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*
Department of Pathology, University of California, San Diego,
California.
Department of Cellular and Molecular Medicine, University of California,
San Diego, California.
Department of Pathology, Stanford University School of Medicine, Stanford,
California.
Correspondence to Dr. Marilyn G. Farquhar, Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA. Phone: 858-534-7711; Fax: 858-534-8549; E-mail: mfarquhar{at}ucsd.edu Dr. Orlando's present address is Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, 915 Camino de Salud, Albuquerque, NM 87131.Dr. Takeda's present address is Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cDNA for podocalyxin recently isolated from human (11), rabbit (12), chicken (13), and rat (1) reveals that it is a type I membrane protein with four potential N-linked and numerous potential O-linked glycosylation sites. Like other sialomucins, the extracellular domain of podocalyxin from various species shows little sequence conservation aside from four conserved cysteine residues and mucin-like structural features. In contrast, the transmembrane and cytoplasmic tail sequences are highly conserved between species, suggesting that these domains have a critical and highly conserved function. However, no studies have yet been reported that describe either potential functions of the cytoplasmic tail or the nature of interacting proteins. By immunoelectron microscopy, we showed previously that the subcellular distribution of podocalyxin in podocytes coincides with that of ezrin, an intracellular actin-linking protein (14). These findings suggested to us that podocalyxin may associate with the extensive actin filament network found in podocyte foot processes. Ezrin is a member of the ezrin-radixin-moesin (ERM) family of actin-binding proteins (15,16,17). ERM family members are known to link cell surface integral membrane proteins with the underlying actin cytoskeleton. Examples of these cell surface proteins include the hyaluronate receptor (CD44) (18,19), sodium-hydrogen exchanger (NHE) (20,21), CD43 (22,23), and cell adhesion molecules (ICAM-1, -2, and -3) (19,24). Regulation of ezrin activity involves phosphorylation by Rho-dependent kinase, a serine/threonine kinase that belongs to the myotonic dystrophy family of kinases (25). Phosphorylation of threonine residues in ezrin activates it and facilitates its associations with membrane proteins and actin filaments (15,26,27).
In the present study, we investigated whether podocalyxin is associated with the actin cytoskeleton and determined whether ezrin forms an integral link between podocalyxin and actin filaments. Here, we show that podocalyxin and ezrin co-localize at the apical plasma membrane of podocyte foot processes and that they form a very stable co-immunoprecipitable complex. Using selective detergent extraction and co-sedimentation assays, we show that podocalyxin is associated with actin filaments and that ezrin mediates this interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies
Anti-podocalyxin monoclonal antibodies (mAb) 5A and 1A, raised against
Triton X-114 detergent-extracted rat glomeruli
(29), reacts with the
ectodomain of rat podocalyxin. Anti-podocalyxin polyclonal antibody (pAb)
(0601) was generated against a synthetic peptide corresponding to the
C-terminal 13 amino acids of rat podocalyxin as described previously
(1). Anti-ezrin pAb was raised
against recombinant human ezrin as described previously
(30,31)
and demonstrates no cross reactivity with moesin and radixin, two closely
related members of the ERM family. Anti-ezrin mAb (3C12) was purchased from
NeoMarkers, Inc. (Fremont, CA). The epitope for mAb 3C12 is reported to be
between amino acids 362 and 585. Anti-actin mAb AC-40
(32) was purchased from Sigma.
Cross-absorbed anti-rabbit Alexa 488 IgG (H+L) and anti-mouse Alexa 594
F(ab')2 were purchased from Molecular Probes, anti-rabbit (10
nm) and anti-mouse (5 nm) gold conjugates were purchased from
Amersham-Pharmacia (Piscataway, NJ), and horseradish
peroxidase—conjugated anti-rabbit and anti-mouse IgG was purchased from
BioRad (Hercules, CA).
