracellular resistance, although further
work is needed to prove this suggestion.
ATP depletion: I. Rearrangement of the actin cytoskeleton 3311
The effect of ATP depletion on the actin
cytoskeleton
The results demonstrate that ATP depletion is associated with
a marked reorganization of the actin cytoskeleton (Fig. 9). The
initial step appears to be the disruption of the cortical actin
network followed by dissolution of the stress fibers. The last
arrangement of actin fibers that appears to be disrupted is the
apical actin ring (Fig. 3). This sequence reverses the organization of actin observed during epithelial morphogenesis, supporting the hypothesis of Bacallao and Fine (1989) for cell
injury (R. Bacallao et al., unpublished data). The time course
for actin disruption is roughly coincident with other events that
appear to be linked to actin microfilaments, as follows: (1) the
disappearance of cortical actin fibers within 20-30 minutes of
ATP depletion coincides with the beginning of internalization
of E-cadherin and possible decrease in cell-cell contacts
(Mandel et al., 1994). (2) The dissolution of stress fibers is
usually accompanied by loss of adhesion to the filter supports.
Whole epithelial sheets often float away from the support after
60 minutes of ATP depletion (results not shown). (3) The disruption of TJ structure after 60 minutes of ATP depletion correlates with the final disappearance of the apical actin ring. It
is tempting to speculate that the coincidence of the latter events
suggests that the linearity of the TJ strands may be determined
by links to the actin cytoskeleton. Disruption of the apical actin
ring may affect these links, leading to the observed aggregation of TJ particles.
The mechanisms leading to actin rearrangement after ATP
depletion are unknown. Our results are similar to those
obtained by Bershadsky and co-workers (1980), who found
that energy depletion caused a gradual disorganization of actincontaining microfilament bundles in fibroblasts. Other investigators have also noted differences in the relative susceptibility
of the actin cytoskeleton to ATP depletion. In endothelial cells
it has been shown that stress fibers are depolymerized
following 20 minutes of ATP depletion whereas the cleavage
furrow formed during mitosis was not depolymerized at this
time (Sanger et al., 1983). Canfield et al. (1991) reported actin
redistribution to a perinuclear location required about 2 hours
of energy depletion in LLC-PK 1 cells. A similar time scale (23 hours) was reported by Hinshaw et al. (1991) to elicit shortening of actin microfilaments in a murine cell line following
ATP depletion. The specificity and complexity of the actin
depolymerization pattern presented in this paper suggest that
this process is not merely a reversal of actin polymerization
caused by ATP depletion. Rather, it is more likely that this
condition activates specific time- and location-dependent actinsevering proteins, as originally suggested by Bershadsky and
Gelfand (1983). Their conclusion was based on the observation that ATP depletion prevented rapid depolymerization
(within 5 minutes) of filamentous actin when fibroblasts were
treated with cytochalasin B and D. Since the time course of
actin depolymerization continued at the normal rate observed
for ATP depletion (20 minutes) it was unlikely that ATP
depletion itself mediated actin depolymerization (Bershadsky
et al., 1980). Our own unpublished observations also show that
in MDCK cells the time course of actin depolymerization of
the stress fibers and cortical actin network after ATP depletion
is longer than the time course of cytochalasin D effects on the
actin cytoskeleton.
It has been shown that actin can be phosphorylated in vitro
by several proteins kinases (Machicao and Wieland, 1985;
Machicao et al., 1983; Erikson et al., 1979). However, only
recent evidence has shown that actin monomers can be phosphorylated in vivo (Schweiger et al., 1992; Howard et al.,
1993). In addition the phosphorylation state of actin correlates
with cell shape changes in Dictyostelium (Schweiger et al.,
1992; Howard et al., 1993). In vitro studies of phorphorylated
actin have demonstrated that phosphorylation of serine
residues of rabbit skeletal muscle actin decreases its affinity
for DNAse I. The phosphorylation state of actin monomers was
not examined in this study and will require additional experiments.
The factors leading to the formation of polymerized actin
aggregates in the cytoplasm after 60 minutes of ATP depletion
are likewise unknown. However, a possible sequence of events
may be deduced from known in vitro properties of actin: ATP
lowers the critical concentration for spontaneous polymerization of G actin, ADP-G actin monomers polymerize at a higher
concentration, and therefore ATP is not an essential cofactor
for actin polymerization (Carlier, 1990). As described above,
actin filament depolymerization may be initially due to
severing of the actin filaments by actin-binding protein(s). The
kinetics of subunit addition/loss now favors depolymerization
since the number of free ends increases and the concentration
of ATP-G actin falls due to a lack of substrate. Eventually the
level of ADP-G actin may rise above its critical concentration
at which point polymerized actin aggregates may form. Recent
work in vitro has shown that ATP-depleted cytosol caused
spontaneous actin polymerization, similarly actin polymerization has been observed following ischemia in vivo (Kurzchalia
et al., 1992; Molitoris et al., 1991).
These phenomena described in cultured cells are similar to
those observed in vivo following ischemic injury. The actin
cytoskeleton was rapidly rearranged in the proximal tubule
with an accompanying loss of the apical brush border (Venkatachalam et al., 1981; Kellerman and Bogusky, 1992). In the
latter study, the actin cytoskeleton of the apical brush border
appeared to be most sensitive to the effects of ATP depletion.
The changes in the cortical actin network were not determined
in these studies. Furthermore, a common observation made by
Molitoris and co-workers (1989, 1991) is the rapid increase in
paracellular permeability following ischemia. These results
suggest that the present observations made in renal cultured
cells may be used to understand the derangements in cytoskeletal and junctional structure that occur in vivo.
The effects of ATP depletion on microtubules
Buendia et al. (1990) have shown that there is a dynamic interaction between the actin cytoskeleton and microtubule network
in MDCK cells. Based on this study, we tested the possibility
that the breakup of the actin filaments and the formation of
actin aggregates was mediated by the microtubule network.
The present results demonstrate that the morphology of the
microtubule network was not affected by ATP depletion. Furthermore, depolymerization of the microtubule network by
nocodazole prior to ATP depletion did not change the time
course of actin depolymerization or its subsequent aggregation.
These results are consistent with those obtained in fibroblasts,
where it has been shown that ATP depletion stabilized the
microtubule cytoskeleton. Fibroblasts depleted of their cyto-
3312 R. Bacallao and others
plasmic ATP did not show any change in the organization of
the microtubule cytoskeleton nor were the microtubules
disrupted when the cells were treated with nocodazole (Bershadsky and Gelfand, 1983). This difference in sensitivity to
the effects of ATP depletion between the actin cytoskeleton
and microtubule network may be a useful method to study
microtubule function in the absence of a normal actin
cytoskeleton.
In summary, this work demonstrates the time course for
actin cytoskeleton disassembly following ATP depletion and
its possible relationship to epithelial tight junctional properties.
There is a rapid loss of TER that does not coincide with visible
alterations in F-actin distribution. The actin cytoskeleton is first
depolymerized along the apical-basal axis of the cell followed
by the basal stress fibers. As the period of ATP depletion
continues, the actin ring located at the apical portion of the cell
breaks up. At this point, TJ structure is also affected with coincident loss of the molecular fence function. This disruption of
the actin cytoskeleton is independent of microtubule organization. These results demonstrate that ATP depletion may be a
useful method to study junctional complex assembly and disassembly in epithelial cells.
The authors thank Dr Leon Fine for his support and encouragement
for this project, Dr Thomas Buell for his help with the TER measurements and Mr Mike Kreman for his technical expertise in the
freeze fracture experiments. The work was supported in part by NIH
grants DK01777 and the American Heart Association Grant in Aid
no. 92016070 (to R.B.) and DK 26816 (to L.J.M.). The authors also
thank Mark Gregory of Leitz America, IL for his generous support.
Steven Napier generously contributed the diagram presented in this
paper. We are grateful to Arthur Toga at UCLA, Department of Neuroscience, for providing generous access to his computer imaging
facilities in general and allowing us to store our data sets in particular.
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(Received 21 September 1993 - Accepted, in revised form,
18 August 1994)