mutant for 2 days. CFP images (excitation with 458 nm laser, emission from 473-515 nm)
were then collected with a water immersion objective Achroplan 40x/0.8W at one airy
unit pinhole on a Zeiss LSM 510 meta confocal microscope.
12
RESULTS
Ezrin T567 phosphorylation in renal proximal tubule and small intestine enterocytes
are higher than that in gastric parietal cells.
Two types of cells well known for the rich brush border microvilli on the apical plasma
membrane, renal proximal tubule cells and small intestinal enterocytes, are a rich source
of ezrin. Our first objective was to determine the relative phosphorylation level on ezrin
T567 (T567-P) in these cells and compare that with parietal cells. For this purpose the
relative levels of total ezrin and of T567-P ezrin were analyzed in isolated gastric glands,
renal proximal tubules and small intestine organoids by Western blot. In order to
approach maximal and minimal levels of T567 phosphorylation the various tissues were
respectively treated with the protein phosphatase inhibitor calyculin A (CLA) or the
metabolic inhibitor sodium azide. The expectation was that inhibition of protein
phosphatases by CLA would increase T567-P level if there were sustained kinase activity.
On the other hand azide shuts off ATP production by the oxidative phosphorylation, thus
depriving the protein kinases of their substrate and causing decreased phosphorylation
level. NaN3 has long been used to achieve chemical anoxia in physiological studies (22,
41).
Western blots of treated and control tissues were probed with anti-T567-P followed by
stripping and reprobing with anti-ezrin (Figure 1). Two proteins were revealed by T567-
P antibody. The band with higher apparent molecular mass (Mr) is T567-P ezrin because
13
it was recognized by a highly specific ezrin antibody. The other band is likely moesin
judging from its slightly lower Mr and subsequent tests. Interestingly, the results for
gastric glands differed markedly from those obtained for proximal tubules and small
intestine. For gastric glands the ezrin T567-P level increased greatly when incubated
with CLA, indicating that T567 phosphorylation level is low in the native condition;
whereas, azide did not produce any detectable effect on ezrin T567-P level, again
reflecting the fact that gastric ezrin carries a low level of T567-P in the steady state. A
contrasting result was observed with renal proximal tubules. CLA had virtually no effect
on ezrin T567-P level, while azide greatly depressed T567-P. The results for small
intestinal enterocytes were similar to those for proximal tubules, indicating that both of
these brush border-rich tissues have a higher steady state level of ezrin T567-P than that
of gastric glands.
Relatively fast turnover of ezrin T567 phosphorylation.
To determine the relative turnover of ezrin T567-P, time-course experiments were
performed to study the changes in ezrin T567-P after the addition of agents that block
phosphorylation or dephosphorylation. For gastric glands, CLA was used because the
phosphorylation level is low in the native state. Changes in the relative level of ezrin
T567-P after addition of CLA are shown in Figure 2A, where it appears that the time for
50% increase of ezrin T567-P was less than a minute for gastric glands. Since ezrin in
proximal tubules is normally highly phosphorylated, we used agents to block
phosphorylation (either azide or the membrane permeable kinase inhibitor staurosporin)
14
and subsequently observed the time course of T567-P depletion from ezrin. In Figure 2B
staurosporin was used to decrease the phosphorylation level. Staurosporin effectively
reduced the phosphorylation of ezrin, with the time required for 50% decrease in T567-P
being about 2-3 min. When doing the similar experiment with azide treatment, the half
time for depletion was found to be somewhat slower (about 4 min, data not shown).
Because of inherent problems with respect to permeation and multiplicity of action for
the various inhibitors these data cannot be taken as definitive values for reaction rates,
but they do indicate that the T567-P turnover is relatively fast in these tissues.