Immunofluorescence Microscopy
Kidneys of Sprague Dawley rats were perfused with Dulbecco's modified
Eagle's medium, followed by 4% paraformaldehyde in 100 mM phosphate buffer (pH
7.4; 15 min). Kidneys were excised and immersion fixed in 4% paraformaldehyde
(45 min) followed by 8% paraformaldehyde (15 min). Samples were cryoprotected
and frozen in liquid nitrogen
(33). Semithin cryosections
(0.5 to 1.0 µm) were cut on a Leica Ultracut UCT microtome (equipped with a
cryoattachment at - 100°C), placed on gelatin-coated microscope slides,
and incubated for 2 h at room temperature with affinity-purified
anti-podocalyxin (0601) and anti-ezrin mAb 3C12, followed by incubation for 1
h with goat anti-rabbit Alexa 488 and anti-mouse Alexa 594. Sections were
observed with a Zeiss Axiophot microscope equipped for epifluorescence. Images
were captured with a CCD camera and SCION Image software and processed with
Adobe Photoshop 5.0. Final images were printed on Kodak Ektatherm XLS paper
using a Kodak ColorEase PS printer.
Immunoelectron Microscopy
Rat kidneys were fixed and prepared as described above for
immunofluorescence. Ultrathin cryosections (approximately 80 nm) were cut,
blocked for 30 min with 10% fetal calf serum in phosphate-buffered saline
(33), and incubated
sequentially with anti-ezrin pAb for 16 h at 4°C followed by
anti-podocalyxin mAb 5A for 2 h at 23°C. Bound primary antibodies were
detected by incubation with anti-rabbit and anti-mouse gold conjugates (5 and
10 nm, respectively; 1:50). Sections then were stained with 2% neutral uranyl
acetate (20 min); adsorption-stained with 0.2% uranyl acetate, 0.2% methyl
cellulose, and 3.2% polyvinyl alcohol; and examined with a JEOL 1200 EX-II
electron microscope.
Glomerular Isolation
All procedures were performed at 4°C. Frozen rat kidneys were dissected
to obtain cortex, and isolated cortex was finely minced and suspended in 20 mM
HEPES (pH 7.4), 150 mM NaCl containing protease inhibitor mix (2 µg/ml
chymostatin, 2 µg/ml leupeptin, 2 µg/ml antipain, 2 µg/ml pepstatin,
10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Material was
gently pressed through a 350-mesh nylon screen followed by a 150-mesh screen.
Filtered material was poured onto a 70-mesh screen and rinsed with cold
phosphate-buffered saline. Glomeruli remaining on top of the screen were
collected, pelleted by centrifugation (500 x g, 5 min,
4°C), and rinsed with cold phosphate-buffered saline. The preparation
consisted of >95% glomeruli when examined with an inverted phase-contrast
microscope.
Cell Culture
MDCK-PC8 and CHO-PC13 cells stably expressing rat podocalyxin were
generated as described previously
(1). Cells were grown in
modified Eagle's medium-Earle salts and Ham's F-12, respectively, supplemented
with 10% fetal calf serum (Hyclone, Logan, UT), 100 µg/ml streptomycin
sulfate, 100 U/ml penicillin G, and 0.5 mg/ml G418 (CalBiochem, San Diego,
CA).
Actin Depolymerization and Texas Red—Phalloidin Staining of
Actin
Cells were grown to approximately 75% confluence on glass coverslips,
incubated for 30 min at 37°C with Dulbecco's modified Eagle's medium alone
or either cytochalasin D (20 µg/ml) or latrunculin B (10 µg/ml) diluted
into Dulbecco's modified Eagle's medium, fixed for 20 min with 2%
paraformaldehyde in PBS, and quenched by incubation for 20 min with 20 mM Tris
(pH 7.4), 150 mM NaCl (TBS), 0.1 M glycine. Cells were then permeabilized for
5 min with 0.05% Triton X-100 in TBS, 0.1 M glycine, and incubated for 1 h
with Texas Red phalloidin (1:100) in TBS, 10% calf serum. After rinsing, cells
were mounted in Gelvatol (Monsanto, St. Louis, MO) containing 1 mg/ml
paraphenylenediamine and examined with a Zeiss Axiophot equipped for
epifluorescence. Images were captured and processed as described above and
printed by use of a Codonics NP-1660 black and white thermal printer.
