From the Forschungsinstitut für Molekulare
Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin, Germany, the ¶ Universita de Bari, Dipartimento di
Fisiologia Generale e Ambientale, Via Amendola 165/A, 70126 Bari,
Italy, the
Institut für Pharmakologie und Toxikologie,
Albert-Ludwigs-Universität Freiburg, Hermann Herder Strasse 5,
79104 Freiburg, and the ** Freie Universität Berlin, Institut
für Pharmakologie, Thielallee 67-73, 14195 Berlin, Germany
Received for publication, November 10, 2000, and in revised form, February 2, 2001
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ABSTRACT |
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Vasopressin regulates water reabsorption
in renal collecting duct principal cells by a
cAMP-dependent translocation of the water channel
aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane.
In the present work primary cultured inner medullary collecting duct
cells were used to study the role of the proteins of the Rho family in
the translocation of AQP2. Clostridium difficile toxin B,
which inhibits all members of the Rho family, Clostridium
limosum C3 toxin, which inactivates only Rho, and the Rho kinase
inhibitor, Y-27632, induced both depolymerization of actin stress
fibers and AQP2 translocation in the absence of vasopressin. The data
suggest an inhibitory role of Rho in this process, whereby constitutive
membrane localization is prevented in resting cells. Expression of
constitutively active RhoA induced formation of actin stress fibers and
abolished AQP2 translocation in response to elevation of intracellular
cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced
both depolymerization of the F-actin cytoskeleton and AQP2
translocation, indicating that depolymerization of F-actin is
sufficient to induce AQP2 translocation. Thus Rho is likely to control
the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.
The antidiuretic hormone arginine-vasopressin
(AVP)1 regulates water
reabsorption in renal collecting duct principal cells by inducing the
translocation of the water channel aquaporin-2 (AQP2) from
intracellular vesicles primarily into the apical cell membrane (shuttle
hypothesis; Refs. 1 and 2). The molecular targets of AVP on the surface
of principal cells are heptahelical vasopressin V2 receptors coupled to
the Gs/adenylyl cyclase system. Activation of this system
by the hormone raises the level of intracellular cAMP and results in
the activation of protein kinase A (PKA) which then phosphorylates its
substrates, one of which is AQP2.
The phosphorylation of AQP2 by PKA and also the anchoring of PKA to
subcellular compartments via protein kinase A anchoring proteins are
prerequisites for AQP2 translocation to the cell membrane (2-5). In
addition, the involvement of a heterotrimeric G protein of the
Gi family in the AQP2 translocation has been demonstrated
in CD8 cells (6).
The cytoskeleton consists of various components, including microtubules
and F-actin, both of which are involved in AVP-mediated changes of
osmotic water permeability (2, 7-9). Microtubule-disrupting drugs like
colchicine and nocodazole inhibit AVP-mediated increases in osmotic
water permeability in renal collecting ducts by 65 and 72%,
respectively (10-13). Disruption of the F-actin cytoskeleton by
cytochalasin B or dihydrocytochalasin B inhibits the AVP-induced increase in osmotic water permeability in toad bladder epithelium by
25-50% (13, 14). The F-actin cytoskeleton also undergoes rearrangements after stimulation of cells with cAMP-elevating agents.
After stimulation with vasopressin, total F-actin decreases in toad
bladders by 20-30% (15) and apical F-actin in rat collecting duct
principal cells by 26% (16). In CD8 cells, F-actin decreases in
response to forskolin (17). The mechanisms underlying these changes of
the F-actin cytoskeleton are not understood, and their influence on the
fusion of AQP2-bearing vesicles with the cell membrane are not known.
Valenti et al. (17) recently suggested that the
rearrangement of apical F-actin might be a prerequisite for promoting
redistribution of AQP2-containing vesicles and a consequence of
phosphorylation and dephosphorylation processes, mediated by PKA and
protein phosphatases 1 and 2A, respectively.