Since moesin existed in a similar amount as ezrin in the brush border of proximal tubules,
it was also of interest to examine the phosphorylation turnover of moesin in these
samples. Thus the blots with proximal tubules were also probed with anti-moesin
antibody. The result shown in Figure 2B indicated that moesin, like ezrin, assumed a
highly phosphorylated steady state in proximal tubule, and the phosphorylation level on
T558 dropped rapidly upon inhibition of kinases with staurosporin. The time required for
half decrease in T558-P being about 2-3 min, similar to that of ezrin T567-P.
Ezrin-cytoskeleton binding is enhanced by phosphorylation
To evaluate T567-P turnover as a regulatory mechanism for ezrin activity in tissues, two
extraction methods were used to study the interactions of ezrin with F-actin and plasma
membrane in renal proximal tubules and gastric glands. Extraction with TX-100 removes
soluble and membrane bound proteins, leaving behind the F-actin cytoskeleton pellet and
15
many actin bound proteins (15). Isolated gastric glands and proximal tubules were taken
fresh as control tissue, or treated with CLA to block protein dephosphorylation and thus
maximize phosphorylation levels, or treated with sodium azide to reduce ATP
phosphorylation levels. The tissues were then extracted with 1% TX-100 for 5 min at room
temperature. Samples of supernatant extracts and cytoskeleton pellet were examined by
Western blots probed for T567-P, total ezrin, and actin.
For gastric glands, total actin was predominantly distributed to the cytoskelton pellet over
the supernatant (~4:1) and was not significantly different between the various
experimental treatments (Figure 3C). This is consistent with previous findings that actin
in gastric glands is predominantly in the filamentous form (2). As expected, treatment
with CLA resulted a large increase (~4-5 fold) in T567-P level compared to control
glands (Figure 3B). Treatment with N3 produced no significant change in T567-P
compared to fresh glands (Figure 3B). For all treatments ezrin T567-P was always
predominantly distributed toward the pellet which was always about 4 times greater than
that extracted into the TX-100 supernatant (Figure 3B). In control glands total ezrin was
distributed between supernatant and pellet in approximately 60:40 ratio (Figure 3A).
Treatment with CLA greatly altered the TX-100 extraction so that the majority of ezrin
(>80%) remained with the pellet (Figure 3A). For glands treated with N3 there was very
little difference from control, either with respect to the relative amount of T567-P or in
the supernatant/pellet distribution ratio of total ezrin (Figure 3A).
16
As in gastric glands, proximal tubular actin was predominantly distributed to the TX-100
pellet for all treatments (Figure 3F). Treatment of proximal tubules with CLA produced no
significant change in the level of total T567-P ezrin (P > 0.05), although there was a tendency
for a shift in T567-P distribution from pellet to supernatant (Figure 3E). After N3-treatment
the levels of T567-P ezrin were significantly (P < 0.01) decreased in the supernatant and
pellet fractions (Figure 3E), and total ezrin was redistributed toward the supernatant
compared to control (Figure 3D). The distribution of T567-P ezrin was always predominant
in the pellet under all conditions for both proximal tubules and gastric glands (cf. Figures 3B
& 3E). The most obvious difference between proximal tubules and gastric glands occurred
in the respective control preparations where the proportion of total ezrin was predominantly
associated with the cytoskeleton for proximal tubules and reversed for gastric glands (cf.
Figures 3A & 3D).
For both gastric glands and renal proximal tubules T567-P ezrin was always more distributed
toward the cytoskeletal pellet than the TX-100 supernatant, consistent with studies on various
cell lines (15, 25) and supporting the notion that T567 phosphorylation exposes the actin
binding site on ezrin. The distribution data also predictably show that when T567
phosphorylation is high (e.g., CLA treatment in gastric glands and steady state control for
proximal tubules), total ezrin is also associated with the cytoskeletal pellet and these trends
are reversed on lowering phosphorylation with N3.
