* Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan; First Department of Internal
Medicine, Nagoya University School of Medicine, Nagoya 466, Japan; § Department of Cell Biology, Graduate School of
Biological Science, Nara Institute of Science and Technology, Ikoma 630-01, Nara, Japan;
Second Department of Internal
Medicine, Osaka University Medical School, Suita, Osaka 565, Japan; ¶ Department of Genetics, Osaka University Medical
School, Suita, Osaka 565, Japan; and ** College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto 606, Japan
Breakdown of microvilli is a common early
event in various types of apoptosis, but its molecular
mechanism and implications remain unclear. ERM
(ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and
function as general actin filament/plasma membrane
cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at
the early phase of Fas ligand (FasL)-induced apoptosis
in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When
the FasL-induced dephosphorylation of ERM proteins
was suppressed by calyculin A, a serine/threonine
protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1-converting enzyme (ICE) protease inhibitors
suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings
indicate that during FasL-induced apoptosis, the ICE
protease cascade was first activated, and then ERM
proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown.
Next, to examine the subsequent events in microvillar
breakdown, we prepared DiO-labeled single-layered
plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells.
On single-layered plasma membranes from nontreated
cells, ERM proteins and actin filaments were densely
detected, whereas those from FasL-treated cells were
free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM
proteins is responsible for the microvillar breakdown at
an early phase of apoptosis and that the depletion of
ERM proteins from plasma membranes results in the
gross dissociation of actin-based cytoskeleton from
plasma membranes. The physiological relevance of this
ERM protein-based microvillar breakdown in apoptosis will be discussed.
APOPTOTIC cell death is an active process, which is a
critical feature of the regulated development of
multicellular organisms (Wyllie et al., 1980 Microvilli are specific sites of actin filament/plasma membrane interaction and are composed of core actin filaments
and several actin-binding proteins, such as villin, fimbrin,
and ERM proteins (Bretscher, 1991 The ERM family consists of three closely related proteins: ezrin, radixin, and moesin (Arpin et al., 1994 ERM proteins function as general cross-linkers between
actin filaments and specific groups of integral membrane
proteins such as CD44, CD43, ICAM-2, etc. (Turunen et
al., 1989 In the present study, to clarify the molecular mechanism
of microvillar disappearance during apoptosis, we examined the behavior of ERM proteins at the early phase of
apoptosis. It is well known that Fas ligand (FasL), a death
factor, binds to Fas, a cell surface receptor, to induce apoptosis (Suda et al., 1993 Cells and Antibodies
Mouse L929 cells expressing recombinant human Fas antigen (LHF) and
mouse MTD-1A cells were cultured in DME supplemented with 10%
FCS. HL-60 cells were cultured in RPMI1640 supplemented with 10% FCS.
Ezrin-, radixin-, and moesin-specific mAbs (M11, R21, and M22) were
raised in rats using recombinant ezrin, radixin, and moesin, respectively,
as antigens (Hirao et al., 1996 Induction of Apoptosis
LHF cells were detached from the dishes by trypsinization and washed
with culture medium. They were then cultured on 2% agar-coated 12-well
plates in 1 ml of culture medium at the concentration of 5 x 105 cells/ml.
To induce apoptosis, FasL was added to the culture medium at the final
concentration of 1 µg/ml (Suda et al., 1993 To induce apoptosis, MTD-1A cells were incubated with 0.5 µM staurosporine for 16 h, and HL-60 cells were cultured for 16 h in the presence
of 50 µM C2 ceramide, 0.5 µM staurosporine, or 5 µg/ml actinomycin D.
Immunofluorescence Microscopy
LHF cells were collected by centrifugation, washed with PBS, and then
placed on poly-L-lysine-coated coverslips for 15 min for attachment. LHF
cells on coverslips or cultured MTD-1A cells were fixed with 3% formaldehyde in PBS for 10 min at room temperature and treated with 0.1% Triton X-100 in PBS for 5 min. After soaking in PBS containing 1% BSA for
10 min, samples were incubated with the first antibody, washed with PBS,
and then incubated with the second antibody (FITC-conjugated goat anti-
rat IgG or FITC-conjugated goat anti-rabbit IgG [Tago, Inc., Burlingame,
CA]) in the presence of 4 Fractionation of Soluble and Insoluble ERM Proteins
Collected LHF cells or HL-60 cells were resuspended and homogenized
by weak sonication in a solution containing 130 mM KCl, 20 mM NaCl, 2 mM
EDTA, 1 mM EGTA, 50 mM Tris-HCl, pH 7.4, 1 mM p-amidino PMSF,
10 µg/ml leupeptin, and 10 µg/ml aprotinin at 4°C. The homogenates were
centrifuged at 100,000 g for 10 min at 4°C to recover the soluble and insoluble fractions in the supernatant and pellet, respectively. Equivalent amounts
of supernatant and pellet were applied to SDS-polyacrylamide gels, electrophoresed, and subsequently subjected to immunoblotting with pAb
TK89, mAb M11, mAb R21, or mAb M22.
Labeling of Cellular Phosphoproteins
and Immunoprecipitation
LHF cells were placed in 2% agar-coated 12-well plates in phosphate-free
DME with 10% FCS. Phosphate-free FCS was prepared by dialyzing
against 0.9% NaCl in 10 mM Hepes buffer, pH 7.4. The cells were cultured for 4 h in the same medium containing 0.5 mCi/ml [32P]orthophosphate (Phosphorous-32; NEN Life Science Products, Boston, MA).