Proteins of the Rho family (Rho, Rac, and Cdc42) of small GTP-binding
proteins are active in their GTP-bound and inactive in their GDP-bound
forms. They participate through their effectors in the organization of
the actin cytoskeleton. As initially shown in fibroblasts, activation
of Rho triggers the assembly of actin stress fibers and formation of
focal adhesions, activation of Rac leads to formation of lamellipodia
and membrane ruffles, and activation of Cdc42 induces surface
protrusions designated filopodia (18-20). In addition, Rho family
proteins are involved in the regulation of vesicle motility (21),
endocytotic events like receptor-mediated endocytosis (22, 23) and
various exocytotic processes. For example, the inactivation of Cdc42
and Rac by Clostridium sordellii lethal toxin inhibits the
Ca2+-triggered release of hexoseaminidase from rat
basophilic leukemia cells (RBL 2H3 cells; Ref. 24). This finding was
confirmed by expression of dominant negative forms of Cdc42 and Rac in
these cells (25). Inhibition of Rho by Clostridium
botulinum C3 toxin prevented Ca2+-triggered
release of hexoseaminidase in permeabilized mast cells by 80% (26).
Conversely, constitutively active RhoA or Rac1 greatly enhanced
Ca2+-induced secretion from permeabilized mast cells
(27).
These findings led to the hypothesis that Rho proteins may be involved
in the control of AQP2 translocation to the cell membrane. A recently
described primary cell culture model of rat IMCD cells (28) was
utilized to test this hypothesis. The cells were incubated with
Clostridium difficile toxin B, which inhibits all members of
the Rho family by glucosylation (Rho at Thr37, Rac and
Cdc42 at Thr35; Ref. 29), or microinjected with
Clostridium limosum-derived C3-fusion toxin, which
inactivates Rho by ADP-ribosylation at Asn41 but affects
neither Rac nor Cdc42 (30). In addition, IMCD cells were incubated with
the Rho kinase inhibitor, Y-27632, and microinjected with a
constitutively active mutant of RhoA. Surprisingly our data suggest
tonic inhibition of AQP2 translocation by Rho, which is relieved by an
increase in cAMP.
Toxins, Phalloidin, Antibodies, and Plasmids--
C.
difficile toxin B (29) and C. limosum-derived C3-fusion
toxin consisting of full-length C. limosum C3 toxin fused to the (inactive) N-terminal part of the actin-ADP-ribosylating C2I component of the C. botulinum binary toxin C2 (C3-fusion
toxin) were prepared as described before (30). Y-27632 was kindly
provided by Welfide Corp. (Osaka, Japan; Refs. 31 and 32).
TRITC-conjugated phalloidin was purchased from Sigma (Deisenhofen,
Germany) and Oregon green coupled to dextran from Molecular Probes
(Leiden, Netherlands). AQP2 was detected with a polyclonal antiserum raised against the C terminus of rat AQP2 (28, 33). Anti-rabbit Cy3-conjugated antibodies were purchased from Dianova (Hamburg, Germany).
A plasmid (pEXV-Myc-V14-RhoA) encoding constitutively active human RhoA
(RhoA-V14) was kindly provided by A. Hall (34). The insert encoding
RhoA-V14 was excized from the plasmid using the restriction enzymes
BamHI and EcoRI, and subcloned into the BglII and EcoRI restriction sites of the plasmid
pEGFP-C1 (CLONTECH, Heidelberg, Germany) to
construct a plasmid encoding a fusion protein (RhoA-V14-GFP) consisting
of RhoA-V14 and the green fluorescent protein (GFP).
Culture of IMCD Cells--
Rat renal inner medullae were the
source of primary cultured IMCD cells (28). Experiments were performed
6 days after seeding. Dibutyryl cAMP (Bt2cAMP), present in
the culture medium for maintenance of AQP2 expression, was removed
16 h prior to experiments with the exception of the experiments in
which the influence of RhoA-V14 expression on AQP2 translocation was determined.
Microinjection of C3-fusion Toxin and Plasmids into IMCD
Cells--
Microinjection was performed as described (35). In brief,
IMCD cells were seeded at a density of 7 × 104 per
cm2 on type IV collagen-coated coverslips with grids.
C3-fusion toxin (40 µg/ml; Ref. 30) was dissolved in
phosphate-buffered saline (137 mM KCl, 2.6 mM
NaCl, 7.8 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3, adjusted to 290 mosmol/kg with 1 M KCl) and co-microinjected (Eppendorf 5171 transjector) into the cytosol with Oregon green coupled to dextran
(2.5 mg/ml) on days 4 or 5 after seeding. The cells were fixed 20 min
after microinjection and processed for immunofluorescence microscopy
(see below).