Ezrin remains bound to membrane in the nonphosphorylated form: biochemical evidence
17
In another extraction method we used digitonin to form pores in the plasma membrane
allowing soluble cytoplasmic proteins to exit while retaining cytoskeleton, membrane
proteins and large protein complexes associated with membranes or cytoskeletal structures
(24). Freshly isolated gastric glands were compared with an experimental set treated with
CLA; control proximal tubules were compared with an experimental set treated with NaN3.
The respective glands and tubules were permeabilized by treatment with digitonin and
extracted for 5 min, either in low salt buffer or normal phosphate buffered saline. Blots of
supernatant extracts and residual pellet fractions were probed for ezrin T567-P, total ezrin,
and actin. Additional gels were run in parallel for Commassie blue staining to ascertain that
cytosolic proteins did leak out after digitonin permeabilization (data not shown).
Similar to earlier experiments, control levels of ezrin T567-P were relatively low in gastric
glands and relatively high in proximal tubules; treatment of glands with CLA caused a large
increase (~4-fold) in T567-P (Figure 4B), and treatment of tubules with N3 caused a large
decrease in T567-P (Figure 4D). When control preparations of either gastric glands or
proximal tubules were permeabilized in low salt buffer relatively little of the total ezrin was
released to the supernatant, e.g., < 20% for glands and <5% for tubules (Figure 4A, C).
There was little change in the pattern of ezrin released from permeabilized gastric glands
after CLA treatment or the pattern of ezrin released by tubules after treatment with N3
(Figure 4A, C). These data contrasted sharply with those for TX-100 cytoskeletal extraction,
where low activity of T567 phosphorylation was correlated with a higher distribution of total
ezrin to the supernatant. Since control glands or N3-treated tubules demonstrate relatively
little ezrin binding to actin cytoskeleton when the bulk of ezrin is in the de-phospho state, the
18
general retention of ezrin in the digitonin-permeabilized preparations appears to be due to
another binding site, likely the N-terminal membrane binding site.
The pattern of ezrin release by digitonin-permeabilized glands was considerably altered when
the extraction medium included relatively high ionic strength (PBS). In PBS the majority of
total ezrin was released by the control glands and the distribution ratio was reversed by the
increased T567-P associated with CLA treatment (Figure 4A). On the other hand for control
proximal tubules with their high steady state level of ezrin T567-P the elevated ionic strength
medium accounted for only a slight loss of total ezrin to the supernatant and this was
reversed when N3 was used to lower T567-P (Figure 4C).
Ezrin remains bound to membrane in the nonphosphorylated form: imaging evidence
To study the localization of nonphosphorylated ezrin in an in situ setting, renal proximal
tubules were either incubated with nitrogen for hypoxia or sodium azide for chemical anoxia
before immunofluorescence staining with ezrin antibody. Phosphorylation on ezrin T567
was found greatly decreased by hypoxia treatment and further decreased by anoxia (NaN3)
treatment (Figure 5A). Nevertheless, proximal tubules in each of the three different
treatments were stained similarly (Figure 5B, C, D). There was always a high degree of co-
localization of ezrin with F-actin. Most of the staining was with the apical brush border
membrane facing the lumen of the tubule; a weak but clear staining of the basal membrane
was also detected; cytoplasm and lateral membrane were not stained. These results indicated
that ezrin is localized to a membrane surface regardless of the phosphorylation level on T567.
19
Similar experiments were performed with small intestine organoids. Samples of normoxia
and anoxia conditions (treated with azide) were compared after double staining for ezrin and
F-actin. Both stains were largely co-localized with the majority of the staining at the brush
border membrane (Figure 6A, B). While faint staining of F-actin is visible on lateral cell
membrane, ezrin seems exclusively localized on the microvilli at the brush border. No
staining was detected on basal membrane for both molecules. Again, no change of
localization occurred when ezrin was dephosphorylated by the azide treatment.
