(Received for publication, November 10, 1995; and in revised form, January 3, 1996)
From the
The highly related actin isoforms are thought to have different
functions. We recently demonstrated a polarized distribution of actin
isoforms in gastric parietal cells and association of gastric ezrin
with the cytoplasmic -actin isoform (Yao, X., Chaponnier, C.,
Gabbiani, G., and Forte, J. G.(1995) Mol. Biol. Cell. 6,
541-557). Here we used ultrastructural immunocytochemistry to
verify that
-actin is located within canalicular microvilli and
the apical cortex of parietal cells, similar to the localization
reported for ezrin. Furthermore, we tested whether ezrin binds
preferentially to cytoplasmic
-actin compared with the skeletal
muscle
-actin isoform. Purified cytoplasmic
-actin (from
erythrocytes) and skeletal
-actin were assembled with gastric
ezrin. Co-sedimentation experiments showed that gastric ezrin
selectively co-pelleted with the
-actin isoform and only very
poorly with
-actin. Binding of erythrocytic
-actin to ezrin
is saturable with a molar ratio of
1:10 (ezrin:actin) and a
dissociation constant
4.6
10
M. In addition, ezrin promoted pyrene-labeled actin
assembly, with predominant effects on filament elongation and a
distinct preference for
-actin compared with
-actin. Given
these isoform-selective associations, we speculate that actin isoforms
might segregate into different functional domains and exert specificity
by interacting with isoform-orientated binding proteins.
Although actin is a highly conserved protein, several distinct
tissue-specific isoforms exist. The actin isoforms are encoded by
separate genes and differ by less than 10% in amino acid sequence (1) and are generally believed to exert different functions.
For example, profilin has different affinities for each of the
cytoplasmic actin isoforms, -actin and
-actin, and for
sarcomeric actin(2, 3) . We showed recently that there
is a polarized distribution of cytoplasmic
- and
-actin
isoforms in gastric parietal cells(4) , consistent with some
difference in functional activity and/or preferential interaction with
localized actin-binding proteins. The
-actin isoform is
concentrated near the apical membrane of all gastric epithelial cells,
including the apical secretory canalicular membrane of parietal cells,
while the
-actin isoform is primarily distributed toward the
basolateral surface, with minor deposition in the region of the
secretory canaliculi of parietal cells.
Ezrin is an actin-binding
protein of the ezrin/radixin/moesin (ERM) family of
cytoskeleton-membrane linker proteins(5) . Within the gastric
epithelium, ezrin has been localized exclusively to the apical
canalicular membrane of parietal cells(6, 7) . Because
of its cytolocalization and stimulation-dependent phosphorylation, an
implied role for ezrin has been suggested in the apical surface
membrane remodeling associated with parietal cell activation via the
protein kinase A pathway(6, 8) . Phosphorylation of
ezrin has also been associated with surface membrane remodeling of A431
cells stimulated by epidermal growth factor(9) , although
activation in this case was via protein tyrosine kinase. Our previous
studies showed that the F-actin which co-localized with ezrin in
parietal cells was primarily comprised of the -actin isoform, and
that the
-actin isoform was preferentially co-immunoprecipitated
with ezrin from extracts of gastric membranes(4) . Recently,
Shuster and Herman (10) reported that ezrin, contained within
lysates of retinal pericytes, bound to immobilized matrices of
cytoplasmic
-actin, but not to the skeletal
-actin isoform.
These authors further demonstrated a co-localization of antibodies
against the
-actin isoform and ezrin in leading lamellae of motile
cells. Thus a body of evidence suggests interaction of ezrin with the
actin cytoskeleton may be specific for the
-actin isoform, but
much controversy remains regarding the nature of the interaction and
whether it is direct or via intermediary proteins.
The purpose of
the present experiments was to test if gastric ezrin exerts
isoform-specific association with cytoplasmic -actin in relatively
simple reconstituted systems. Accordingly, we separately assembled
skeletal
-actin and cytoplasmic
-actin in the presence of
gastric ezrin and subjected them to centrifugation. Gastric ezrin
selectively co-sedimented with cytoplasmic
-actin compared with
skeletal
-actin, which is consistent with an earlier observation
that showed a weak interaction between skeletal
-actin and
intestinal ezrin(11) , and supports the notion of isoform
specificity. Further characterization of ezrin-actin interaction by
using erythrocytic
-actin revealed that gastric ezrin binds to
actin in a saturable manner with a molar ratio of about 1:10
(ezrin:actin) and a dissociation constant (K
)
4.6
10
M for the ezrin-actin binding relationship.
