Normal Development of Mice and Unimpaired Cell Adhesion/Cell
Motility/Actin-based Cytoskeleton without Compensatory
Up-regulation of Ezrin or Radixin in Moesin Gene Knockout*
Yoshinori
Doi
§,
Masahiko
Itoh
,
Shigenobu
Yonemura
,
Satoru
Ishihara
,
Hiroshi
Takano¶
,
Tetsuo
Noda¶
,
Shoichiro
Tsukita
, and
Sachiko
Tsukita
**
From the
Department of Cell Biology, Faculty of
Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, the
§ Second Department of Internal Medicine, Osaka University,
Medical School, Suita, Osaka 565, Japan, the ¶ Department of Cell
Biology, Cancer Institute, Toshima-ku, Tokyo 170, Japan, the
Department of Molecular Genetics, Tohoku University School of
Medicine, Sendai 980, Japan, and the ** College of Medical Technology,
Kyoto University, Sakyo-ku, Kyoto 606, Japan
 |
ABSTRACT |
Ezrin/radixin/moesin (ERM) proteins are general
cross-linkers between the plasma membrane and actin filaments. Because
their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand,
experiments at the cellular level and in vitro have
suggested their functional redundancy. To assess the possible unique
functions of ERM proteins in vivo, the moesin gene located
on the X chromosome was disrupted by gene targeting in embryonic stem
cells. Male mice hemizygous for the mutation as well as homozygous
females were completely devoid of moesin but developed normally and
were fertile, with no obvious histological abnormalities in any of the
tissues examined. In the tissues of the mutant mice, moesin completely
disappeared without affecting the expression levels or subcellular
distribution of ezrin and radixin. Also, in platelets, fibroblasts, and
mast cells isolated from moesin-deficient mice, targeted disruption of
the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal
contact formation of fibroblasts, and microvillar formation of mast
cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level.
 |
INTRODUCTION |
Three closely related proteins, ezrin, radixin, and moesin
constitute a gene family called the
ERM1 family. Ezrin, radixin,
and moesin were identified in different directions (1-7), but
isolation and sequencing of their cDNAs revealed that they were
closely related (amino acid sequence identity of 70-80% in the mouse)
(8-12). Independently of the lines of study on ERM proteins, another
ERM-like protein was identified as a tumor suppressor or hereditary
neurofibromatosis type 2 and named merlin
(moesin/ezrin/radixin-like
protein) or schwannomin (13, 14).
It is now widely accepted that ERM proteins function as general
cross-linkers between plasma membranes and actin filaments (for reviews
see Refs. 15-19). The highly conserved NH2-terminal half
of ERM proteins directly binds to the cytoplasmic domains of integral
membrane proteins such as CD44, CD43, ICAM-1, and ICAM-2 (20-24). On
the other hand, ERM proteins directly interact with actin filaments
(25-28). The co-existence of plasma membrane- and actin
filament-binding domains in individual molecules may allow ERM proteins
to function as plasma membrane/actin filament cross-linkers.
Furthermore, ERM proteins are also thought to be involved in plasma
membrane/actin filament cross-linkage through hetero- and/or
homo-dimerization (29-32) and through binding to EBP (ERM-binding
phosphoprotein) 50/NHE-RF (regulatory cofactor of
Na+-H+ exchanger) (33, 34). There is
accumulating evidence that the cross-linking activity of ERM proteins
is regulated by the Rho-dependent signaling pathway through
binding to Rho-GDP dissociation inhibitor and/or Rho-dependent
phosphorylation (23, 35-38).
One of the important questions regarding ERM proteins that have not yet
been addressed is the extent to which ezrin, radixin, and moesin are
functionally redundant. Targeted disruption of ERM protein genes would
be one of the most direct ways to approach this issue. Among the ERM
proteins, we expected moesin to be rather functionally unique, partly
because this molecule lacks the polyproline stretch found in both ezrin
and radixin and partly because only moesin is not tyrosine
phosphorylated by the epidermal growth factor receptor (39). Therefore,
to address the redundancy problem in ERM proteins we generated mice
with a targeted null mutation of the moesin gene located on the X chromosome.
 |
EXPERIMENTAL PROCEDURES |
Antibodies--
Rat anti-ezrin, radixin, and moesin mAbs (M11,
R21, and M22, respectively) (40) and rabbit anti-ezrin pAb (TK90) (41) were specific for respective antigens, whereas rabbit anti-ERM pAbs
(TK89 and TK88) (41) recognized COOH- and NH2-terminal halves of all ERM proteins, respectively. Rabbit anti-radixin pAb (I1)
(6) recognized all ERM proteins on immunoblotting but recognized only
radixin on immunofluorescence microscopy. Mouse anti-ERM mAb (CR22)
reacted strongly with moesin (11). Mouse anti-vinculin mAb (Sigma) were purchased.