Immunoprecipitations and Immunoblotting
In some cases, before immunoprecipitations, MDCK-PC8 and CHO-PC13 cells
were treated with A. ureafaciens sialidase, as described previously
(1), or incubated at 37°C
for 4 h with Y-27632 at the indicated concentrations diluted into their
respective growth media. For immunoprecipitation, proteins from isolated rat
kidney glomeruli or MDCK-PC8 or CHO-PC13 were solubilized with 1% Triton X-100
in TBS and protease inhibitor mix. Detergent insoluble material was removed by
centrifugation (12,000 x g, 5 min, 4°C), and soluble
proteins were incubated with a mix of anti-podocalyxin mAb 5A and 1A (3 µg
each) and protein G-agarose for 16 h at 4°C. Immunoprecipitates were
washed with TBS containing 1% Triton X-100 (3x) followed by TBS
(1x), solubilized in Laemmli sample buffer supplemented with 4%
ß-mercaptoethanol and heated at 95°C for 3 min. For immunoblotting,
antibody-bound proteins were resolved by sodium dodecyl
sulfate—polyacrylamide gel electrophoresis and electrotransferred to
polyvinylidene difluoride membranes using a wet tank transfer method (BioRad).
Membranes were blocked with TBS, 0.1% Tween-20, and 5% calf serum for 30 min
at 23°C and incubated with either anti-podocalyxin or anti-ezrin (1:4000)
antisera or anti-actin mAb AC40 (100 ng/ml) in blocking buffer for 1 h at
23°C. Membranes were washed 3 times (10 min) with TBS, 0.1% Tween-20, 1%
calf serum, and bound antibodies were detected with species-specific
horseradish peroxidase—conjugated secondary antibodies, followed by
chemiluminescence detection according to the manufacturer's instructions
(Pierce).
Selective Detergent Extraction and Co-Sedimentation Analysis
Cortex was removed from frozen rat kidneys, finely minced, re-suspended in
20 mM HEPES (pH 7.4) and 150 mM NaCl, and processed using a TissueMizer
(Tekmar, Cincinnati, OH) equipped with 0.5-cm rotary blades (3 min). Cortical
homogenate was incubated for 3 h at 4°C with an equal volume of 10 mM
imidazole (pH 7.2), 75 mM KCl, 5 mM MgCl2, 1 mM
ethyleneglycoltetraacetic acid containing 2% (1% final concentration) of
Triton X-100 (TX-100),
3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate hydrate, Tween-20,
sodium dodecyl sulfate, or N-lauroyl sarcosine and protease inhibitor mix. In
some cases, 0.6 M KI was added to the solubilization step for actin
depolymerization
(34,35,36).
Detergent-solubilized proteins were centrifuged at 100,000 x g
at 4°C for 45 min. Pelleted material was resuspended to identical volume
as the supernatant, and proteins were analyzed by immunoblotting as described
above with anti-podocalyxin, anti-ezrin, and anti-actin antibodies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because podocalyxin and ezrin are co-expressed in podocytes, we next determined whether they co-localize along the apical plasma membrane of podocyte foot processes by performing double immunogold labeling of ultrathin cryosections from rat kidney. Podocalyxin and ezrin were found to be concentrated along the cell body and the apical plasma membrane of the foot processes above the slit diaphragms (Figure 2).
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Podocalyxin and Ezrin Are Associated in Glomerular Epithelial
Cells
Co-localization of podocalyxin and ezrin at the apical surface of podocytes
prompted us to examine whether these two proteins are associated in a
molecular complex and can be co-immunoprecipitated. Toward this end, proteins
were detergent-extracted from isolated rat kidney glomeruli and subjected to
immunoprecipitation with anti-podocalyxin mAb, followed by immunoblotting with
anti-ezrin pAb. As shown in Figure
3, ezrin could be co-immunoprecipitated with podocalyxin,
indicating that it is complexed with podocalyxin in glomeruli. After
immunoprecipitation with mAb 20B to megalin, another kidney antigen present in
podocytes and the proximal tubule brush border, no ezrin could be detected in
the precipitates, thus demonstrating the specificity of ezrin's interaction
with podocalyxin.