The labeled cells were lysed and incubated for 5 min in 0.1 ml of solubilization buffer (1% SDS, 10 mM Tris-HCl, pH 7.4, 10 mM Na3VO4, 1 mM
Na2MoO4, and 10 mM p-amidinoPMSF). After addition of 0.9 ml of IP
buffer (10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% NaDOC, 150 mM
NaCl, 2 mM EDTA, 1 mM EGTA, 5 mM NaF, 1 mM Na3VO4, 0.1 mM
Na2MoO4, 1 mM p-amidinoPMSF, and 1 µg/ml leupeptin), the lysate
stood on ice for an additional 10 min and was centrifuged at 50,000 g for 20 min. The supernatant was immunoprecipitated with 20 µl of protein G-Sepharose 4B (Zymed Laboratories, Inc., South San Francisco, CA) conjugated with pAb TK89, pAb T90, or mAb M22. Sepharose 4B-bound immune complexes were washed five times with IP buffer containing 0.1%
SDS. Immune complexes were then eluted by boiling in SDS-PAGE sample buffer and resolved by SDS-PAGE. After transferring from gels onto
nitrocellulose sheets, the 32P signals were analyzed by autoradiography
(Fujix Bioimage Analyzer Bas 200 System; Fuji Photofilm Corp., Tokyo,
Japan), and ERM proteins were detected by immunoblotting with pAb
TK89.
Phosphoamino Acid Analysis
Antiezrin pAb (TK90) or antimoesin mAb (M22) immunoprecipitates
from 32P-labeled LHF cells with or without 1-h FasL treatment were resolved by SDS-PAGE and transferred onto polyvinyl difluoride membranes (Immobilon; Millipore Corp., Bedford, MA). Phosphoamino acids were analyzed based on the method of Boyle et al. (1991) ICE-like or CPP32-like Protease Inhibitor Treatment
LHF cells were labeled with [32P]orthophosphate for 3 h, and then Ac-YVAD-cho or Ac-DEVD-cho (Takara Shuzo Co., Ltd., Kyoto, Japan)
was added at the final concentration of 300 µM. After 1 h incubation, 1 µg/ml of FasL was added. At 3 h after the FasL stimulation, the phosphorylation level of ERM proteins was examined as described above.
Gel Electrophoresis and Immunoblotting
One-dimensional SDS-PAGE (7.5%) was performed, based on the
method of Laemmli (1970) Analysis of Chromosomal DNA
Cells (1 x 106) were washed with PBS and incubated in 15 µl of 10 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.5% SDS, and 1 µg/ml
proteinase K at 50°C for 30 min. Then, 3 µl of 1 mg/ml RNase A was
added and incubated for an additional 1 h. The digested samples were
added to the wells of a 2% agarose gel. After electrophoresis, the DNA
was visualized by UV illumination after ethidium bromide staining.
Single-layered Plasma Membrane Preparation
LHF cells were cultured in 2% agar-coat plates and vitally labeled for 1.5 h
with DiO (170 µg/ml) (Hong and Hume, 1986 Translocation of ERM Proteins from Plasma
Membranes to Cytoplasm at an Early Phase of
Fas-mediated Apoptosis
To examine whether ERM proteins are involved in the microvillar disappearance commonly observed in various
types of apoptosis, mouse L929 fibroblast transfectants expressing human Fas (LHF) were cultured on agar-coated
dishes (Yonehara et al., 1989
Using this system, we first examined the behavior of radixin at the early stage of Fas-mediated apoptosis by immunofluorescence microscopy. In LHF cells before FasL
stimulation, radixin was concentrated at the plasma membranes, especially in microvilli, and were also distributed
rather diffusely in the cytoplasm (Fig. 2). Within 1 h after
FasL stimulation, marked deformation of plasma membranes such as bleb formation was observed. Concomitantly, the concentration of radixin on the plasma membranes faded. At 2-3 h after FasL stimulation, radixin was
distributed evenly in the cytoplasm, and DAPI staining
identified condensed and fragmented nuclei. As shown in
Fig. 3, ezrin and moesin showed the same behavior as radixin at the early stage of Fas-mediated apoptosis of LHF cells.
The cytoplasmic translocation of ERM proteins shown
by immunofluorescence microscopy was also detected biochemically (Fig. 4). At various times after FasL stimulation, LHF cells were homogenized in physiological solution and divided into soluble and insoluble fractions by
centrifugation. Equivalent amounts of the soluble and insoluble fractions were resolved by SDS-PAGE, and the
amount of ERM proteins in each fraction was estimated
by immunoblotting with pAb TK89 that recognized all
ERM proteins. As shown in Fig. 4 a, in the absence of
FasL, ~50% of ERM proteins were recovered in the insoluble fraction. FasL stimulation gradually decreased the
amount of insoluble ERM proteins, and at 1, 2, and 3 h after FasL stimulation, ~35, 15, and 5% of total ERM proteins were recovered in the insoluble fraction, respectively. The cytoplasmic translocation of ERM proteins was
also detected by immunoblotting with ezrin-, radixin-, or
moesin-specific antibodies (Fig. 4 b). We then concluded that the microvillar ERM proteins translocate from the
plasma membrane to the cytoplasm at an early stage of
Fas-mediated apoptosis.
The apoptosis-associated cytoplasmic translocation of
ERM proteins was also observed in cultured mouse epithelial cells (MTD-1A) and human promyelocytic leukemic cells (HL-60), although the fairly slow and nonsynchronized induction of apoptosis in these cells prevented
analysis of the early events of apoptosis in detail. As
shown in Fig. 5 A, when apoptosis was induced in MTD-1A
cells by 16 h incubation with staurosporine, radixin as well
as ezrin and moesin were translocated from apical microvilli and lateral cell-cell contact sites to the cytoplasm.