Construction of Targeting Vector--
Mouse moesin genomic
clones were isolated from a 129/Sv mouse genomic library using mouse
moesin cDNA (nucleotides 1-315) (11) as a probe. The targeting
vector (see Fig. 1A) was constructed by standard recombinant
DNA techniques; the 0.7-kb PstI-KpnI fragment containing the 3' part of exon 3 was deleted and replaced by a loxP-neo
cassette in which loxP sequences (42, 43) flank phosphoglycerate kinase-neo cassette (44) oriented in the opposite orientation to moesin
transcription. In addition, a splicing acceptor (SA) sequence (45, 46)
and a polyadenylation signal (PA) [XhoI-PstI fragment of pCAGGS (47)] were ligated 3' of this cassette. The coding
region of the diphtheria toxin A gene driven by the MCl promoter (DT-A)
was then ligated downstream from the vector construct for negative
selection against random integration of the vector (48). As shown in
Fig. 1, the final vector construct consists of a 7.7-kb 5'-homologous
region, loxP/phosphoglycerate kinase-neo/loxP/SA/PA, a 1.5-kb
3'-homologous region, and DT-A.
Disruption of Moesin Gene in Embryonic Stem Cells and Generation
of Moesin-deficient Mice--
Embryonic stem (ES) cells were
electroporated with 20 µg of linearized targeting vector DNA using a
Bio-Rad Gene Pulser at 0.25 V and 960 microfarad. Cells were plated on
feeder cells in normal growth medium for 36-48 h, followed by
selection with 175 µg/ml G418. After 8-10 days, G418-resistant
colonies were picked up. The colonies were screened individually by
cleaving genomic DNA (7 µg) with BamHI and by probing the
Southern blots with the 230-bp cDNA sequence in exons 4 and 5 downstream from the 3' homologous region (3' probe; see Fig.
1A). Correct targeting was confirmed by Southern blotting of
BstXI-digested genomic DNA with the 50-bp cDNA in exon 2 upstream from the 5' homologous region (5' probe; see Fig.
1A). Targeted clones were also checked for single
integration by hybridization with a neo probe. Two correctly targeted
ES cell clones (clones 145 and 199) were expanded and injected into the blastocysts from C57BL/6 mice, which were then transferred into the
uteri of pseudopregnant ICR recipients. Male chimeras with extensive ES
cell contributions to their coats were bred with C57BL/6 female mice.
Tail DNA from agouti F1 offspring was genotyped by Southern blotting
analysis. F1 heterozygous females and F1 wild-type males were interbred
and the littermates were genotyped.
Immunoblotting--
Protein extracts from ES cells (+/Y,
/Y)
and various tissues of wild-type and moesin-deficient mice were
separated by SDS-polyacrylamide gel electrophoresis (10%) and then
electrophoretically transferred from gels onto nitrocellulose
membranes, followed by incubation with antibodies. For antibody
detection, a blotting detection kit with biotinylated immunoglobulin
and streptavidine-conjugated alkaline phosphatase (Amersham Pharmacia
Biotech) was used.
Immunofluorescence Microscopy--
Immunofluorescence microscopy
of frozen tissue sections (10 µm) and embryonic fibroblasts were
performed as described previously (49). They were examined using
confocal imaging system (Bio-Rad) equipped with a Zeiss Axiophot II
photomicroscope (Carl Zeiss, Oberkochen, Germany).
Platelet Aggregation Assay--
Blood was collected by cardiac
puncture on sodium citrate (0.38%), mixed with an equal volume of
suspension buffer (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.38% sodium
citrate, pH 7.4) and then centrifuged at 120 × g for
10 min at room temperature. The supernatant was collected as
platelet-rich plasma, which was diluted to 3 × 105
platelets/µl with plasma. Platelet aggregation was assayed as described previously (50).