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Podocalyxin Is Associated with Actin Filaments
Because ezrin is known to mediate interactions between cell surface
proteins and the actin cytoskeleton, we next examined whether podocalyxin is
associated with actin filaments. Actin filaments are stable structures that
are resistant to mild detergent extraction. After detergent treatment, actin
filaments as well as tightly associated proteins remain insoluble and are
pelleted by centrifugation at 100,000 x g. Therefore, we
extracted rat kidney membranes with both mild nondenaturing detergents (Triton
X-100, 3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propane-sulfonate hydrate,
or Tween-20) or with harsh denaturing detergents (sodium dodecyl sulfate or
N-lauroyl sarcosine), centrifuged the extracts at 100,000 x g,
and immunoblotted the resulting detergent soluble (supernatant) and insoluble
(pellet) fractions with anti-podocalyxin and antiactin antibodies. As
expected, actin was found primarily in the insoluble fraction when membranes
were extracted with mild detergents and in the soluble fraction when extracted
with denaturing detergents (Figure
4). A significant fraction of the podocalyxin co-sedimented with
actin in the insoluble fraction after mild detergent extraction, whereas all
of the podocalyxin together with actin was found in the soluble fraction after
extraction with denaturing detergents.
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We next examined the distribution of podocalyxin and ezrin in detergent-soluble and -insoluble fractions before and after actin depolymerization, to determine whether the co-sedimentation of podocalyxin with actin was dependent on intact actin filaments. Rat kidney cortical homogenates were extracted with mild detergents in the absence or presence of 0.6 M KI, which was shown previously to depolymerize actin (34,35,36). In the absence of KI, both podocalyxin and ezrin co-sedimented with actin (Figure 5). However, in the presence of KI actin, podocalyxin and ezrin were found in the soluble fraction. These data indicate that sedimentation of podocalyxin requires intact actin microfilaments and that podocalyxin is associated with the actin cytoskeleton through complex formation with ezrin.
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Interaction between Podocalyxin and Ezrin Is Not Dependent on Intact
Actin Filaments
We next addressed whether the in vivo interaction between
podocalyxin and ezrin is dependent on polymerized actin filaments. To this
end, MDCK-PC8 and CHO-PC13 cells stably expressing rat podocalyxin
(1) were treated with the actin
depolymerizing agents cytochalasin D or latrunculin B, followed by
immunoprecipitation with anti-podocalyxin and immunoblotting with anti-ezrin.
As expected, treating MDCK-PC8 or CHO-PC13 cells with either cytochalasin D or
latrunculin B resulted in complete disruption of the actin cytoskeleton, as
seen by Texas Red—phalloidin staining
(Figure 6A). Without treatment,
ezrin was found to co-precipitate with podocalyxin from both MDCK-PC8 or
CHO-PC13 cells (Figure 6B),
indicating that exogenous expression of podocalyxin in these cell types is
sufficient for interactions with endogenous ezrin and that this interaction
does not require co-expression of additional podocyte-specific proteins. Cells
treated with either cytochalasin D or latrunculin B showed no significant
reduction in the amounts of ezrin co-precipitating with podocalyxin. These
results indicate that the association between podocalyxin and ezrin in
vivo is maintained after actin depolymerization and therefore is not
dependent on intact actin microfilaments.
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Interaction between Podocalyxin and Ezrin Is Not Affected by
Sialidase Treatment or Rho-Kinase Inactivation
Events that alter extracellular domains of cell surface proteins often
affect their intracellular interactions with cytosolic proteins. Because the
polyanionic charge of podocalyxin is responsible for podocalyxin's
anti-adhesion properties (1),
we examined whether removal of sialic acid would result in loss of its
association with ezrin. Toward this end, MDCK-PC8 or CHO-PC13 cells were
treated with sialidase and subjected to co-immunoprecipitation analysis. As
shown in Figure 7A, sialidase
treatment of podocalyxin had no quantitative effect on the amount of ezrin
that co-precipitated with podocalyxin. Thus, interactions between ezrin and
the cytoplasmic tail of podocalyxin in transfected MDCK or CHO cells are
independent of the ionic charge on podocalyxin's ectodomain.