The plasmid DNA encoding RhoA-V14-GFP (see above) was dissolved in the
same buffer as C3-fusion toxin and microinjected into the nuclei of
IMCD cells (100-200 copies/cell). Dibutyryl cAMP was permanently
present during the procedure. After injection, the cells were kept
under culture conditions (see above) for another 3 h to allow
expression of the plasmids and subsequently processed for
immunofluorescence microscopy (see below).
Immunofluorescence Microscopy and Quantification of
Immunofluorescence Intensities--
For immunofluorescence microscopy
IMCD cells were grown on coverslips. AQP2 was detected by confocal
laser scanning microscopy (LSM 410; Carl Zeiss, Jena, Germany; Leica
DMLB microscope with Sensicam 12 Bitled CCD camera, Bensheim, Germany)
using specific antibodies and Cy3-conjugated anti-rabbit secondary
antibodies (4, 28). RhoA-V14-GFP expression was visualized by detection of GFP fluorescence using laser scanning microscopy.
For visualization of F-actin, IMCD cells were grown, fixed, and
permeabilized as described for the detection of AQP2 (4, 28). They were
incubated with TRITC-conjugated phalloidin (100 µg/ml) for 30 min and
subsequently washed with phosphate-buffered saline as described (4,
28). Fluorescence signals were detected by conventional epifluorescence
microscopy (Leica DMLB microscope with a Sensicam 12 Bitled CCD camera,
Bensheim, Germany).
For quantification of the effects of AVP, toxins, and RhoA-V14-GFP on
AQP2 localization, the ratio of intracellular/cell membrane fluorescence signal intensities was calculated as described previously (4). For all groups, mean and S.E. values were calculated. Statistical
analyses were performed using the Student's t test and
one-way analysis of variance (4).
The Effect of Bacterial Toxins and the Rho Kinase Inhibitor,
Y-27632, on the F-actin Cytoskeleton of IMCD Cells--
Prior to
investigating the role of GTP-binding proteins of the Rho family in the
translocation of AQP2, bacterial toxins which affect the activity of
these proteins were tested for their ability to alter
F-actin-containing cytoskeletal structures, i.e. actin stress fibers (Fig. 1). For this purpose
IMCD cells were incubated with toxin B (4 µg/ml) for 3.5 h.
C3-fusion toxin was microinjected (40 µg/ml) and the cells were
prepared for detection of F-actin 20 min after microinjection. Cells
were co-microinjected with Oregon green coupled to dextran (2.5 mg/ml).
In addition, cells were incubated with Y-27632 (100 µM,
1 h). After these treatments the cells were fixed, permeabilized,
and F-actin was detected by incubation with TRITC-conjugated phalloidin
(0.1 mg/ml, 30 min). F-actin was subsequently visualized by
epifluorescence microscopy (Fig. 1).
Stimulation of IMCD cells with AVP (100 nM, 1 h)
induced a slight decrease in F-actin compared with non-stimulated
control cells (Fig. 1). A detailed analysis by optical sections of 1 µm through the cells indicated a decrease mainly of the cortical F-actin (data not shown; Ref. 16). In toxin B-, C3-fusion toxin-, and
Y-27632-treated IMCD cells, the phalloidin staining was strongly reduced, indicating depolymerization of F-actin. At later time points
(toxin B >4 h; C3-fusion toxin >1 h), the cells rounded up and
detached from the surface of the culture dish (data not shown).
Incubation with toxin B, Y-27632, or microinjection of C3-fusion toxin
resulted in an apparently stronger staining of the cortical F-actin.
This impression may be due to the disappearance of intracellular
F-actin, blunting visualization of cortical F-actin in untreated cells.
These data show that the toxins in the concentrations applied are
effective in altering F-actin-containing structures, i.e. the actin stress fibers. Within the time frame indicated, shape changes
were not detected in the majority of cells. Therefore these
concentrations of the toxins were applied in subsequent experiments.
Inhibition of Proteins of the Rho Family by Toxin B Induces
Translocation of AQP2 into IMCD Cell Membranes in the Absence of
Vasopressin--
Having established the effectiveness of toxin B in
modulating F-actin-containing structures, the toxin was applied to
investigate whether proteins of the Rho family are involved in the
regulation of AQP2 translocation in IMCD cells (Fig.