For assembly experiments, actin was used at a
concentration of 5 µM. Because some polymerization of
actin occurs during the freezing of samples, thawed actin was spun at
312,000 g for 40 min before initiating assembly.
Polymerization was initiated at the following final conditions: 5
mM Tris, pH 7.5, 0.5 mM ATP, 2 mM MgCl
, 10 mM KCl, and 0.2 mM DTT.
Fluorescence was monitored continuously: excitation wavelength =
355 nm; emission wavelength = 407 nm. Purified yeast cofilin was
used as a control according to Moon et al.(18) .
Figure 1:
-Actin is heavily localized to
apical canalicular surfaces of resting parietal cells. Thin sections
were stained with affinity-purified anti-
-actin antibody followed
by colloidal gold-conjugated (5 nm) goat anti-rabbit IgG. Immunostained
grids were incubated with silver enhancer solution followed by
post-staining with uranyl acetate. The micrograph shows a cross-section
through a gastric gland including several parietal cells (PC)
and mucous neck cells (MNC) surrounding the gland lumen. There
is a dense distribution of enhanced gold particles along the apical
surfaces (Ap) of all epithelial cells, especially within
parietal cells along the intracellular canaliculi (IC), which
are invaginations of the apical surface membrane coursing throughout
the parietal cell. Gold particles are also seen along the basolateral
region (Bl) of parietal cells, with a somewhat lower density
than along canaliculi. Large mitochondria throughout the cytoplasm are
characteristic of parietal cells; numerous cytoplasmic tubulovesicles
and canalicular microvilli, also characteristic of parietal cells, are
not easily visualized at this magnification. Bar marker is 2
µm.
Figure 2:
-Actin is primarily localized to
apical canalicular surfaces of secreting parietal cells. Gastric glands
stimulated with histamine plus isobutylmethylxanthine were prepared and
immunostained as described in Fig. 1. Enhanced gold particles
are clearly seen along the dilated intracellular canaliculi (IC) of parietal cells and near the apical surfaces (Ap) of neighboring chief cells (CC). There is
somewhat less deposition of gold particles at basolateral surfaces (Bl) of parietal cells and virtually no basolateral staining
of chief cells. In these stimulated parietal cells, the mitochondria
are highly concentrated in the cytoplasm due to the fusion of
tubulovesicles to the canalicular membrane, and the canalicular spaces
are extended and readily seen. The bar marker is 2
µm.
Figure 3:
Magnified view shows that -actin is
heavily localized to apical and canalicular microvilli of parietal
cells. Secreting gastric glands were processed as described in Fig. 1. Thin sections were stained with anti-
-actin
antibody followed by colloidal gold-conjugated goat anti-rabbit IgG (5
nm); no silver enhancement was applied. The gold particles are
primarily distributed to the apical microvilli (mv) and to the
cortical region (cor) subadjacent to the apical membrane. Some
gold particles are also seen near lateral membrane folds (lat). Bar marker is 0.5
µm.
Figure 4:
Chromatographic purification of gastric
ezrin on S-Sepharose. Ezrin was extracted from gastric mucosal
homogenates, precipitated by 75%
(NH)
SO
, and partially purified by
successive chromatography on hydroxyapatite and Q-Sepharose, as
described under ``Materials and Methods.'' The ezrin peak
(identified by dot blot) eluting from Q-Sepharose was applied as
starting material (SM) to a column of S-Sepharose and eluted
with a 20 mM to 1 M NaCl gradient (tube numbers
indicated above). Eluting fractions were subjected to
SDS-polyacrylamide gel electrophoresis and stained by Coomassie Blue.
One peak, eluting in the range of 450-550 mM NaCl (tubes
18-22), was rich in ezrin but also had several additional
peptides, including some ezrin hydrolytic products (identified by
separate Western blot). The second ezrin peak eluting in the range of
625-675 mM NaCl (tubes 26-28) was virtually free
of contaminating peptides except for some small molecular weight
peptides migrating near the dye front. This second highly purified peak
of ezrin was used for subsequent binding studies. Molecular weight
standards (mw) are shown to the left.