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Rho-kinase—mediated phosphorylation of ezrin is necessary to activate ezrin to serve as a link between membrane proteins and actin filaments. Thus, if the association between podocalyxin and ezrin is in dynamic equilibrium, inhibition of Rho-kinase might be expected to lead to accumulation of inactive ezrin and reverse its association with podocalyxin. To examine more closely the dynamic stability of the interaction, we incubated MDCK-PC8 and CHO-PC13 cells with various concentrations of the potent Rho-kinase inhibitor Y-27632 (28) and quantified the association of podocalyxin and ezrin by co-immunoprecipitation analysis. When cells were treated with 40 µM Y-27632, extensive disruption and fragmentation of the actin cytoskeleton were seen in both cell lines as visualized by Texas Red—phalloidin staining (data not shown); however, no quantitative change was seen in the amount of ezrin that co-precipitated with podocalyxin (Figure 7B). These results indicate that once formed, interactions between podocalyxin and ezrin are very stable and not reversed by inactivation of Rho-kinase. Although previous studies showed that Rho-kinase is necessary for ezrin activation, this is the first demonstration that inhibition of Rho-kinase is not sufficient for inactivation of ezrin once it is complexed with a membrane protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we showed that ezrin, an ERM family member known to link several plasma membrane proteins to the actin cytoskeleton, is concentrated along the apical plasma membrane of podocyte foot processes (14). In the present study, we found that podocalyxin and ezrin co-localize in this location by immunofluorescence and immunogold labeling. In addition, we found that podocalyxin and ezrin can be co-immunoprecipitated and thus are associated as a complex in podocytes. An association with ezrin suggests that podocalyxin may interact indirectly with the actin cytoskeleton (16,17,39). Using selective detergent extraction and differential centrifugation, we showed that podocalyxin co-sediments with actin filaments. Moreover, when actin filaments are depolymerized, both podocalyxin and ezrin no longer sediment, indicating that podocalyxin is associated with actin filaments through interactions with ezrin. Taken together, these data indicate that podocalyxin is linked to the actin cytoskeleton in glomerular epithelial cells and likely contributes to podocyte foot process morphology through complex formation with ezrin. This study also describes the first reported function of the highly conserved cytoplasmic domain of podocalyxin.
ERM family members, including ezrin, are characterized by their structural similarities to erythrocyte band 4.1 membrane protein (16,17). An N-terminal FERM protein module is common to all family members and is thought to serve as an anchor to their associated membrane proteins (40,41). Ezrin also contains a C-terminal actin binding motif that is essential for linking membrane proteins to the cytoskeleton (42,43). Regulation of ERM proteins and their associations with membrane proteins is thought to occur through intramolecular masking of protein—protein interaction sites (15,17). The inactive form of these proteins typically is found either as monomers with their N-terminal FERM domain tightly associated with their C-terminal domain or as oligomers with intermolecular head-to-tail associations (44). In ezrin, Rho-kinase—mediated threonine phosphorylation weakens head-to-tail associations and unmasks membrane protein and F-actin binding sites (27). Once activated, ezrin is able to link plasma membrane proteins with the underlying actin cytoskeleton. Whether ezrin first engages the plasma membrane proteins followed by actin binding or is first assembled onto actin filaments followed by recruitment of membrane proteins is unknown. However, we show that complex formation between podocalyxin and ezrin is very stable and, once formed, is unaffected by actin depolymerization or Rho-kinase inhibition. The stability of this interaction suggests that the cytoplasmic tail of podocalyxin serves as a scaffold for preassembly of activated ezrin, and this in turn is followed by attachment to actin filaments. Alternatively, rather than a direct interaction between ezrin and podocalyxin, other ezrin-binding regulatory proteins, such as NHERF (21,45), may contribute to the assembly of ezrin and podocalyxin and their subsequent attachment to actin filaments. Consistent with this, we recently identified a direct interaction between podocalyxin and NHERF (46). However, the topographical relationship among podocalyxin, ezrin, and NHERF is unknown.
Loss of the characteristic foot process organization of podocytes accompanies a number of glomerular disorders associated with severely compromised glomerular filtration. Loss of foot process structure and function can be mimicked by treating glomeruli with cytochalasin (47), thus demonstrating the importance of intact actin cytoskeleton to podocyte morphology and glomerular function. Our demonstration that podocalyxin is associated with actin filaments, together with our previous work showing the role of its ectodomain as an anti-adhesin (1), strongly implicates podocalyxin as a major contributor to the development and maintenance of foot process morphology. It follows that structural and/or functional defects in either podocalyxin or associated cytoskeletal linker proteins, such as ezrin, may contribute to glomerular disorders and thus serve as viable targets for future studies.
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