Unregulated membrane projections with the expression of T567D mutant ezrin
Although ezrin in renal proximal tubule is in a highly phosphorylated form, fast and high
turnover of the T567-P still exists. Thus, it was of interest to determine if this
phosphorylation turnover is required for its normal function. For this purpose, isolated renal
tubule cells were cultured on Matrigel coated coverslips and infected with recombinant
adenovirus expressing CFP-tagged constructs of ezrin, including wild-type, the T567A
mutant, or the T567D mutant. After 48 hr of infection, expression of the three CFP-tagged
ezrin constructs was directly examined by fluorescence microscopy. In addition, uninfected
control cells were stained for both ezrin and F-actin. As seen in Figure 7A, these control
cells maintain cell surface microvilli of about 3 μm length after two days in culture. Just like
the freshly prepared proximal tubules, the majority of ezrin and F-actin was found to be on
the microvillar membranes. The ezrin T567-P level in these cultured tubule cells was also
examined. As shown in Figure 7B, with similar amount of ezrin, the T567-P level in the
20
control sample is similar to that in the CLA treated sample; while the azide treated sample
showed a diminished level of ezrin T567-P. These results indicate that the ezrin activity is
kept similar in these cultured cells compared to the freshly isolated tubules.
Live cell imaging of CFP fluorescence (Figure 8A) showed that wild-type ezrin had a similar
staining pattern to that of native ezrin. A similar staining pattern was also observed for cells
expressing the ezrin T567A mutant (Figure 8B). However, the distribution and appearance
of the T567D mutant differed from wild-type or T567A mutant, although it was still
localized on cell surface membrane structures (Figure 8C). T567D mutant ezrin usually
tended to accumulate at one end of a cell, compared to wild-type or the T567A mutant which
had a more even distribution at cell surface. An even more marked difference was that cells
expressing T567D mutant often carried cell surface projections longer than 5 μm (many of
them longer than 10 μm) while cells expressing wild-type or T567A mutant carried
microvilli of the more normal 2-3 μm length.
21
DISCUSSION
The steady state of ezrin phosphorylation in renal proximal tubules and small intestinal
enterocytes is higher than that in gastric parietal cells.
Ezrin was identified as a major component on the apical canalicular membrane in gastric
parietal cells stimulated to secrete acid (18, 39). Studies with the ezrin gene knockdown in
mice demonstrated that ezrin is required for the stimulation-associated elaboration of the
microvilli on the apical canalicular membrane of parietal cells (38). Based on the relatively
high turnover of phosphorylation on ezrin T567 and the drastic change in its binding affinity
to filamentous actin associated with that phosphorylation, a rolling motor model was
hypothesized to describe how ezrin repositions membrane along the filamentous actin length,
thus maintaining appropriate tensile force between membrane and cytoskeleton during
dynamic membrane turnover associated with parietal cell secretion (49). At present no other
theory explains how ezrin works in other cells. Thus we tested the rolling motor model,
focusing on other microvilli-rich cells: renal proximal tubule cells and enterocytes of small
intestine.
Ezrin from both renal proximal tubule cells and intestinal enterocytes were found to be
highly phosphorylated, compared to the low steady state level of phosphorylation in the non-
secreting gastric parietal cells (Figure 1). Both the renal proximal tubule cells and small
intestinal enterocytes are known for the presence of their characteristic apical brush border
membranes, which are densely packed with uniformly sized microvilli. In contrast, the
22
microvilli found in non-secreting gastric parietal cells normally exist in various lengths and
sparse distribution (13). Upon stimulation, there is a massive membrane recruitment from
intracellular tubulovesicles onto the apical canalicular membrane, forming a much denser
distribution of elongated apical microvilli (13). Previously, we reported a significant
increase of ezrin phosphorylation (40), and specifically phosphorylation of ezrin on T567
(49), in gastric parietal cells when these cells were physiologically stimulated. Thus, a
higher steady state level of ezrin T567-P seems to be correlated with a denser distribution of
microvilli, whether from the same cell (e.g., the different physiological conditions of parietal
cell), or from different types of tissue.