2). Incubations with toxin B were carried
out as described above. The distribution of AQP2 was determined by
laser scanning immunofluorescence microscopic analyses using specific
antisera. Fig. 2 shows a mainly intracellular distribution of AQP2 in
untreated IMCD cells (control). After stimulation of the cells with
AVP, AQP2 staining was mainly observed at the basolateral cell membrane
as shown by both xy and xz scans (see also: Refs.
4 and 28). Incubation of the cells with toxin B alone was sufficient to
invoke a strong redistribution of AQP2 (Fig. 2). Addition of AVP (100 nM) 1 h prior to the cessation of toxin B treatment
resulted in a further translocation of AQP2 (Fig. 2), as is evident
from the further decrease in intracellular fluorescence.
To quantify the effects of AVP and toxin B, intracellular and cell
membrane fluorescence signal intensities were related to nuclear
fluorescence signal intensities by analysis of laser scanning microscopic images, and the ratios of intracellular/cell membrane fluorescence signal intensities were determined (Fig.
3; Ref. 4). A ratio >1, indicating a
predominant intracellular localization of AQP2, was obtained for
control cells (1.75 ± 0.06; mean ± S.E.). Ratios <1,
statistically different from untreated control cells, were obtained for
AVP-stimulated cells (0.53 ± 0.03) and for cells incubated with
toxin B in the absence of AVP (0.70 ± 0.01), indicating a
predominant localization of AQP2 at the cell membrane. These results
suggest an inhibitory role of Rho proteins, whereby constitutive membrane localization of AQP2 is prevented in resting IMCD cells. The
ratio of intracellular/cell membrane fluorescence signal intensities after stimulation of toxin B-pretreated cells was 0.54 ± 0.02. This ratio was significantly different from both untreated control cells and toxin B-pretreated cells.
Inhibition of Rho or Rho Kinases Induces Redistribution of AQP2
into IMCD Cell Membranes--
To identify the protein of the Rho
family involved in the translocation of AQP2, C3-fusion toxin, which
specifically inactivates Rho, was used (see "Experimental
Procedures"; Ref. 30). The toxin was microinjected into the cytosol
together with Oregon green coupled to dextran. The dye was employed for
identification of microinjected cells and had no effect on the
intracellular localization of AQP2 (data not shown). Non-injected
control cells surrounding the microinjected ones show a predominantly
intracellular localization of AQP2 (Fig. 2). In microinjected cells,
however, AQP2 staining at the cell membrane is considerably increased, indicating a redistribution of AQP2. Likewise, inhibition of the downstream effectors of Rho, the Rho kinases, by Y-27632 (31, 32)
induced a translocation of AQP2 to the cell membrane (Fig. 2).
Quantitative analysis of the effect of C3-fusion toxin and Y-27632 on
IMCD cells (Fig. 3) yielded ratios of intracellular/cell membrane
fluorescence signal intensities of 0.94 ± 0.04 and 0.77 ± 0.05, respectively, which were significantly different from those
obtained for control cells. These data further support the notion that
a Rho-dependent pathway prevents a constitutive membrane localization of AQP2 in resting IMCD cells.
Activated Rho Abolishes cAMP-mediated Translocation of AQP2 into
IMCD Cell Membranes--
The effects of Rho inactivation described
above prompted us to study the effect of constitutively active Rho on
AQP2 redistribution in response to elevation of intracellular cAMP. For
this purpose, IMCD cells were microinjected with a plasmid encoding a
fusion protein (RhoA-V14-GFP) of constitutively active RhoA (RhoA-V14) and the green fluorescent protein (GFP; 100-200 copies/cell). RhoA-V14-expressing cells were visualized by the detection of GFP
fluorescence. The activity of RhoA-V14 was assayed by its ability to
induce actin stress fiber formation 3 h after microinjection of
the cells (Fig. 4).