To determine whether gastric ezrin stably binds to
actin filaments in an isoform-specific manner, we assayed the ability
of ezrin to co-sediment with skeletal muscle -actin and RBC
cytoplasmic
-actin. Gastric ezrin was incubated with the
respective actin isoforms under polymerizing conditions for 2 h, and
the filaments were pelleted by centrifugation at 312,000
g for 40 min. Actin filaments longer than 10 subunits in length will
sediment under these conditions(19) . The Coomassie
Blue-stained gel in Fig. 5A shows that gastric ezrin did not
appear in the pellet when incubated without actin and pelleted very
poorly when incubated with
-actin. However, ezrin obviously
appeared in the pellet when
-actin was polymerized (Fig. 5B). The S1 tryptic fragment of myosin II, a known
actin-binding protein that decorates actin filaments side-wise, was
used as a control. The myosin subfragment sedimented with either the
skeletal
-actin or the RBC
-actin without isoform specificity
and with an apparent molar ratio of 1:1 (myosin/actin). It is also
apparent that neither gastric ezrin nor the myosin subfragment affected
the amount of either
- or
-actin that sedimented.
Figure 5:
Gastric ezrin is preferentially
co-pelleted with the -actin isoform, but not the skeletal
-actin isoform. Samples of ezrin alone, actin alone, actin plus
ezrin, and actin plus myosin S1 subfragment were incubated for 2 h in
polymerization buffer and centrifuged as described under
``Materials and Methods.'' Equal volumes of supernatant (S) and pellet (P) fractions were resolved by
electrophoresis and proteins visualized by Coomassie blue staining. A, skeletal
-actin (5 µM) was incubated with
1 µM ezrin or 5 µM myosin subfragment, as
indicated. B, RBC
-actin (5 µM) was
incubated with 1 µM ezrin or 5 µM myosin
subfragment, as indicated. In the myosin-containing lanes, the 95-kDa
band is the S1 fragment, while the lower molecular mass band (
70
kDa) is probably derived from the further tryptic degradation of the S1
fragment.
Figure 6:
Gastric ezrin co-sediments with the
-actin isoform in a saturable manner. Samples of ezrin alone,
actin alone, or varied molar ratios of ezrin/actin were incubated for 2
h in polymerization buffer and centrifuged as described under
``Materials and Methods.'' Equal volumes of supernatant (S) and pellet (P) fractions were resolved by
electrophoresis and visualized by Coomassie Blue. A, mixtures
of ezrin/actin where ezrin concentration was fixed (1 µM)
and actin varied (5-100 µM). B, mixtures of
ezrin/actin where ezrin was increased (0.05-2 µM) at
a fixed actin concentration (5 µM). Individual
concentrations of ezrin and actin are
indicated.
Figure 7:
Summary of ezrin binding with -actin.
Ezrin was varied over the range shown while
-actin concentration
was constant at 5 µM. Ezrin band densities were measured
in the supernatants and pellet, and the ezrin bound was calculated as
the fractional amount of ezrin in the pellet. Data are the mean
± S.E. from four separate experiments, two derived from
Coomassie Blue-stained protein and two from Western blots probed for
ezrin.
Figure 8:
Ezrin
differentially alters the polymerization kinetics of -actin and
-actin isoforms. Aliquots of pyrene-labeled skeletal
-actin (A) or RBC
-actin (B) were preincubated alone,
with purified yeast cofilin, or with several concentrations of purified
gastric ezrin as described under ``Materials and Methods.''
Assembly was initiated at zero time by polymerization medium to a final
concentration of 2 mM MgCl
, 10 mM KCl,
and 0.5 mM ATP. Assembly was followed by changes in
fluorescence of the pyrene-labeled actins over time: Excitation
= 355 nm; emission = 407 nm. In all cases the final
concentration of actin was 5 µM. Assembly of either
-actin or
-actin alone is shown by solid lines.
Incubation with cofilin at 1:16 molar ratio (cofilin/actin) is shown by dotted lines. Incubations with various ezrin/actin molar
ratios (as indicated) are shown by the dashed
lines.
When gastric
ezrin was included with the pyrene-labeled skeletal -actin, the
curve shifted slightly to the left in the late stage of polymerization,
but not in the initial nucleation stage, suggesting that ezrin might
bind to the actin filaments or possibly promote the assembly (Fig. 8A). An increased molar ratio of ezrin/actin
further shifted the curve to the left.