The 16-h incubation with C2 ceramide, staurosporine, or
actinomycin D induced apoptosis in HL-60 cells, and in all
cases the cytoplasmic translocation of ERM proteins was
clearly detected (Fig. 5 B).
FasL-induced Dephosphorylation of ERM Proteins
Coupled with Their Cytoplasmic Translocation
We next examined what types of modification of ERM
proteins occur at the time of their cytoplasmic translocation at the early stage of the Fas-mediated apoptosis. The
possible degradation of ERM proteins was first postulated
since ICE proteases are known to be commonly activated
as an early event of apoptosis. As shown in Fig. 4, however, ERM translocation was not associated with ERM
degradation. We next examined the phosphorylation levels of ERM proteins since some reports suggested that
phosphorylated ERM proteins are concentrated at the
plasma membrane. LHF cells were metabolically labeled
with [32P]orthophosphate and stimulated with FasL. At
various times after FasL stimulation, ERM proteins were
recovered by immunoprecipitation with pAb TK89 that
recognized all ERM proteins and analyzed by autoradiography (Fig. 6 a). Before stimulation, ERM proteins were fairly highly phosphorylated, and FasL markedly decreased their phosphorylation level within 1 h. This downregulation continued up to the completion of apoptosis.
To determine which types of phosphoamino acid residues were dephosphorylated, we analyzed phosphoamino
acids of ezrin and moesin after 0 and 1 h of incubation with
FasL using LHF cells that were metabolically labeled with
[32P]orthophosphate. Ezrin and moesin were recovered by
immunoprecipitation with their specific antibodies, pAb
TK90 and mAb M22, respectively. Radixin, however, was
not purified from LHF cells by immunoprecipitation, partly because the available radixin-specific mAb R21 was
not potent for immunoprecipitation, and partly because
the expression level of radixin was fairly low in LHF cells.
Recovered ezrin and moesin were phosphorylated and dephosphorylated before and 1 h after FasL stimulation, respectively (Fig. 6 b). As shown in Fig. 6 c, without FasL
stimulation, serine/threonine as well as tyrosine residues
of ezrin and moesin were phosphorylated. Among these
phosphoamino acids, phosphothreonine appeared to be preferentially dephosphorylated after 1 h of incubation
with FasL, although the levels of phosphorylation of serine
and tyrosine residues were also partially decreased. These
observations suggested that dephosphorylation on threonine residues of ERM proteins is primarily accompanied
by their cytoplasmic translocation during Fas-mediated
apoptosis.
To clarify whether the dephosphorylation of ERM proteins is coupled with their cytoplasmic translocation, and,
if so, which is located upstream in the FasL-triggered signaling pathway, we stimulated LHF cells with FasL in the
presence of 300 nM calyculin A, a potent serine/threonine
phosphatase inhibitor (Morana et al., 1996
Relationship between Dephosphorylation of ERM
Proteins and Activation of ICE Protease Cascade
We next examined whether the dephosphorylation of
ERM proteins is located upstream or downstream of the
ICE protease cascade. As previously reported, in the presence of 300 µM Ac-YVAD-cho or Ac-DEVD-cho (ICE-like or CPP32-like protease-specific inhibitors, respectively),
LHF cells resisted FasL-induced apoptosis (data not shown)
(Nicholson et al., 1995
Subsequent Events in the Cytoplasmic Translocation of
ERM Proteins during FasL-induced Apoptosis
The question then arose as to what type of cellular event is
evoked by the cytoplasmic translocation of ERM proteins,
i.e., the disappearance of microvilli, in apoptosis. Considering that ERM proteins function as general cross-linkers
between plasma membranes and actin filaments, it was expected that gross dissociation of the actin-based cytoskeleton and plasma membranes occur as a result of the cytoplasmic translocation of ERM proteins. To evaluate this
expectation, we prepared DiO-labeled single-layered plasma
membranes from FasL-treated and nontreated LHF cells
(Tsukita et al., 1984
Next, the single-layered plasma membranes were stained
with rhodamine phalloidin to visualize actin filaments. As
shown in Fig. 10, the membranes prepared from the nonstimulated cells were associated with dense actin filaments,
whereas those from the FasL-stimulated cells were almost
free of actin filaments. Furthermore, calyculin A suppressed
this FasL-induced dissociation of actin filaments from
plasma membranes. We then concluded that FasL stimulation induced the gross dissociation of the actin-based cytoskeleton from plasma membranes through the cytoplasmic translocation of ERM proteins.