Subsequently the effect of the microinjection of the plasmid
encoding RhoA-V14-GFP on the localization of AQP2 was investigated in
stimulated cells (i.e. in the presence of
Bt2cAMP). Fig. 5 shows two
microinjected IMCD cells expressing RhoA-V14-GFP (left panel) surrounded by non-injected cells. In non-injected cells, AQP2 is predominantly localized at the cell membrane (AQP2,
middle panel). In contrast, microinjected cells in the same
field exhibit a mainly intracellular localization of AQP2. Expression
of GFP alone had no effect on the localization of AQP2 (data not
shown). The overlay of both signals is shown in the right
panel.
Quantitative analysis of the effect of RhoA-V14 expression in IMCD
cells (Fig. 3) showed an intracellular/cell membrane fluorescence signal intensity ratio of 1.89 ± 0.17. This value was not
significantly different from that obtained for non-stimulated cells,
indicating that constitutively active RhoA abolished AVP-mediated
translocation of AQP2. It is therefore concluded that constitutively
active RhoA-V14 prevents the translocation of AQP2 to the IMCD cell membrane.
Depolymerization of the F-actin Cytoskeleton by Cytochalasin D
Induces Translocation of AQP2 into IMCD Cell Membranes--
The data
presented above show the involvement of Rho in the translocation of
AQP2. To test whether this proceeds independently of the F-actin
cytoskeleton or whether this is possibly mediated by Rho via the
regulation of the F-actin-containing network, F-actin was depolymerized
by incubation of the cells with cytochalasin D (2 µM) for
6, 11, 16, 25, and 30 min. This treatment induced a gradual decrease in
F-actin content over 0-25 min (Fig. 6). After 16 min, a pronounced F-actin depolymerization was induced, and cell shape changes were observed. At 25 min, the cortical F-actin
appeared thickened and the cells were clearly shrunken. The apparently
stronger staining of the cortical F-actin may be due to the
disappearance of intracellular F-actin which in the untreated control
cells blunts visualization of cortical F-actin. After 30 min, F-actin
acquired a punctate appearance, and holes in the monolayer
appeared.
At early time points, i.e. when cytochalasin D-mediated
F-actin depolymerization had commenced (6-11 min), AQP2 translocated to the cell membrane (Fig. 7). The ratios
of intracellular/membrane signal intensities were 0.87 ± 0.03 at
6 min and 0.67 ± 0.05 at 11 min, indicating a continuing
translocation of AQP2 to the cell membrane (Fig. 3). The intracellular
staining of AQP2 after 16 min of incubation with cytochalasin D
appeared stronger compared with that at 11 min (Fig. 7); the ratio of
intracellular/cell membrane signal intensities was 0.84 ± 0.05 (Fig. 3). As described above, longer incubation with cytochalasin D
(25-30 min) severely affected the IMCD cells even to the point of cell
death. Therefore no conclusive data could be obtained regarding the
intracellular localization of AQP2 at these time points.
These results clearly indicate that partial depolymerization of F-actin
by cytochalasin D is sufficient to induce translocation of AQP2 to the
cell membrane. These data are consistent with the hypothesis that Rho
influences AQP2 translocation via regulation of F-actin-containing
cytoskeletal structures.
Previous studies using toad bladders have demonstrated the
involvement of the F-actin cytoskeleton in AVP-mediated increases in
osmotic water permeability (13, 14). In addition, the reorganization of
the F-actin cytoskeleton by AVP is a consistent finding in both
amphibian urinary bladder epithelia and mammalian renal principal cells
(2, 16). The small GTP-binding proteins of the Rho family (Rho, Rac,
and Cdc42) are involved in the regulation of the F-actin cytoskeleton.
The aim of this study was to investigate their possible involvement in
the translocation of AQP2 to the cell membrane. Inactivation of all
members of the proteins of the Rho family by incubation of IMCD cells
with toxin B or selective inactivation of Rho by C3-fusion toxin
induced the translocation of AQP2 in the absence of AVP. In addition,
inhibition of effectors of Rho, the Rho kinases, induced a
translocation of AQP2 to the cell membrane to a similar extent as
C3-fusion toxin in the absence of AVP. Conversely, expression of a
constitutively active mutant of RhoA (RhoA-V14) abolished the
cAMP-mediated translocation of AQP2. These findings indicate that Rho
plays an inhibitory role in the AQP2 shuttle. The inhibitory role of
Rho in a cAMP-triggered exocytotic event (AQP2 translocation) is in
contrast to the stimulatory role of Rho in Ca2+-triggered
exocytosis in RBL 2H3 and mast cells (see Introduction).