The addition of ezrin also
promoted the assembly of pyrene-labeled -actin, with effects that
were more pronounced than for skeletal
-actin (Fig. 8B). Ezrin reduced the time for assembly to
achieve a steady state in a concentration-dependent manner, e.g. at a molar ratio of 1:10 (ezrin:actin) the time to achieve steady
state decreased from
60 to
30 min. The profile of ezrin on
RBC
-actin assembly is somewhat like that of cofilin, that is,
ezrin does not seem to modulate the lag associated with the nucleation
phase while it promotes the filament elongation phase as evidenced by
increased rate of assembly after the initial lag phase. Thus the
pyrene-labeled actin assembly assay indicated that ezrin might directly
interact with actin isoforms, with a distinct preference for RBC
-actin compared with skeletal
-actin.
Two different actin isoforms have been identified within
parietal cells, cytoplasmic -actin and cytoplasmic
-actin,
which are polarized to the apical and basolateral membranes,
respectively(4) . In addition these studies demonstrated a
preferential interaction between ezrin and the
-actin isoform
extracted from native parietal cells. In the present studies, we
extended our earlier finding by using immunoelectron microscopy to
localize
-actin to the apical microvilli and in vitro reconstitution of ezrin-
-actin interaction using purified
gastric ezrin and erythrocytic
-actin.
Using immunoelectron
microscopy, we localized -actin to the canalicular surface and
apical microvilli where gastric ezrin is enriched(6) . Despite
the dramatic elongation of apical microvilli and dilation of the
canalicular lumina of parietal cells during acid secretion, the
immunogold labeling of
-actin did not reveal obvious
stimulation-mediated redistribution of
-actin. In fact, a recent
report on the state of actin in resting and stimulated gastric glands
using the DNase I assay did not reveal any significant change in either
filamentous or monomeric actin pool(21) , suggesting that
stimulation-mediated elongation of microvilli could be due mainly to an
invagination of plasma membrane resulting from the fusion of
tubulovesicular membrane. It is possible that the stability and
integrity of microfilaments are required for providing a structural
support for the dynamic extension of apical plasma membrane and growth
of microvilli.
The three-dimensional structure of cytoplasmic
-actin was recently solved in complex with profilin(22) .
Although its primary sequence is generally similar to skeletal
-actin, cytoplasmic
-actin displays several structural
differences. These include: the N-terminal conformation of
-actin
bearing a turn rather than the helical structure in skeletal
-actin, distinct rotational differences within subdomains of the
isoforms, and differences in side chain orientation at residues
38-52. Physiological interpretation of these structural
differences is still under debate(23) , but functional
distinctions for the interaction of actin isoforms with actin binding
proteins have been reported. Larsson and Lindberg (2) showed
that cytoplasmic
-actin and
-actin have higher affinity to
bind profilin (K
10
M) than that of sarcomeric actin (K
4
10
M) and that
profilin interaction with the non-muscle isoforms was regulated by
Mg
. Rozycki et al.(3) further
demonstrated that profilin preferentially binds to cytoplasmic
-actin compared with
-actin. The increased ratio of
- to
-actin isoforms during the course of co-purifying ezrin and actin
by immunoprecipitation hinted that
-actin filaments might
preferentially bind to ezrin(4) . Bretscher observed (11) that ezrin co-sedimented with filamentous skeletal muscle
-actin in vitro only at low ionic strength. Our studies
with the comparison of two different actin isoforms clearly show that
gastric ezrin preferentially binds to cytoplasmic
-actin while
ezrin binds poorly to the skeletal
-actin.
A recent series of
studies has provided further information concerning the structural and
functional relevance of closely related ERM protein family members.
Martin et al.(24) showed that the N-terminal domain
of ezrin inhibits the functional activity of cell surface protrusion
exerted by the C-terminal domain. These authors further mapped the
inhibitory domain to the first 115 N-terminal residues. A similar
interaction between the N- and C-terminal domains was also observed by
Henry et al.(25) in their study of the functional
relevance of radixin. Gary and Bretscher (26, 27) carried out a meticulous characterization of
homotypic and heterotypic interaction among the ERM protein family
members, specifically ezrin and moesin. They established conditions
under which there is heterotypic interaction between the N-terminal
domain (amino acids 1-296) and C-terminal domain (amino acids
479-585) to form dimers and possibly oligomers. These terminal
``interactive domains'' were clearly demonstrated in an
extensive set of test probings using fusion protein constructs of the
respective N- and C-terminal interactive domains. However, for the
full-length fusion protein, or for isolated native ezrin, they
interpreted their results to suggest that the monomer exists in a form
in which the C-terminal interactive domain is masked by a folding that
also partially obscures the N-terminal interactive domain. When
full-length ezrin was denatured the C-terminal interactive domain
became accessible, but the N-terminal domain was inactivated by
denaturation. Because the actin binding domain of ezrin is near the C
terminus(28) , and the apparent masking of the C-terminal
interactive domain in the full-length monomer, Gary and Bretscher
offered these data as a basis to explain the lack of ezrin association
with -actin filaments in vitro(11) . It is
possible that modification (e.g. phosphorylation) and
accessory proteins might perturb this masking process and expose the
F-actin binding site in the C-terminal domain. In fact, purified ezrin
from gastric mucosa contains multiple spots as resolved by
two-dimensional electrophoresis, which suggests that native gastric
ezrin might contain a pool of phosphoezrin and/or multiple isoforms.