T567 phosphorylation turnover is required for ezrin function in renal proximal tubule
cells.
The prompt decrease in ezrin T567-P with azide treatment in renal proximal tubule cells
and small intestine enterocytes indicates that continuous kinase activity is needed to
maintain the high level of phosphorylation on ezrin T567. The importance of T567-P
turnover for the regulation of ezrin function in renal proximal tubule cells is
demonstrated with the expression of the T567D mutant, which mimics permanent
phosphorylation of T567, not allowing the turnover of ezrin between phospho and
dephospho forms. When T567D mutant ezrin was over-expressed in primary kidney cell
cultures, surface membrane structures were abnormally long and tended to accumulate at
a small area on the plasma membrane, where the T567D mutant was localized. This
morphological change obviously affected the fine structure of microvilli and may even
23
disrupt the polarized distribution of the membrane proteins. Since the precise
localization of channels and pumps on the plasma membrane(s) is essential for the
functional transport of nutrients from the glomerular filtrate back into circulation, it is
conceivable that the abnormal morphology of the renal proximal tubule cells would result
in a decreased efficiency of function. Unfortunately, this experiment cannot be done with
the primary cultures of proximal tubule cells expressing T567D mutant since the cells do
not maintain their polarity in culture. However, experiments done with a polarized
kidney epithelial cell line NRK-52E demonstrated an abnormal relocation of Na,K-
ATPase onto the apical membrane upon treatment with RhoA (23), which is known to
induce phosphorylation of ERM proteins on the conserved T567/T564/T558 site (25, 33).
The T567-P high turnover mechanism for regulation of ezrin activity is thus not limited
to gastric parietal cells, but also applies to ezrin in renal proximal tubule cells and small
intestine enterocytes. In addition, moesin, which is expressed at relatively high levels in
renal proximal tubules, was also subjected to the high turnover regulation on the
phosphorylation of the conserved T558 (Figure 2B). Thus it may be that this high
turnover mechanism is a universal one for the regulation of all ERM protein activity.
Ezrin brings membrane to cytoskeleton
Studies with transformed cell lines often described ezrin as a diffusely localized, or
cytosolic, protein, in its inactive form (4, 5, 42). Since ezrin has increased membrane and
cytoskeleton binding affinity upon activation (by phosphorylation), the major function of
24
ezrin was believed to be “linking F-actin to membrane” (6). However, our examination
of the state and localization of ezrin in three freshly isolated tissues suggests a
modification of this concept. Phosphorylation on T567 was observed to induce tighter
membrane binding of ezrin in gastric parietal cells (49), as expected from many other
studies (12, 19, 45), since the membrane binding N-terminus is partially masked by the
C-terminus in the non-phosphorylated N-C binding conformation (14, 48). However,
ezrin in its non-phosphorylated form still localizes to the microvilli-rich apical membrane
of parietal cells as shown previously (49) and here in Figure 4A. Similarly, in renal
proximal tubule cells and small intestine enterocytes, the dephosphorylated form of ezrin
remains localized on the brush border microvilli-rich membrane (Figures 5 & 6).
Binding analyses showed that dephosphorylation by anoxia treatment would only slightly
shift the distribution of ezrin toward the cytosolic pool, which is very small (Figure 4C).
Severe alterations in the cytoskeleton and the repolarization of many membrane proteins
occurs rapidly after ischemia and reperfusion in the kidney (26-29). Although the
mechanism of these transitions is not completely understood, it is known that ischemia
alone does not cause the rearrangement of F-actin cytoskeleton. While reperfusion after
40 min of ischemic treatment caused significant decrease in F-actin, the remaining F-
actin signal still largely remained on the brush border membrane (7). Our procedures to
induce anoxia or hypoxia, and the observed results, were obviously similar to the renal
ischemia treatments without reperfusion.