The inhibitory role of Rho in cAMP-triggered AQP2 translocation was
confirmed using the permanent rabbit cortical collecting duct cell line
CD8, stably transfected with rat
AQP2.2 These cells respond to
forskolin by translocation of AQP2 into the apical cell membrane (6,
17, 36). Inhibition of Rho induced the translocation of AQP2 to the
membrane of resting CD8 cells and expression of constitutively active
Rho prevented the cAMP-triggered translocation.
Compared with AVP, toxin B, C3-fusion toxin, and Y-27632 induced an
incomplete translocation of AQP2. This effect may be explained by a
partial inactivation of the inhibitory Rho family-dependent signaling pathway. This assumption is supported by the observation that
longer incubations with the toxins (toxin B > 4 h; C3-fusion toxin > 1 h) induced cell shape changes and detachment of
the cells from the culture dishes, indicating that the Rho
family-dependent pathway is not completely inhibited by the
toxins at earlier time points.
In IMCD cells, toxin B caused a 2-fold increase in osmotic water
permeability (Pf). This was shown by applying a new technique
using laser scanning reflection microscopy, established for determining
osmotic water permeability coefficient changes in single adherent cells
(37). These data confirm the inhibitory role of the proteins of the Rho
family in the translocation of AQP2 and show that this inhibition is
functionally relevant.
Incubation of IMCD cells with toxin B or Y-27632 and
microinjection of C3-fusion toxin resulted not only in AQP2
translocation but also in depolymerization of F-actin containing stress
fibers (Fig. 1). Similarly, cytochalasin D caused both depolymerization of the F-actin cytoskeleton and translocation of AQP2 within 6-11 min
of incubation (Figs. 6 and 7). Thus depolymerization of the F-actin
cytoskeleton is sufficient to induce the translocation of AQP2, and the
F-actin cytoskeleton apparently prevents translocation of AQP2-bearing
vesicles to the cell membrane of resting cells. Its depolymerization
allows AQP2-bearing vesicles to translocate to the cell membrane. The
molecular basis of this observation may be explained by different
hypotheses. It is feasible that AQP2-bearing vesicles are linked
directly to the F-actin cytoskeleton as postulated by Brown et
al. (9). Alternatively an intermediate protein may be required, as
postulated by Umenishi et al. (38).
There are a few cell types in which the disassembly of the F-actin
cytoskeleton can trigger exocytosis. However, the role of Rho has not
been investigated in this context. In pancreatic acinar cells, the
introduction of the actin monomer-binding protein The results presented here may be explained by the following
model: stimulation of IMCD cells with AVP results in an increase in
intracellular cAMP, which in turn activates PKA. One of the substrates
of PKA is Rho, which is phosphorylated at Ser188 (41). The
phosphorylation of Rho decreases the binding of RhoA to Rho kinases
(42). The subsequent attenuation of Rho activity would favor
depolymerization of the F-actin cytoskeleton and allow translocation of
AQP2 into the cell membrane. Consistent with this model, our results
show that stimulation of IMCD cells with AVP induces a depolymerization
of the F-actin cytoskeleton (Fig. 1). Induction of polymerization of
F-actin-containing stress fibers by expression of constitutively
active RhoA (RhoA-V14) abolished the translocation of AQP2 in response
to continuous stimulation of IMCD cells with Bt2cAMP (Fig.
5). In addition, incubation of IMCD cells with cytotoxic necrotizing
factor I, derived from pathogenic Escherichia coli, which
activates all members of the Rho family by deamidation (29), strongly
induced the formation of actin stress fibers and inhibited AVP-mediated
AQP2 translocation (data not shown). The increase in F-actin content
may either increase the binding of AQP2-bearing vesicles to the F-actin
cytoskeleton or the F-actin cytoskeleton might become a barrier for
these vesicles similar to the barrier function of cortical F-actin in
other exocytotic processes. For example, in chromaffin cells,
pancreatic acinar cells and mast cells, cortical F-actin disassembly is
considered a prerequisite for exocytosis (39, 43-45). The model
proposed above is further supported by the finding that RhoA and RhoB
are present in particulate fractions prepared from non-stimulated IMCD
cells (data not shown). Since activation of Rho is accompanied by a
translocation from the cytosol to membranes, this finding may indicate
that RhoA and RhoB are either active or in the process of activation
(46-48).
The only other system in which Rho appears to inhibit exocytosis via
F-actin is the bovine chromaffin cell (45). Gasman et al.
(49, 50) described a vesicle-associated signaling cascade in resting
chromaffin cells in which the heterotrimeric G protein Go
activates phosphatidylinositol 4-kinase via Rho. Activation of
phosphatidylinositol 4-kinase may stabilize F-actin, thereby excluding
secretory vesicles from exocytosis. For two reasons it is tempting to
speculate that a similar mechanism underlies the inhibition of the AQP2
translocation: (i) mastoparan-sensitive heterotrimeric G proteins of
the Go/i family have been co-purified with AQP2-bearing
vesicles from CD8 cells (6), and, (ii) the finding that Rho proteins
are present in IMCD and CD8 cell fractions enriched for AQP2-bearing
vesicles (data not shown). Final proof that Rho and
phosphatidylinositol 4-kinase reside on AQP2-bearing vesicles will
require analysis of the protein pattern of immuno-isolated vesicles
using AQP2-, Rho-, and phosphatidylinositol 4-kinase-specific antibodies. In summary, the data presented here show that active Rho
prevents AQP2 translocation from intracellular vesicles to the cell
membrane, presumably by stimulating the formation of the F-actin cytoskeleton.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The effect of bacterial toxins and the Rho
kinase inhibitor, Y-27632, on the F-actin cytoskeleton in IMCD
cells. IMCD cells were left untreated (control), incubated with
AVP (100 nM, 1 h) or with C. difficile
toxin B (4 µg/ml, 3.5 h) which inhibits all proteins of the Rho
family. Incubations with the Rho kinase inhibitor, Y-27632, were
carried out for 1 h (100 µM). C. limosum
C3-fusion toxin (40 µg/ml, 20 min; Ref. 30) which specifically
inhibits Rho was microinjected. Microinjected cells were visualized by
co-microinjected Oregon green coupled to dextran (2.5 mg/ml). After
incubations for the indicated lengths of time cells were fixed,
permeabilized, and incubated with TRITC-conjugated phalloidin (0.1 mg/ml). Phalloidin fluorescence was detected by epifluorescence
microscopy. Scale bars, 20 µm.
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Fig. 2.
Localization of AQP2 in IMCD cells. IMCD
cells were either left untreated (control), incubated with AVP (100 nM, 1 h), toxin B (4 µg/ml, 3.5 h), or Y-27632
(100 µM, 1 h). C3-fusion toxin (40 µg/ml, 20 min)
was microinjected. Microinjected cells were visualized by
co-microinjected Oregon green coupled to dextran (2.5 mg/ml). AVP (100 nM) was added to toxin B-pretreated cells 1 h prior to
cessation of toxin treatment. After incubations for the indicated
lengths of time, the cells were fixed, permeabilized, and incubated with anti-AQP2 and
secondary cy3-conjugated anti-rabbit antibodies. AQP2
immunofluorescence was detected by laser scanning microscopy. The upper
part of each panel represents xy scans; the lower part
represents xz scans along the white line in the
xy image. Scale bars, 20 µm.
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Fig. 3.
Quantitative analysis of the effects of
bacterial toxins, Y-27632, cytochalasin D and RhoA-V14 on the
localization of AQP2 in IMCD cells. IMCD cells were treated as
indicated in Figs. 1, 2, and 4-7. Immunofluorescence signals were
detected by laser scanning microscopy (4). The intracellular and cell
membrane immunofluorescence signal intensities were determined and
related to nuclear signal intensities (n = 25 for
untreated control, n = 14 for + AVP, n = 41 for toxin B AVP, n = 24 for toxin B + AVP,
n = 27 for C3-fusion toxin, n = 24 for
Y-27632, n = 9 for Rho-V14; n = 15 for
cytochalasin D for 6 and 11 min, n = 12 for
cytochalasin D for 16 min; 8-bit scale; mean ± S.E.; three
independent experiments). The ratios of intracellular/cell membrane
fluorescence signal intensities were calculated. Ratios >1 indicate a
predominantly intracellular localization of AQP2 and ratios <1 a
predominant localization at the cell membrane. Values significantly
different from untreated control cells are indicated
(asterisks, p < 0.001).
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Fig. 4.
The effect of the expression of a
constitutively active RhoA mutant (RhoA-V14) on the F-actin
cytoskeleton in IMCD cells. IMCD cells were cultured in the
presence of Bt2cAMP and microinjected with a plasmid
(100-200 copies/cell) encoding RhoA-V14 fused to the GFP. RhoA-V14
expression was analyzed 3 h after microinjection by detection of
GFP fluorescence (epifluorescence microscopy; RhoA-V14-GFP; left
panel). Thereafter, the cells were fixed, permeabilized, and
incubated with TRITC-conjugated phalloidin (right panel) to
visualize the F-actin cytoskeleton (see legend to Fig. 1). TRITC
fluorescence was detected by epifluorescence microscopy. Scale
bars, 20 µm.
View larger version (38K):
[in a new window]
Fig. 5.
Localization of AQP2 in IMCD cells expressing
a constitutively active mutant of RhoA (RhoA-V14). IMCD
cells were cultured in the presence of Bt2cAMP and
microinjected with a plasmid encoding RhoA-V14 fused to the GFP (see
Fig. 4). RhoA-V14 expression was analyzed 3 h after microinjection
by detection of GFP fluorescence (epifluorescence microscopy;
RhoA-V14-GFP; left panel). Thereafter, the cells were fixed,
permeabilized, incubated with anti-AQP2 and secondary cy3-conjugated
anti-rabbit antibodies. AQP2 immunofluorescence was detected by
epifluorescence microscopy. The overlay of GFP and AQP2 fluorescence
signals is shown in the right panel. Scale bars, 20 µm.
View larger version (99K):
[in a new window]
Fig. 6.
The effect of cytochalasin D on the F-actin
cytoskeleton in IMCD cells. IMCD cells were left untreated (0 min)
or incubated with cytochalasin D (2 µM) which induces
depolymerization of the F-actin cytoskeleton. At the time points
indicated, the cells were fixed, permeabilized, and incubated with
TRITC-conjugated phalloidin ((0.1 mg/ml) to visualize the F-actin
cytoskeleton (see legend to Fig. 1). TRITC fluorescence was detected by
epifluorescence microscopy. Scale bars, 20 µm.
View larger version (70K):
[in a new window]
Fig. 7.
The effect of cytochalasin D on the
localization of AQP2 in IMCD cells. IMCD cells were incubated
without (control) or with AVP (100 nM, 1 h)
or cytochalasin D (2 µM) for the indicated lengths of
time. After incubations, cells were fixed, permeabilized, incubated
with anti-AQP2 and secondary Cy3-conjugated anti-rabbit antibodies.
AQP2 immunofluorescence was visualized by laser scanning microscopy.
The upper part of each panel represents xy scans;
the lower part represents xz scans along the
white line in the xy image. Scale
bars, 20 µ m.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thymosin, which
induces limited F-actin depolymerization, into permeabilized cells,
results in rapid amylase release without applying additional stimuli
(39). In alveolar epithelial type II cells (AET2 cells), C2 toxin,
which depolymerizes the F-actin cytoskeleton, increased basal
surfactant secretion (40).
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ACKNOWLEDGEMENTS |
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We are grateful to H. Barth for preparing C3-fusion toxin, A. Geelhaar, C. Bouchaala, and B. Oczko for excellent technical assistance and J. Dickson for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Ro 597/6 and the Fond der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Tel.: 49-30-94793-260; Fax: 49-30-94793-109; E-mail: klussmann@fmp-berlin.de.
Published, JBC Papers in Press, February 13, 2001, DOI 1.1074/jbc.M010270200
2 G. Tamma, E. Klussmann, K. Aktories, M. Svelto, W. Rosenthal, and G. Valenti, manuscript submitted.
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ABBREVIATIONS |
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The abbreviations used are: AVP, arginine-vasopressin; AQP2, aquaporin-2; Bt2cAMP, dibutyryl cAMP; IMCD, inner medullary collecting duct; GFP, green fluorescent protein; PKA, protein kinase A; TRITC, tetramethylrhodamine isothiocyanate; Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Rho kinase inhibitor).
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