Fibroblast Culture and Cell Motility Assay--
Embryonic
fibroblast cells were obtained from E14.5 wild-type (+/Y, +/+) and
moesin-deficient (
/Y,
/
) embryos and cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum on
fibronectin-coated dishes (IWAKI, Chiba, Japan) for 7 days. Cell
movement was recorded using a time lapse video system (LVR-3000N; SONY,
Tokyo, Japan) at 37 °C under a × 10 phase contrast objective lens.
Mast Cell Culture--
Mast cells were isolated according to the
technique described previously (51). Briefly, bone marrow was carefully
taken out from the femurs of wild-type or moesin-deficient mice and suspended in
-minimum essential medium containing 10% fetal calf serum and 40 units/ml recombinant murine interleukin-3. The cell suspensions were placed on culture dishes for 7 days, and nonadherent cells were transferred into fresh medium in new culture dishes every 4 days. After 6-7 weeks of culture, homogeneous populations (>90%) of
bone marrow-derived mast cells were obtained. They were placed on
poly-L-lysine coated coverslips and then fixed with 2.5%
glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate
buffer (pH 7.2), and processed for the scanning electron microscopic observation.
 |
RESULTS |
Targeted Disruption of Moesin Gene in Embryonic Stem
Cells--
The mouse genomic clone used for construction of the
targeting vector is shown in Fig.
1A. Judging from the structure
of the human moesin gene consisting of 12 exons (52), this mouse clone was thought to contain exons 2-5. The targeting vector was designed with the expectation that homologous recombination between the vector
and the moesin gene would result in deletion of a 3' part of exon 3. Because the numbers of nucleotides in exon 1 (12 bp from ATG), exon 2 (84 bp) and exon 3 (96 bp) are multiples of 3, it was possible that an
aberrant transcript would be produced from the targeted allele by
skipping the disrupted exon 3. To minimize this possibility, a SA and
PA were placed at the 3' end of LoxP-neo cassette.

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Fig. 1.
Targeted disruption of the mouse moesin gene
in ES cells. Panel A, schematic representation of wild-type
allele, targeting vector, and targeted allele of the moesin gene. The
targeting vector contained the loxP/phosphoglycerate kinase-neo/loxP
cassette and SA/PA in its middle portion to delete a 3' part of exon 3 (Ex3). DT-A, diphtheria toxin A gene;
bars, 5' and 3' probes for Southern blotting. The
BamHI fragments detected by the 3' probe (BamHI
digest) and BstXI fragments detected by the 5' probe
(BstXI digest) from wild-type and targeted alleles are also
shown. B, BamHI; Bs, BstXI;
P, PstI; K, KpnI.
Panel B, Southern blot analysis of genomic DNA from
wild-type cells (wild (+/Y)) and two independent clones of
targeted ES cells (145 ( /Y) and 199 ( /Y)).
Using the 3' probe, BamHI-digested fragments from wild-type
and targeted alleles yielded 9.2- and 5.0-kb bands, respectively
(3' probe). The correct integration of the vector
was confirmed by Southern blotting of BstXI digest with 5'
probe (5' probe). Targeted clones were checked
for single integration by hybridization with a neo probe (neo
probe). Panel C, immunoblotting analysis of the
expression of ERM proteins in wild-type clones (wild (+/Y))
and targeted ES cell clones (145 (-/Y) and 199 (-/Y)). Anti-ERM pAb TK89, TK88 recognized COOH- and
NH2-terminal halves of all ERM family members, whereas mAb
M11, R21, and M22 were specific for ezrin, radixin, and moesin,
respectively. Anti-mouse ERM mAb CR22 reacted strongly with moesin.
Targeted ES cells completely lacked moesin expression.
Arrows indicate ezrin (E), radixin
(R), and moesin (M).
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The linearized targeting vector was introduced into J1 ES cells by
electroporation, and cells were selected with G418. To screen for
homologous recombination events, DNA from resistant clones were
subjected to Southern blotting analysis with a probe corresponding to a
sequence 3' of the recombination site (3' probe). The wild-type moesin
allele displayed a 9.2-kb band on Southern blotting of
BamHI-digested DNA, whereas the disrupted locus showed a
5.0-kb band (Fig. 1B). Correct targeting was confirmed by
Southern blotting with a 5' probe, and targeted clones were also
checked for single integration by hybridization with a neo probe. Of
430 G418-resistant clones examined, two had undergone a single
homologous recombination event (clones 145 and 199) (Fig.
1B). As the moesin gene is located on the X chromosome (52,
53) and ES cells were established from male mice, the moesin single
knockout cells were expected to be deficient in moesin. Immunoblotting
analysis was then performed using two different pAbs, TK88 and TK89,
which recognized NH2- and COOH-terminal halves of all ERM
proteins, respectively (Fig. 1C). In wild-type ES cells,
both pAbs recognized three bands around 80 kDa, which corresponded to
ezrin, radixin, and moesin from the top, whereas both clones 145 and
199 lacked the moesin band in TK88 as well as TK89 immunoblotting. In
these moesin single knockout cells, additional bands did not appear at
the smaller molecular mass region in TK88 or TK89 immunoblotting, confirming that the moesin single knockout cells are deficient in
moesin. This was further confirmed by the immunoblotting with ezrin-,
radixin-, and moesin-specific mAbs (M22 and CR22 recognized distinct
epitopes of the COOH-terminal half of moesin) (Fig. 1C). Theoretically, of course, the possibility cannot be completely excluded
that only the short fragment of moesin molecule, which cannot be
detected with pAbs (TK88 or TK89) or mAbs (M22 or CR22), is expressed
in small amounts in the moesin single knockout cells. It is not,
however, likely that these small fragments of moesin, if any, can work
as functional ERM proteins.
Generation of Moesin Null Mice--
Clones 145 and 199 were
injected into C57BL/6 recipient blastocysts, and male chimeric mice
were crossed with C57BL/6 females. F1 female agouti pups, which were
expected to be heterozygous for the mutant moesin allele, were then
crossed with wild-type males and littermates were genotyped using the
3' probe (Fig. 2A). This
interbreeding yielded offspring at the expected Mendelian segregation
ratio, 1:1:1:1 of wild-type male (+/Y), wild-type female (+/+),
heterozygous female (+/
), and hemizygous male (
/Y). Moesin-deficient male mice (
/Y) developed and grew normally in the
laboratory environment and showed no differences in weight, size, or
reproductive ability from wild-type mice at least up to 12 months old.
Furthermore, interbreeding between F2 hemizygous males (
/Y) and F2
heterozygous females (+/
) yielded F3 homozygous females (
/
),
which also showed normal development, growth, and reproductive
capacity, at least up to 9 months old (data not shown). Various tissues
were examined histologically in hematoxylin-eosin-stained sections of
moesin-deficient mice (
/Y and
/
), and no significant abnormalities were detected (date not shown). Most of the following analyses were performed using hemizygous male mice (
/Y).

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Fig. 2.
Moesin-deficient mice. A,
genotypes of F2 mice. Genomic DNA was isolated from tails, and
BamHI digests were hybridized with the 3' probe. In addition
to wild-type male (+/Y) and female (+/+) mice,
males ( /Y), and females (-/+), hemi- and
heterozygous, respectively, for null mutation of the moesin gene were
identified. B, expression of ERM proteins in various tissues
of wild-type mice (+/Y) and moesin knockout
( /Y) male mice. Tissues were homogenized in SDS sample
buffer, and each lysate was applied onto SDS-polyacrylamide gel
electrophoresis at the same amount of total protein (15 µg/lane),
followed by immunoblotting with anti-ERM pAb T89. T89 pAb recognized
all ERM family members (see Fig. 1C). Arrows
indicate ezrin (E), radixin (R), and moesin
(M).
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Expression Levels and Subcellular Distributions of Ezrin and
Radixin in Moesin-deficient Mice--
We first examined whether the
expression levels of ezrin and radixin were elevated in
moesin-deficient mice in a compensatory manner by immunoblotting with
anti-ERM pAb TK89 that recognized COOH-terminal halves of all ERM
family members (Fig. 2B). As reported previously, in
wild-type mice most tissues co-expressed ezrin, radixin, and moesin in
various expression ratios, but the liver and intestine lacked ezrin and
radixin expression, respectively. Unexpectedly, in moesin-deficient
mice, neither ezrin nor radixin expression was affected; no
compensatory up-regulation of ezrin or radixin was detected in any of
the tissues examined. Immunoblotting with ezrin-, radixin-, and
moesin-specific mAbs also confirmed this conclusion, and anti-ERM pAb
TK88 that recognized NH2-terminal halves of all ERM
proteins gave the same results without additional bands at the lower
molecular mass region (date not shown).
We next examined the subcellular distributions of ezrin and radixin in
various tissues of moesin-deficient mice. When frozen sections of the
kidney from wild-type mice were doubly stained with anti-ezrin pAb
(TK90)/anti-moesin mAb (M22) (Fig.
3A) or anti-radixin pAb
(I1)/anti-moesin mAb (M22) (data not shown), intense ezrin, radixin,
and moesin signals were detected from apical surfaces of proximal
tubules and glomeruli. As reported previously, in endothelial cells of
blood vessels, moesin was abundantly detected, whereas the expression
levels of ezrin and radixin were rather low. In the kidney of
moesin-deficient mice, in which the moesin signal was not detected by
immunofluorescence microscopy, the subcellular distributions of ezrin
(Fig. 3A) and radixin (date not shown) did not appear to be
affected. In the liver of wild-type mice, the only ERM proteins
expressed were radixin and moesin. In this tissue, moesin expression
was mostly restricted to sinusoidal endothelial cells (Fig.
3B, panel b), whereas radixin was detected
abundantly in bile canaliculi and in small amounts in sinusoidal
endothelial cells (Fig. 3B, panel a). In the
liver of moesin-deficient mice, the subcellular distribution of radixin
did not appear to be affected (Fig. 3B, panels c
and d), and no induction of ezrin was detected (data not
shown). Further intensive immunofluorescence microscopic analyses led
us to conclude that targeted disruption of the moesin gene did not
affect either the expression levels or subcellular distribution of
ezrin or radixin in any of the tissues examined. Because we detected no abnormalities in moesin-deficient mice at the tissue level, we next
examined several types of cells isolated from moesin-deficient mice, in
which moesin was reported to be important for physiological functions.

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Fig. 3.
Subcellular distribution of ERM proteins in
kidney and liver of wild-type and moesin knockout male mice.
A, frozen sections of kidney from wild-type mice
(a and b) and moesin knockout mice (c
and d) were doubly stained with ezrin-specific pAb TK90
(a and c) and moesin-specific mAb M22
(b and d). Arrowheads, apical surfaces
of proximal tubules; asterisks, glomeruli;
arrows, endothelial cells. B, frozen sections of
liver from wild-type mice (a and b) and moesin
knockout mice (c and d) were doubly stained with
radixin-specific pAb I1 (a and c)/moesin-specific
mAb M22 (b and d). Arrows, sinusoidal
endothelial cells; arrowheads, bile canaliculi;
bar, 10 µm.
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Aggregation of Moesin-deficient Platelets--
Among the ERM
proteins, human platelets were reported to predominantly express
moesin, and moesin was suggested to be involved in their aggregation
process (54). Therefore, we first examined the aggregation activity of
platelets isolated from moesin-deficient mice. Upon immunoblotting of
wild-type mouse platelets, in addition to large amounts of moesin,
significant amounts of ezrin and radixin were also detected (Fig.
4A). In platelets collected
from moesin-deficient mice, the moesin band disappeared completely,
leaving relatively small amounts of ezrin and radixin without
compensatory up-regulation.

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Fig. 4.
Moesin-deficient platelets.
A, immunoblotting of platelets isolated from wild-type mice
(+/Y) and moesin knockout ( /Y) male mice as
well as human platelets with anti-ERM pAb TK89. In human platelets,
moesin was abundantly detected in addition to small amounts of radixin.
In wild-type mouse platelets, moesin was predominant, but ezrin as well
as radixin were detected also at significant levels. B,
platelet aggregation assay. Aggregation of platelets was initiated by
the addition of thrombin (0.0125, 0.025, and 0.1 units/ml), ADP (0.5 µM), or collagen (2 and 5 µg/ml), and the extent of
aggregation was measured as the turbidity change with a platelet
aggregometer.
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We then compared the aggregation ability of platelets between
wild-type and moesin-deficient mice (Fig. 4B). Wild-type and moesin-deficient platelets were collected, and they were subjected to
the aggregation assay; aggregation was initiated by addition of various
concentrations of thrombin, ADP, or collagen, and the extent of
aggregation was measured as the turbidity change with a platelet
aggregometer. Unexpectedly, as shown in Fig.
4B, no differences were
detected between wild-type and moesin-deficient platelets. These
observations led us to conclude that in platelets targeted
disruption of the moesin gene did not affect their aggregation activity
without any compensatory up-regulation of ezrin or radixin.

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Fig. 5.
Moesin-deficient fibroblasts.
Fibroblasts were cultured from wild-type embryos (A and
C) and moesin knockout embryos (B and
D) and stained with rhodamine-phalloidin (A and
B) or anti-vinculin mAb (C and D).
Targeted disruption of the moesin gene did not affect the morphology,
number of stress fibers, or focal contacts of cultured embryonic
fibroblasts. The migration rates of these cells were examined on
coverslips using a time lapse video system (E). There were
no significant differences in migration rate between wild-type mice
(+/Y and +/+) and moesin-deficient mice ( /Y and / )
(p < 0.005). Bar, 50 µm.
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Stress Fiber/Focal Contact Formation and Cell Motility of
Moesin-deficient Fibroblasts--
Moesin was reported to be required
for the Rho-dependent formation of actin stress fibers and
focal contacts in fibroblasts (36). We then isolated and cultured
embryonic fibroblasts from wild-type as well as moesin-deficient mice
and stained them with rhodamine-phalloidin or anti-vinculin mAb. As
shown in Fig. 5 (A-D), even in moesin-deficient
fibroblasts, well developed stress fibers with vinculin-positive focal
contacts were observed, indicating that targeted disruption of the
moesin gene did not affect stress fiber/focal contact formation in
fibroblasts. We next compared the migration rate of moesin-deficient
fibroblasts on coverslips with that of wild-type fibroblasts using a
time lapse video system. Again, however, no statistically significant
difference was detected by Student's t test (p < 0.005) (Fig. 5E). The mean migration speeds of
moesin-deficient and wild-type fibroblasts on coverslips were
31.45 ± 1.44 and 29.05 ± 1.52 µm/h,
respectively (n = 100).
Microvillar Formation in Moesin-deficient Mast Cells--
Finally,
we examined microvillar formation in moesin-deficient cells, in which
ERM proteins were reported to be directly involved through experiments
with antisense oligonucleotides (40). To obtain a homogeneous
population of mast cells bearing well developed microvilli, we cultured
bone marrow isolated from wild-type as well as moesin-deficient mice in
medium containing interleukin-3 for 6-7 weeks (51). Immunoblotting
with anti-ERM pAb TK89 revealed that wild-type mast cells expressed
large amounts of moesin, small amounts of radixin, and only trace
amounts of ezrin and that targeted disruption of the moesin gene did
not elevate the expression levels of ezrin or radixin (Fig.
6A). However, as shown in Fig.
6B, no significant differences were detected in the length
or number of microvilli between the wild-type and moesin-deficient mast cells on scanning electron microscopy.

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Fig. 6.
Moesin-deficient mast cells. Homogenous
populations (>90%) of mast cells were isolated and cultured from
wild-type mice (+/+) or moesin-deficient ( / ) female mice
as described under "Experimental Procedures." A,
immunoblotting analysis with anti-ERM pAb TK89. In wild-type mast
cells, moesin was predominant. Arrows indicate ezrin,
radixin, and moesin from top to bottom.
B, scanning electron microscopy. Bar, 20 µm.
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 |
DISCUSSION |
ERM (ezrin/radixin/moesin) proteins have been implicated as
general cross-linkers between the plasma membrane and actin filaments (for reviews see Refs. 15-19). In this study, we generated male and
female mice hemi- and homozygous, respectively, for a null mutation in
the moesin gene located on the X chromosome. Surprisingly, the mutant
mice exhibited no obvious abnormalities in appearance or fertility, and
a systemic histological scan of mutant tissues revealed no
abnormalities. Our results clearly demonstrated that moesin is not
required for normal mouse development or for survival in the laboratory
environment. This is surprising in view of the degree of conservation
of the moesin gene, for example, the occurrence of a moesin gene in
Drosophila (55, 56) and the tissue-specific regulated
expression of this gene (57-59).
To date immunofluorescence microscopy and immunoblotting analyses have
revealed that the ratio of the levels of ezrin, radixin, and moesin
expression in individual cells varies between different tissues in
wild-type mice. Furthermore, ezrin, radixin, and moesin are not always
colocalized. From these in situ observations in wild-type
mice, ezrin, radixin, and moesin have been suggested to have specific
functions. However, experiments in vitro or at the cellular
level have not clearly identified differences in function between these
molecules. When the expression of any one or two ERM proteins was
selectively suppressed by antisense oligonucleotides, no phenotypic
changes were detected, and cell-cell/cell-matrix adhesion and
microvillar formation were affected only when expression of all family
members was suppressed (40). Furthermore, the NH2-terminal
halves of all ERM proteins directly bound to the cytoplasmic domains of
CD44, Rho-GDP dissociation inhibitor, and PIP2 (23, 35) and
formed both homo- and heterodimers (29-31).
The present results favor the notion that ERM proteins are functionally
redundant also at the whole body level. Interestingly, in
moesin-deficient mice as well as isolated moesin-deficient cells,
targeted disruption of the moesin gene did not induce compensatory up-regulation of the other members of the ERM family. In any of the
tissues examined in mutant mice, no changes were detected in the
expression levels or subcellular distributions of ezrin or radixin.
More importantly, in isolated moesin-deficient cells such as platelets
and mast cells, in which moesin was predominantly expressed in
wild-type cells, moesin completely disappeared, leaving relatively
small amounts of ezrin and radixin. The aggregation activity of
moesin-deficient platelets and the microvillar formation of
moesin-deficient mast cells were not affected. Considering that ERM
proteins were thought to be directly involved in platelet aggregation
(54) and microvillar formation (4, 31, 40, 60, 61), we
concluded that only a small fraction of total ERM proteins in wild-type
platelets and mast cells are sufficient for their physiological
functions. Similar observations, i.e. no phenotypic changes
in knockout mice without up-regulation of other family members, has
been reported in various systems. For example, mice devoid of
components of intermediate-sized filaments such as vimentin and glial
fibrillary acidic protein developed normally without compensatory
expression of other intermediate filament components (62, 63).
Another issue that we should discuss here is the function of moesin in
intracellular signaling. We proposed that the cross-linking activities
of ERM proteins were regulated by the Rho-dependent signaling pathway through direct binding to Rho-GDP dissociation inhibitor and/or through Rho-dependent posphorylation (23, 35-38). Ezrin and radixin bound to Rho-GDP dissociation inhibitor with similar
binding constants to moesin and were phosphorylated by Rho kinase with
similar efficiency to moesin in vitro. Mackay et
al. (36) found that moesin was required for the
Rho-dependent formation of stress fibers and focal contacts
in permeabilized Swiss 3T3 cells. Also in this case, moesin was able to
be replaced by ezrin and radixin. From the present results, we
concluded that the functions of ERM proteins in the
Rho-dependent signaling pathway are redundant also at the
whole body level.
In conclusion, the present observations were consistent with the notion
that ERM proteins are functionally redundant. The possibility cannot be
excluded that a more distantly related protein to moesin in the band
4.1 superfamily functionally compensates for the lack of moesin.
Further generation of mutant mice lacking ERM proteins, both singly and
in combination, will lead to a better understanding of the
physiological relevance of the occurrence of these three closely
related proteins.
 |
ACKNOWLEDGEMENTS |
We thank Dr. S. Nishikawa (Department
of Molecular Genetics, Kyoto University) for helpful
discussions. We thank Dr. T. Murata and Dr. F. Ushikubi (Department of
Cell Pharmacology, Kyoto University) for technical advice regarding
platelet aggregation assay. Y. Doi thanks Dr. Y. Matsuzawa (Second
Department of Internal Medicine, Osaka University) for providing him
with the opportunity for this study.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-Aid for Cancer
Research and a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science and Culture of Japan.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: Dept. of Cell
Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel.: 81-75-753-4373; Fax: 81-75-753-4660; E-mail: atsukita{at}mfour.med.kyoto-u.ac.jp.
The abbreviations used are:
ERM, ezrin/radixin/moesin; ES, embryonic stem; mAb, monoclonal antibody; pAb, polyclonal antibody; kb, kilobase(s); SA, splicing acceptor; PA, polyadenylation signal; bp, base pair(s).
 |
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