The binding of T567 unphosphorylated ezrin to the membrane seems to be mediated by
phosphatidyl inositol-4, 5-biphosphate (PIP2). Tsukita and her colleagues (44) found that
25
treatment of two cell lines A431 and MDCK II with staurosporin could decrease the T567-P
without affecting the membrane localization of ezrin. However, when the PIP2 levels in
A431 cells were decreased by microinjection of C3 transferase (which blocks the activation
of Rho and PI4,5-kinase), ezrin was found dephosphorylated on T567 and diffused away
from microvillus membranes. The PIP2 level in MDCK II did not respond to C3 transferase,
but could be blocked with neomycin, which also caused a decreased T567-P level and
diffusive staining of ezrin. The dependence of ezrin function on PIP2 binding was also
reported by Arpin’s group (12). Their results with the LLC-PK1 cell line indicated that ezrin
binding to PIP2, through its NH2-terminal domain, is required for membrane localization and
T567 phosphorylation. It may well be that the membrane binding site on ezrin has some
dependence on ionic bonding as the depletion of ezrin in permeabilized glands and tubules
was increased by high salt (Fig. 3), consistent with previous observations in gastric glands
(18), and as might be inferred by cytosolic divalent ion competition for ezrin release in
glands (43) and in LLC-PK1 cells (12); however, in the latter case the authors have suggested
that increased cytosolic Ca2+ might catalyze PIP2 hydrolysis.
In contrast to the continuous binding to the membrane, the binding of ezrin to F-actin was
greatly enhanced when the C-terminal actin-binding site was unmasked by phosphorylation
at T567. Mandel et al. demonstrated that dissociation of ezrin from actin cytoskeleton
occurred in renal proximal tubules upon anoxia treatment (9). They later showed evidence
that anoxia treatment induced ezrin dephosphorylation by 2D electrophoresis (8). Very
likely this dephosphorylation occurs on T567, although we cannot exclude the possibility of
other Ser/Thr phosphorylation site(s). By F-actin co-sedimentation assays (49) and Triton
26
extraction analyses (Figure 3), dissociation of ezrin from F-actin was found to be induced by
dephosphorylation on T567 in gastric parietal cells. Similar data were obtained from many
other cell models (12, 20, 30, 31, 37, 45, 49).
A relatively constant membrane binding and the ever changing F-actin binding gives the
ezrin molecule the power to associate and re-associate membrane and cytoskeleton, thus
enabling a dynamic reposition of membrane along the filamentous actin length, in a fashion
similar to a rolling molecular motor attached to cell membrane. A cartoon depicting the
relaxation-reattachment hypothesis to promote dynamic rearrangement within defined
surface morphology is shown in Figure 9. Such a model is logical and essential for surface
plasticity in view of the growth and repositioning required for cytoskeleton expansion, as
well as the re-establishment of membrane surface forces as processes of recruitment and
endocytosis occur. This model also explains why blocking moesin dephosphorylation with
CLA caused the impairment of neutrophil motility (46), which is crucial to effective host
defenses against microorganisms.
In summary, higher steady state phosphorylation levels on ezrin T567 were detected with
freshly isolated renal proximal tubule cells and small intestine enterocytes, as compared to
gastric parietal cells. However, high levels of phosphorylation did not exempt ezrin in these
tissues from the high turnover regulation on T567-P, which assures a relaxation change in the
association of ezrin between the F-actin-bound and -unbound state, without necessarily
affecting the binding of ezrin to membrane. Interruption of the regulation of high turnover of
T567-P, such as introduction of the ezrin T567D phosphorylation mimic, has been observed
27
to cause abnormal growth and rearrangement of membrane projections (15, 33), sometimes
causing local changes in polarity (23, 47), and likely to interfere with the normal membrane
transport processes of the affected cells.
ACKNOWLEDGEMENT
The authors thank Holly L. Aaron of the Berkeley Molecular Imaging Center for her
professional assistance in obtaining confocal images with Zeiss 510 META LSM microscope.
GRANT
This work was supported by a grant from the National Institutes of Health, 5RO1DK10141-
42.