Since there are three consensus phosphorylation sites for protein
kinase A in ezrin (Ser
, Thr
, and
Thr
), and protein kinase A-mediated phosphorylation has
been implicated in hormone-stimulated acid secretion(8) , it is
conceivable that the phosphorylation might alter the intramolecular
masking effect and expose the C-terminal domain for actin binding. In
fact, phosphorylation of ezrin by epidermal growth factor receptor
tyrosine kinase triggered the dimerization, although the functional
activity of this dimer is unknown at present(27) .
Alternatively, ezrin isoforms might form functional oligomers that
might exert F-actin-binding action.
Shuster and Herman (10) recently reported that ezrin preferentially binds to an
affinity column made of filamentous erythrocytic -actin, but not
to a skeletal
-actin. Because these authors were unable to
reconstitute erythrocytic
-actin-ezrin interaction in
vitro, they concluded that their observed interaction was
indirect, and suggested that a 73-kDa polypeptide served to link the
interaction between ezrin and filaments of
-actin isoform,
although other candidates were also possible. Data presented here would
argue against a requirement for the 73-kDa component, since there was
no such peptide present in our reaction mixtures. On the other hand, it
is not possible to rule out the participation of a low molecular weight
component in the ezrin-
-actin filament interaction.
There are
some apparent contradictions concerning the nature of ezrin-actin
interaction. Based on the displacement of ezrin from actin filaments by
cytochalasin D, Shuster and Herman (10) proposed that ezrin
binds to the barbed ends of actin filaments. Using a blot overlay
assay, Pestonjamasp et al.(29) found that immobilized
ezrin bound to actin filaments and the binding was minimized by myosin
S1 subfragment but not by gelsolin or capping protein, suggesting that
binding occurs at the filament sides and not at barbed ends. Moreover,
the parallel localizations of ezrin and F-actin at the light and EM
levels (6, 7, 11) are consistent with side
binding. Our studies of ezrin on pyrene-labeled actin assembly in
vitro also favors the idea that ezrin interacts with actin
filaments along the side since ezrin dose-dependently promotes actin
assembly, which is typically seen for a side-binding polypeptide,
myosin S1 fragment(30, 17) . Recently, Turunen et
al.(28) revealed that the region 558-578 in ezrin,
moesin, and radixin shows high sequence homology with the actin binding
site of CapZ -subunit(31) . Despite the fact that both
radixin and CapZ
-subunit bind to the barbed ends of actin
filaments(31, 32) , it is not clear whether the
homologous region which exerts actin binding for ezrin is also
responsible for the end-binding in the case of CapZ and radixin. Recent
studies showed that EF1
, a transcription factor, has typical
barbed end capping activity in addition to the known actin filament
side-binding property(33) . Thus, we cannot rule out the
possibility that ezrin might interact with the ends of actin filaments
at present.
In summary, our immunoelectron microscopic data show
cytoplasmic -actin is indeed located to the apical microvilli and
terminal web of parietal cells, in a pattern identical to what has been
reported for ezrin(4, 6, 7) . We have
verified that gastric ezrin is preferentially associated with
cytoplasmic
-actin filaments in vitro, compared with
skeletal
-actin, and that gastric ezrin binds to
-actin
filaments in a saturable manner. Finally, based on the pyrene-labeled
actin assembly data, we speculate that ezrin might modulate the
elongation phase of the actin assembly. Because of the isopreferential
associations demonstrated in this study and morphological separations
seen in several systems(34, 35, 36) , we
suggest that actin isoforms might segregate into different functional
domains and exert their specificities by interacting with
isoform-orientated actin-binding proteins.