Microvillar disappearance is one of the early common
events in various types of apoptosis. In this study, using
FasL-induced apoptosis in LHF cells, we found that
plasma membrane-bound ERM proteins, ubiquitously expressed microvillar proteins, are translocated to the cytoplasm concomitantly with microvillar disappearance (within
1 h of incubation with FasL). Considering that the depletion of ERM proteins from living cells by antisense oglionucleotide treatment resulted in the complete disappearance of
microvilli from the cell surface (Takeuchi et al., 1994b The first question is how activated proteases induce the
dephosphorylation of ERM proteins and their subsequent
cytoplasmic translocation. In most types of cells, approximately half of the ERM protein is distributed in the cytoplasm (Sato et al., 1992 The second question was the subsequent event of the cytoplasmic translocation of ERM proteins, i.e., the breakdown of microvilli at the early phase of apoptosis. ERM
proteins are general cross-linkers between actin filaments
and the plasma membranes. In this sense, microvilli can be
regarded as membrane domains that are specialized for
the ERM protein-based interaction of actin filaments with the plasma membrane. Thus the depletion of ERM proteins
from the plasma membrane, i.e., the breakdown of microvilli, is expected to result in the gross dissociation of actin
filaments from the plasma membrane. The DiO-stained
single-layered membrane preparations developed in this
study allowed us to evaluate this expectation and led us to
conclude that at an early phase of FasL-induced apoptosis,
the actin-based cytoskeleton and plasma membrane are completely dissociated, leaving actin filament-free plasma
membranes. Many actin filament-plasma membrane cross-linkers other than ERM proteins have been identified, but
such gross dissociation of actin filaments and plasma membranes has not been observed by the downregulation of
their expression, suggesting the predominant role of ERM
proteins in the regulation of actin filament/plasma membrane interaction. Furthermore, this gross dissociation is consistent with our previous results obtained with antisense oligonucleotides that the suppression of ERM protein
expression affected several independent cellular events, such
as cell-cell adhesion, cell-matrix adhesion, and microvillar
formation, where actin filament/plasma membrane interactions may play important roles (Takeuchi et al., 1994b During apoptosis, breakdown of microvilli has been reported to be followed by marked changes in cytoskeletal
organization. Several cytoskeletal proteins and related signaling molecules, such as actin, fodrin, Gas2, and protein
kinase C Considering that microvillar breakdown is a common
early event in apoptosis, it is likely that the gross dissociation between actin filaments and plasma membranes occurs
commonly at an early phase of various types of apoptosis.
Actually, the apoptosis-associated cytoplasmic translocation of ERM proteins was observed not only in LHF cells
but also in cultured epithelial cells (MTD-1A) and promyelocytic leukemic cells (HL-60). Furthermore, this gross dissociation is located downstream of activation of the
ICE protease cascade, which is commonly activated in apoptosis (Enari et al., 1996; Nagata
and Goldstein, 1995
; Chinnaiyan and Dixit, 1996
; Fraser
and Evans, 1996
; Jacobson, 1997
; Nagata, 1997
). Apoptosis is characterized by marked morphological alterations of the nucleus, such as chromatin condensation. Various
types of stimuli are known to cause apoptosis, and irrespective of stimuli, apoptotic cell death is usually accompanied by the activation of interleukin-1
-converting enzyme (ICE)1 family members of cysteine proteases
followed by the fragmentation of nuclear DNA into oligonucleosomal-sized units (Enari et al., 1995
; Los et al., 1995
;
Alnemri et al., 1996
). Relatively few apoptosis-related
substrates for the ICE family members have been reported, and our knowledge of the roles of these substrates
in DNA fragmentation is still limited (Chinnaiyan and
Dixit, 1996
; Fraser and Evans, 1996
; Jacobson, 1997
; Nagata, 1997
). Plasma membranes and the cytoskeleton also
undergo marked morphological changes during apoptosis.
Among these changes, the disappearance of microvilli has
been recognized as one of the common early events of apoptosis, although its molecular mechanism and physiological implications in apoptosis are unknown.
; Sato et al., 1992
; Arpin
et al., 1994
; Tsukita et al., 1997a
,b). At least one member
of the ERM family is found in microvilli in all types of
cells, whereas the expression and distribution of villin and
fimbrin are restricted to some specific types of cell. When
the expression of ERM proteins was suppressed by antisense oligonucleotides, microvillar structures completely disappeared from the cell surface, indicating that these
proteins play a key role in microvillar formation in general
(Takeuchi et al., 1994b
).
; Tsukita
et al., 1997a
,b). Sequence analyses of cDNAs have revealed
that the amino acid sequence identity among ERM proteins is 70-80% (Gould et al., 1989
; Funayama et al., 1991
;
Lankes and Furthmayr, 1991
; Sato et al., 1992
; Tsukita et
al., 1997a
,b). The sequences of their amino-terminal halves
are highly conserved (~85% identity) and homologous to
the amino-terminal ends of some membrane-associated proteins such as band 4.1 protein, talin, and merlin/schwannomin (a tumor suppressor molecule for neurofibromatosis type II) (Rouleau et al., 1993
; Trofatter et al., 1993
), indicating that the ERM family is included in the band 4.1 superfamily (Takeuchi et al., 1994a
).
; Yonemura et al., 1993
; Tsukita et al., 1994
; Helander et al., 1996
; Tsukita et al., 1997a
,b). Their carboxy-
and amino-terminal halves bind to actin filaments and the
cytoplasmic domains of integral membrane proteins, respectively. Several lines of evidence indicate that the
amino- and carboxy-terminal halves of ERM proteins suppress the functions of their carboxy- and amino-terminal
halves, respectively, by binding to each other in a head-
to-tail manner (Henry et al., 1995
; Magendantz et al., 1995
;
Martin et al., 1995
; Hirao et al., 1996
; Tsukita et al., 1997a
,b).
In the cytoplasm, this head-to-tail association results in
folded monomers or homotypic/heterotypic oligomers of
ERM proteins, which are inactivated as plasma membrane/actin filament cross-linkers. Some signals have been
suggested to disrupt this intramolecular or intermolecular
head-to-tail interaction to expose both membrane- and actin filament-binding domains, activating ERM proteins to
function as cross-linkers. Recently, the Rho signaling pathway has been implicated in this activation process of ERM proteins (Hirao et al., 1996
; Tsukita et al., 1997a
,b). Serine/ threonine as well as tyrosine phosphorylation have also
been suggested to be involved in the regulation of ERM
protein function (Urushidani et al., 1989
; Berryman et al.,
1995
; Jiang et al., 1995
; Nakamura et al., 1995
).
; Nagata and Goldstein, 1995
). Here,
we induced apoptosis by adding human FasL to mouse L
fibroblasts expressing human Fas (LHF cells). In this system, within 1 h of incubation FasL induced not only the
translocation of ERM proteins from the plasma membrane (microvilli) to the cytoplasm but also the dephosphorylation of ERM proteins. Close analyses using phosphatase inhibitors or ICE protease inhibitors revealed that
the ICE protease cascade is first activated by FasL stimulation, and then ERM proteins are dephosphorylated, followed by the cytoplasmic translocation of ERM proteins.
Furthermore, we found that the cytoplasmic translocation
of ERM proteins resulted in the gross dissociation of the
actin-based cytoskeletal components from the plasma
membrane. These findings clarified the molecular mechanism of microvilli disappearance at the early phase of apoptosis, providing a clue to understanding its physiological
relevance.
Materials and Methods
). Two pAbs, TK89 and TK90, were raised in
rabbits against synthesized peptides corresponding to the mouse radixin
and ezrin sequences (amino acids 551-570 and 480-489), respectively.
TK89 detected all ERM proteins, whereas T90 was specific for ezrin.
). In some experiments, calyculin A (Research Biochemicals International, Natick, MA) was added to
the culture medium with FasL at the final concentration of 300 nM (Morana et al., 1996
).
,6
-diamidino-2-phenylindole (DAPI). Samples
were then washed with PBS and examined using a fluorescence microscope (Axiophoto photomicroscope; Carl Zeiss, Oberkochen, Germany).
with minor
modifications. 32P-labeled phosphorylated ezrin or moesin bands were excised from membranes and hydrolyzed in 200 µl of 6 M HCl at 110°C for
60 min. The hydrolysates were lyophilized using a Speed Vac Concentrator (Savant Instruments, Hicksville, NY) and resuspended in 6 µl of pH
3.5 buffer (5% glacial acetic acid, 0.5% pyridine) containing cold phosphoamino acid standards. The samples were then spotted onto silica gel-
coated thin-layer chromatography plates and resolved by electrophoresis
at 1,300 V for 1.3 h using pH 3.5 buffer at 4°C (model Multiphor II; Pharmacia Biotech AB, Uppsala, Sweden). The positions of 32P-labeled phosphoamino acids were determined by autoradiography, and cold phosphoamino acid standards were visualized by ninhydrin staining.
. After electrophoresis, proteins were electrophoretically transferred from gels onto nitrocellulose membranes, which
were then incubated with the first antibody. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG and the
appropriate substrates as described by the manufacturer (Amersham International, Buckinghamshire, UK).
). The FasL-treated (1 h) or
nontreated cells were collected by centrifugation, washed with PBS, and
placed on poly-L-lysine-coated coverslips for 15 min for attachment.
Then, cells were subjected to jet streams of 5-10 ml of buffer containing
10 mM Hepes, pH 7.4, 1 mM MgCl2, and 50 mM KCl using a 10-ml syringe
with a 23-gauge needle (Tsukita et al., 1984
). The isolated plasma membranes on the glass were fixed and stained with rhodamine phalloidin or
with pAb TK89 followed by FITC-conjugated goat anti-rat IgG (Tago,
Inc., Burlingame, CA). Samples were then washed with PBS and examined using a fluorescence microscope (Axiophoto photomicroscope; Carl
Zeiss).
Results
; Itoh et al., 1991
; Nagata and
Golstein, 1995), and the apoptosis was initiated by addition of recombinant human FasL (Suda et al., 1993
; Nagata and Golstein, 1995). On agar-coated dishes, LHF cells were not spread out but rounded, and they were killed by
apoptosis within 3 h as determined by DNA fragmentation
ladder formation, retaining their spherical shape (Fig. 1).
The fairly quick and synchronized induction of apoptosis
in this system allowed us to analyze the cellular events at
the early stage of apoptosis in detail. Furthermore, since
LHF cells retained their spherical shape without attachment to the dish, this system was advantageous for morphological analysis of the apoptotic cellular changes as well as for biochemical analyses.
Fig. 1.
FasL-induced apoptosis in LHF cells. After incubation of LHF cells with
FasL for 0, 1, 2, and 3 h, the
chromosomal DNA was extracted and analyzed by gel
electrophoresis. Judging from
the DNA fragmentation ladder formation, apoptosis was
induced within 2-3 h.
[View Larger Version of this Image (77K GIF file)]
Fig. 2.
Behavior of radixin in LHF cells at 0 h (a and b), 1 h (c
and d), 2 h (e and f), and 3 h (g and h) after FasL stimulation. LHF cells were doubly stained with antiradixin mAb, R21 (a, c, e,
and g) and DAPI (b, d, f, and h). After 1 h of incubation, radixin
translocated from the plasma membrane to the cytoplasm, and
the nuclear change, such as chromatin condensation, began to be
observed after 2 h of incubation. Bar, 20 µm.
[View Larger Version of this Image (69K GIF file)]
Fig. 3.
Behavior of ezrin and moesin in LHF cells at 0 h (a and
c) and 2 h (b and d) after FasL stimulation. LHF cells were
stained with ezrin-specific pAb TK90 (a and b) or moesin-specific
mAb M22 (c and d). Behavior of ezrin and moesin during apoptosis was the same as that of radixin (see Fig. 2). Bar, 20 µm.
[View Larger Version of this Image (59K GIF file)]
Fig. 4.
Solubility of ERM proteins in LHF cells at 0, 1, 2, and 3 h
after addition of FasL. LHF cells were homogenized in physiological saline and centrifuged to separate the soluble and insoluble fractions into the supernatant (S) and pellet (P), respectively.
Equivalent amounts of supernatant and pellet were applied to
SDS-PAGE and subsequently subjected to immunoblotting.
To detect all members of ERM proteins, pAb TK89 was used (a).
To detect ezrin, radixin, and moesin separately, mAb M11, mAb
R21, and mAb M22 were used, respectively (b). ERM proteins
translocated from the insoluble (P) to the soluble fraction (S) as
apoptosis proceeded. Arrows in a indicate ezrin, radixin, and
moesin, respectively, from the top.
[View Larger Version of this Image (30K GIF file)]
Fig. 5.
Behavior of ERM proteins during apoptosis in mouse
epithelial cells (MTD-1A) and human promyelocytic leukemic
cells (HL-60). (A) Immunofluorescence micrographs of MTD-1A
cells at 0 h (a and b) and 16 h (c and d) incubation with staurosporine. MTD-1A cells were doubly stained with antiradixin mAb
R21 (a and c) and DAPI (b and d). After 16 h of incubation, radixin translocated from apical microvilli and lateral cell-cell contact sites to the cytoplasm, and nuclear changes such as chromatin
condensation were observed. Ezrin and moesin showed the same
behavior as radixin (data not shown). (B) Solubility of ERM proteins in HL-60 cells at 0 (control) and 16 h incubation with C2 ceramide, staurosporine, or actinomycin D. HL-60 cells were homogenized in physiological saline and centrifuged to separate the
soluble and insoluble fractions into the supernatant (S) and pellet
(P), respectively. Equivalent amounts of supernatant and pellet
were applied to SDS-PAGE and subsequently subjected to immunoblotting with pAb TK89 capable of recognizing all ERM proteins. ERM proteins translocated from the insoluble (P) to the soluble fraction (S) as apoptosis proceeded. Arrows indicate ezrin, radixin, and moesin, respectively, from the top. Bar, 20 µm.
[View Larger Versions of these Images (111 + 39K GIF file)]
Fig. 6.
Dephosphorylation of ERM proteins in LHF cells during Fas-mediated apoptosis. (a) Time course of apoptosis-associated dephosphorylation of ERM proteins. LHF cells were metabolically labeled with [32P]orthophosphate. At 0, 1, 2, and 3 h
after FasL stimulation, the cell lysate was immunoprecipitated
with anti-ERM pAb, TK89, and analyzed by autoradiography
([32P]) and immunoblotting ([Blotting]). Equal amounts of protein were applied to each lane as revealed in accompanying immunoblots with anti-ERM pAb, TK89. The phosphorylation
level of ERM proteins markedly decreased after a 1-h incubation.
Arrows indicate ezrin, radixin, and moesin, respectively, from the
top. (b) Autoradiography of antiezrin pAb (TK90) or antimoesin
mAb (M22) immunoprecipitates from 32P-labeled LHF cells. Immunoprecipitated ezrin and moesin were phosphorylated and dephosphorylated before and 1 h after FasL stimulation, respectively (arrows). (c) Phosphoamino acid analysis. 32P-labeled
phosphorylated ezrin or moesin bands (arrows in b) were excised
from membranes and processed for phosphoamino acid analysis. The positions of phosphoserine (p-S), phosphothreonine (p-T), and phosphotyrosine (p-Y) were determined by autoradiography
through comparison with the ninhydrin staining profiles on unlabeled phosphoamino acid standards. In both ezrin and moesin,
phosphothreonine appeared to be preferentially dephosphorylated after 1 h of incubation with FasL, although the levels of
phosphoserine and phosphotyrosine were also decreased.
[View Larger Version of this Image (56K GIF file)]
). As shown in
Fig. 7 a, calyculin A suppressed the FasL-induced dephosphorylation of ERM proteins at 1 h of incubation, conversely elevating the phosphorylation level of ERM proteins. This
suppression continued up to 3 h after FasL stimulation,
and the phosphorylation level of ERM proteins remained
at the high level. Then, we examined the effects of calyculin A on the FasL-induced cytoplasmic translocation of
ERM proteins. Immunoblotting of soluble and insoluble
fractions from FasL/calyculin A-incubated LHF cells revealed that ~90% of ERM proteins were recovered in the
insoluble fraction at 1 h of incubation, which continued for
at least 4 h, indicating that the FasL-induced cytoplasmic
translocation of ERM proteins was suppressed by calyculin A (Fig. 7 b). In agreement with these observations,
when these FasL/calyculin A-incubated cells were examined by immunofluorescence microscopy using anti-ERM
antibodies, ERM proteins were shown to be concentrated
at the plasma membrane (Fig. 7 c). These findings indicated that FasL-induced dephosphorylation of ERM proteins is coupled with their cytoplasmic translocation and
that the former is located upstream of the latter.
Fig. 7.
Effects of calyculin A on FasL-induced changes of
ERM proteins in LHF cells. (a) Suppression of FasL-induced dephosphorylation of ERM proteins by calyculin A. LHF cells were
metabolically labeled with [32P]orthophosphate. At 0, 1, 2, and 3 h
after FasL stimulation in the presence of calyculin A, the cell lysate was immunoprecipitated with anti-ERM pAb, TK89, and
then analyzed by autoradiography ([32P]) and immunoblotting
([Blotting]). Equal amounts of protein were applied to each lane
as revealed in accompanying immunoblots with anti-ERM pAb,
TK89. The FasL-induced dephosphorylation of ERM proteins
(see Fig. 6 a) was suppressed, while conversely the phosphorylation level was elevated. Arrows indicate ezrin, radixin, and
moesin, respectively, from the top. (b) Suppression of the FasL-induced cytoplasmic translocation of ERM proteins by calyculin A. Calyculin A was added to LHF cells together with FasL, and, after incubations for 1, 2, 3, and 4 h, ERM proteins were partitioned into soluble (S) and insoluble (P) fractions as described in
Fig. 4 a. Immunoblots with anti-ERM pAb, TK89, revealed that
FasL-induced cytoplasmic translocation of ERM proteins was
suppressed and that conversely calyculin A increased the
amounts of insoluble ERM proteins. Arrows indicate ezrin, radixin, moesin, respectively, from the top. (c) Antiradixin mAb
immunofluorescence micrograph of LHF cells after 2 h of incubation of FasL and calyculin A. Radixin still remained on plasma
membranes. Ezrin and moesin showed the same changes as radixin (data not shown). Bar, 20 µm.
[View Larger Version of this Image (45K GIF file)]
). Under these conditions, i.e., when
the ICE protease cascade was completely suppressed, the
phosphorylation level of ERM proteins was analyzed after
FasL stimulation. LHF cells were metabolically labeled
with [32P]orthophosphate and then stimulated with FasL
in the presence of 300 µM Ac-YVAD-cho or Ac-DEVD-cho. After incubation for 3 h, ERM proteins were recovered by immunoprecipitation, and their phosphorylation
level was examined by autoradiography. As shown in Fig.
8, the FasL-induced dephosphorylation of ERM proteins was mostly suppressed by either Ac-YVAD-cho or Ac-DEVD-cho, indicating that the dephosphorylation of ERM
proteins is located downstream of the FasL-induced activation of ICE proteases.
Fig. 8.
Suppression of FasL-induced dephosphorylation of
ERM proteins by ICE protease inhibitors. LHF cells were metabolically labeled with [32P]orthophosphate for 3 h, and then ICE
protease inhibitors (Ac-YVAD-cho or Ac-DEVD-cho) were
added at 300 µM. After 1 h incubation, FasL was added. At 3 h of
incubation, the cell lysate was immunoprecipitated with anti-ERM pAb, TK89, and analyzed by autoradiography ([32P]) and
immunoblotting ([Blotting]). Equal amounts of protein were applied to each lane as revealed in accompanying immunoblots with
anti-ERM pAb, TK89. The FasL-induced dephosphorylation of ERM proteins was suppressed by both inhibitors. Arrows indicate ezrin, radixin, and moesin, respectively, from the top.
[View Larger Version of this Image (19K GIF file)]
; Hong and Hume, 1986
). Briefly, free-floating LHF cells were incubated with DiO to vitally label
their plasma membrane proper, stimulated with FasL, and
then attached to poly-L-lysine-coated coverslips. In control experiments, without FasL stimulation, cells were attached to poly-L-lysine-coated coverslips. At 1 h of incubation, cells were ruptured with a jet stream of hypotonic solution, and the remnant single-layered plasma membranes with the cytoplasmic side upward on the glass were
stained with anti-ERM antibodies. As shown in Fig. 9, a
and b, when the single-layered plasma membranes were
prepared from nontreated cells, they were double positive
with DiO and anti-ERM antibodies. In contrast, FasL-treated cells left various sizes of DiO-positive plasma
membranes on the cover glasses, almost free of ERM proteins
(Fig. 9, c and d). This finding confirmed the FasL-induced
cytoplasmic translocation of ERM proteins. Also, in this system the suppression of the cytoplasmic translocation of ERM
proteins by calyculin A was observed (Fig. 9, e and f).
Fig. 9.
Detection of ERM proteins on single-layered plasma membranes isolated on cover
glasses. After vital labeling of plasma membranes proper with DiO, single-layered plasma
membranes with the cytoplasmic surface freely
exposed were prepared from nontreated (a and
b), FasL-1 h-treated (c and d), and FasL/calyculin A-1 h-treated (e and f) LHF cells as described in Materials and Methods. These preparations were immunofluorescently labeled with
anti-ERM pAb, TK89. Each single-layered
plasma membrane was detected by DiO signal
(a, c, and e). ERM signal was intense from
plasma membranes of nontreated cells (b),
whereas it was undetectable from those of FasL-treated cells (d). Calyculin A suppressed the
FasL-induced depletion of ERM proteins from
plasma membranes (f). Bar, 20 µm.
[View Larger Version of this Image (109K GIF file)]
Fig. 10.
Detection of actin filaments on the single-layered
plasma membranes isolated on cover glasses. After vital labeling
of plasma membranes proper with DiO, single-layered plasma
membranes with the cytoplasmic surface freely exposed were
prepared from nontreated (a, b, g, and h), FasL-1 h-treated (c
and d), and FasL/calyculin A-1 h-treated (e and f) LHF cells as
described in Materials and Methods. These preparations were labeled with rhodamine phalloidin to visualize actin filaments (b, d,
f, and h). Each single-layered plasma membrane was detected by
DiO signal (a, c, e, and g). Actin filaments were densely associated
with plasma membranes of nontreated cells (b and h), whereas
they were undetectable from those of FasL-treated cells (d). Calyculin A suppressed the FasL-induced dissociation of actin filaments from plasma membranes (f). Bars: (a-f) 10 µm; (g and h)
15 µm.
[View Larger Version of this Image (57K GIF file)]
Discussion
), we
conclude that in FasL-induced apoptosis the cytoplasmic
translocation of ERM proteins, i.e., the depletion of ERM
proteins from plasma membranes, is directly responsible
for microvillar disappearance. Furthermore, in this study
we analyzed the upstream and downstream events of the
microvillar disappearance, i.e., the cytoplasmic translocation of ERM proteins, in the FasL-triggered death signaling pathway, and our conclusions were as follows. FasL
stimulation upregulates the ICE protease cascade as recently clarified, which facilitated the dephosphorylation of
ERM proteins. This dephosphorylation event is required
for ERM proteins to translocate from the plasma membrane to the cytoplasm, which induces gross dissociation of actin filaments from the plasma membrane.
). As described in the introduction,
these cytoplasmic soluble ERM proteins take an inactive
form, in which the amino- and carboxy-terminal halves
mutually suppress their functions through head-to-tail association (Henry et al., 1995
; Magendantz et al., 1995
;
Martin et al., 1995
). Some kinds of activation signals may
release this mutual suppression by disrupting the intramolecular or intermolecular head-to-tail interaction, allowing
the activated ERM proteins to bind to the plasma membranes and actin filaments simultaneously (Arpin et al.,
1994
; Tsukita et al., 1997a
,b). The serine/threonine phosphorylation of ERM proteins has been reported to cause
their translocation from the cytoplasm to the plasma membrane (Urushidani et al., 1989
; Chen 1995; Nakamura et
al., 1995
). These observations are consistent with our
present finding that the dephosphorylation of ERM proteins is coupled with their cytoplasmic translocation during apoptosis, although dephosphorylation was detected
not only at serine/threonine but also at tyrosine residues.
Ezrin has been reported to be a good in vivo substrate for
tyrosine kinases, but its physiological relevance remains
unclear (Bretscher, 1989
; Hunter and Cooper, 1981
). As
during apoptosis, phosphothreonine of ERM proteins appeared to be preferentially dephosphorylated as compared
to phosphoserine and phosphotyrosine, and as calyculin A, a potent serine/threonine phosphatase inhibitor, suppressed the apoptosis-associated dephosphorylation of
ERM proteins, it is likely that the FasL-activated ICE protease cascade directly or indirectly downregulates some
serine/threonine kinase or activates some serine/threonine
phosphatase, resulting in the dephosphorylation of ERM
proteins. On the other hand, Rho, a small GTP-binding
protein, was reported to regulate the activation of ERM
proteins through its downstream signaling molecules (Hirao
et al., 1996
). It is thus also possible that the FasL-activated
ICE protease cascade affects the activity of ERM proteins
through the Rho-dependent signaling pathway. In this
context, it is interesting that D4-GDI, a hematopoietic cell-specific homologue of Rho-GDI, is cleaved by ICE proteases in FasL- and staurosporine-induced apoptosis of
T-cell lines (Songqing et al., 1996
).
).
, have been identified as potential targets for ICE
proteases (Brancolini et al., 1995
; Chinnaiyan and Dixit, 1996
;
Fraser and Evans, 1996
). These targets may play synchronized
roles together with the dephosphorylation of ERM proteins,
resulting in marked changes in cytoskeletal organization.
). This raises questions regarding
the physiological relevance of this gross dissociation in apoptosis in general and whether this apoptosis-associated cytoplasmic event is independent from the nuclear events
such as chromatin condensation and DNA fragmentation. It is noteworthy that calyculin A significantly suppressed
the FasL-induced apoptosis of LHF cells under the conditions used in this study (data not shown). Calyculin A has
also been reported to delay the apoptotic process in lymphoma cells induced by gamma-irradiation, tetrandrine,
bistratene A, cisplatin, and etoposide (Song and Lavin, 1993
;
Morana et al., 1996
). Considering that calyculin A retained
ERM proteins on plasma membranes, it is tempting to speculate that the ERM-based tight association of the actin-based cytoskeleton with the plasma membranes makes
cells resistant to death signals, i.e., that destruction of
ERM-based actin filament/plasma membrane interactions
is required for the apoptotic changes in the nucleus. This
speculation is similar to the recent observation that mutated lamin, a nuclear skeletal protein that is resistant to
ICE protease-dependent proteolysis, delayed apoptosis
(Rao et al., 1996
). Of course, it is possible that calyculin A
does not suppress the nuclear apoptosis through ERM
proteins, but through other proteins directly involved in
the nuclear apoptosis. Further analysis of the physiological
relevance of the gross dissociation of actin filaments from
plasma membranes, i.e., the disappearance of microvilli, in
apoptosis will lead to a better understanding of the total
picture of the molecular mechanism of apoptosis.
Received for publication 24 April 1997 and in revised form 13 August 1997.
Address all correspondence to Sachiko Tsukita, Ph.D., Department 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.jpWe thank all the members of our laboratory (Department of Cell Biology, Faculty of Medicine, Kyoto University) for their helpful discussions. T. Kondo thanks Prof. H. Saito (Nagoya University) for providing him with the opportunity to work in the Department of Cell Biology, Faculty of Medicine, Kyoto University.
This work was supported in part by a Grant-in-Aid for Scientific Research (B) (to Sa. Tsukita), a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science, and Culture of Japan (to Sh. Tsukita).
DAPI, 4,6
-diamidino-2-phenylindole;
ERM, ezrin/radixin/moesin;
FasL, Fas ligand;
ICE, interleukin-1
-converting enzyme.
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