28
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FIGURE LEGENDS
Figure 1. Differential phosphorylation level on Thr567 of ezrin in small intestine
epithelial cells (SI), kidney proximal tubule cells (K) and gastric glands (GG). Small
intestine organoids, kidney proximal tubules and gastric glands were isolated from rabbit
by the collagenase digestion methods described in M&M. These tissues were mock
treated with vehicle (ctrl), treated with sodium azide (N3) or calyculin A (CLA). Western
blot analyses on these samples were done with anti-T567-P and then stripped and
reprobed with anti-ezrin. The arrowhead indicates the position of ezrin T567-P. The
band at the slightly lower Mr is moesin T558-P.
Figure 2. Turnover of ezrin T567 phosphorylation in gastric glands and renal proximal
tubules. A. Gastric glands were treated with calyculin A (CLA); B. proximal tubules
were treated with staurosporin. At various time points, as indicated on top of the lanes,
samples were taken and boiled in SDS-PAGE loading buffer. Samples were then
analyzed by western blot with anti-T567-P, stripped and reprobed with anti-ezrin. For
proximal tubule samples, the blot was further probed with anti-moesin antibody without
stripping.
Figure 3. Enhanced ezrin binding to F-actin upon phosphorylation on ezrin T567 in both
gastric glands (A, B, C) and renal proximal tubules (D, E, F). Control samples (ctrl) and
samples treated with calyculin A (CLA) or sodium azide (N3) were extracted with Triton
X-100 buffer for 5 min at room temperature. The extractions were separated from the
33
pellets by centrifugation. Supernatant (S) and pellet (P) fractions were subjected to
Western blot analyses with anti-ezrin (A, D), anti-T567-P (B, E), and anti-actin (C, F).
Ezrin data are presented as the % of total ezrin distributed between the S and P. The
T567-P data are presented as the % of T567-P compared to the S plus P in CLA-treated
sample for both gastric gland and proximal tubules. The actin data are shown as the
relative distribution of actin between S and P. Density data are plotted as the mean
±SEM; for gastric glands N = 4; for proximal tubules N = 5.
Figure 4. Obstinate binding of ezrin to membrane in gastric glands (A, B) and renal
proximal tubules (C, D). Tissues were treated with either CLA (gastric glands) or with
NaN3 (renal proximal tubules) to obtain phosphorylation levels different from the native
conditions (ctrl). Samples were then treated with digitonin at room temperature for 5 min
to permeabilize the cells. Permeabilizations were performed both in low salt buffer
(Sucrose) or normal phosphate buffered saline (NaCl). Samples were separated into
supernatant (S) and pellet (P) fractions for Western blot analyses. Blots were probed
with anti-ezrin (A, C) and anti-T567P (B, D). Ezrin data are presented as the % of total
ezrin distributed between the S and P. The T567-P data are presented as the % of T567-P
compared to the S plus P in CLA-treated sample for gastric glands, and compared to the
S plus P of the control sample for proximal tubules. Density data are plotted as the mean
±SEM; for gastric glands N = 3; for proximal tubules N = 5.
Figure 5. The pattern of ezrin immunostaining is unchanged in kidney proximal tubule
cells upon hypoxia or anoxia treatment. Proximal tubules from rabbit kidney were
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incubated in nitrogen saturated media (hypoxia treatment, H) for 60 min or incubated
with sodium azide (chemical anoxia, N3) for 30 min. Control samples (ctrl) were
incubated in oxygen saturated media for 60 min. A fraction of each sample was analyzed
by Western blot with anti-T567-P, stripped, and reprobed with anti-ezrin (A). The rest of
the samples (control (B), hypoxia (C) and anoxia (D)) were stained with FITC-phalloidin
(left image) and mouse-anti-ezrin (center image); the right hand image is DIC. For each
treatment higher magnifications are shown for the area in the respective blue boxes. BL:
