* Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan; and Takai
Biotimer Project, ERATO, Japan Science and Technology Corporation, JCR Pharmaceuticals Co., Ltd., Nishi-ku, Kobe
651-22, Japan
The Rho small G protein family, consisting
of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin
cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We
prepared MDCK cell lines stably expressing each of
dominant active mutants of RhoA (sMDCK-RhoDA),
Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1
(sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN)
and analyzed cell adhesion in these cell lines. The actin
filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared
with those in wild-type MDCK cells. Both E-cadherin
and -catenin, adherens junctional proteins, at the
cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in
sMDCK-RacDN cells. The detergent solubility assay
indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells,
whereas it slightly decreased in sMDCK-RacDN cells,
compared with that in wild-type MDCK cells. In
sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells,
neither of these proteins at the cell-cell adhesion sites
was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed
that sMDCK-RacDA cells tightly made contact with
each other throughout the lateral membranes, whereas
wild-type MDCK and sMDCK-RacDN cells tightly and
linearly made contact at the apical area of the lateral
membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell-
cell adhesion. Microinjection of C3 into wild-type
MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction,
but microinjection of C3 into sMDCK-RacDA cells
showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell-
cell adhesion and the tight junction, but not essential
for the Rac subfamily-regulated, cadherin-based cell-
cell adhesion.
THE Rho family belongs to the small G protein superfamily and consists of the Rho, Rac, and Cdc42
subfamilies (for reviews see Hall, 1994 Most of these cell functions are closely related to the
cytoskeleton system, particularly the actin cytoskeleton.
The actin cytoskeleton is known to play an important role
also in cell-cell adhesion. In epithelial cells, cells are linked
together by a junctional complex comprised of adherens
junctions, tight junctions, and desmosomes. Adherens and
tight junctions are linked to actin filaments, whereas desmosomes are linked to intermediate filaments (for reviews
see Madara, 1988 As to the regulation of cell-cell adhesion by the Rho
family, the Rho subfamily regulates the formation of the
tight junction in polarized epithelial cells (Nusrat et al.,
1995 Materials and Chemicals
MDCK cells were supplied by W. Birchmeier (Max-Delbruck-Center for
Molecular Medicine, Berlin, Germany). The cDNA of RhoA was provided by P. Madaule (Kyoto University, Kyoto, Japan). The cDNAs of
V12Rac1, with a mutation of amino acid 12 from Gly to Val, and N17Rac1,
with a mutation of amino acid 17 from Thr to Asn, were provided by A. Hall (University College London, London, England). The cDNA of Cdc42
was provided by P. Polakis (Onyx Pharmaceuticals, Richmond, CA). Mutagenesis of Gly to Val at codon 14 of RhoA (V14RhoA), Gly to Val at
codon 12 of Cdc42 (V12Cdc42), or Thr to Asn at codon 17 of Cdc42
(N17Cdc42) was carried out by site-directed mutagenesis. The pSR Construction of Expression Plasmids of Rho, Rac, and
Cdc42 Mutants
The pSR Cell Culture, Transfection, and Microinjection
MDCK cells were maintained at 37°C in a humidified atmosphere of
10% CO2 and 90% air in DME containing 10% FCS (GIBCO BRL, Grand Island, NY), penicillin (100 U/ml), and streptomycin (100 µg/ml).
Transfection of pSR Immunoblotting and Immunoprecipitation
myc-Tagged proteins, E-cadherin, and Detergent Extraction of E-Cadherin
Detergent extraction of E-cadherin from MDCK cells was performed as
described (Nagafuchi and Takeichi, 1988 Immunofluorescence Microscopy
For localization of actin filaments, myc-tagged proteins, and ZO-1, immunofluorescence microscopy was performed as described (Kotani et al.,
1997 Electron Microscopy
Cells cultured on the poly-L-lysine-coated celldesk LF1 (Sumitomo Bake,
Akita, Japan) were fixed with 2.5% glutaraldehyde in PBS for 2 h, followed by postfixation with 1% OsO4 in 0.1 M cacodylate-HCl, pH 7.4, for
1 h. The samples were dehydrated in a graded series of ethanol, embedded
in Epon812, and examined using an electron microscope (H-7100; Hitachi,
Tokyo, Japan).
Immunoelectron microscopy using the silver enhancement technique
was done as described (Mizoguchi et al., 1994 Preparation of MDCK Cell Lines Stably Expressing
Various Rho, Rac, and Cdc42 Mutants
The pSR
myc-V14RhoA and -V12Rac1 were also detected in the
immunoprecipitated samples from the cell lysates of all the
other clones of sMDCK-RhoDA and -RacDA, although
they were not detected directly from the cell lysates (data
not shown). The reason why cell clones expressing high
levels of both myc-V14RhoA and -V12Rac1 could not be
obtained is not known. However, because the expression of a dominant active or negative mutant of Rho1p causes
lethality to the cells in the yeast Saccharomyces cerevisiae
(Tanaka, K., and Y. Takai, unpublished observations).
Therefore, these proteins at higher expression levels may
cause lethality to the cells. myc-N17Rac1 was detected
from the cell lysates of the other clone of sMDCK-RacDN, and the expression level of myc-N17Rac1 in this clone was similar to that in sMDCK-RacDN-2 (data not
shown). myc-V12Cdc42 was detected in the immunoprecipitated samples from the cell lysates of the other clone of
sMDCK-Cdc42DA, but it was not detected directly from
the cell lysates, indicating that the expression level of myc-V12Cdc42 was less than that in sMDCK-Cdc42DA-2 cells
(data not shown). myc-N17Cdc42 was detected in both the
cell lysates and the immunoprecipitated samples of all the
other clones of sMDCK-Cdc42DN, although the expression
levels of myc-N17Cdc42 varied among these clones (data
not shown).
Localization of the Actin Cytoskeleton and Cell
Shape in MDCK Cell Lines Stably Expressing Various
Rho, Rac, and Cdc42 Mutants
We first analyzed the localization of the actin cytoskeleton
by staining with rhodamine-phalloidin in wild-type and
transformant cell lines. Confocal microscopic images at the
basal levels showed that weak formation of stress fibers and
weak localization of actin filaments at the cell-cell adhesion sites were observed in wild-type MDCK, sMDCK-RacDN-2, -Cdc42DA-2, and -Cdc42DN-6 cells, whereas
prominent stress fibers were observed in sMDCK-RhoDA-5 cells (Fig. 2, a, b, and d-f). sMDCK-RacDA-1 cells possessed weak stress fibers like wild-type MDCK cells, but
the actin filaments at the cell-cell adhesion sites markedly
increased in sMDCK-RacDA-1 cells (Fig. 2 c). Confocal
microscopic images at the apical, junctional levels showed
that actin filaments were localized at the cell-cell adhesion
sites in wild-type MDCK, sMDCK-RhoDA-5, -Cdc42DA-2,
and -Cdc42DN-6 cells to a similar degree (Fig. 2, g, h, k,
and l), whereas sMDCK-RacDA-1 cells possessed denser
and thicker actin filaments at the cell-cell adhesion sites
than the other cell lines, and sMDCK-RacDN-2 cells possessed weaker and less distinct actin filaments at the same
area (Fig. 2, i and j). These changes of the actin filaments
at the cell-cell adhesion sites were also visualized in the
vertical sections (Fig. 2, m-r). The actin filaments at the
cell-cell adhesion sites were observed at the lateral membranes in wild-type MDCK cells, and they tended to be
more condensed at the apical regions (Fig. 2 m). The staining pattern of the actin filaments at the cell-cell adhesion
sites in sMDCK-RhoDA-5, -Cdc42DA-2, and -Cdc42DN-6
cells resembled that in wild-type MDCK cells (Fig. 2, n, q,
and r). However, in sMDCK-RacDA-1 cells, the staining of
the actin filaments at the cell-cell adhesion sites was dense
and thick, and the increased staining was observed throughout the lateral membranes (Fig. 2 o). In contrast, the staining of the actin filaments at the cell-cell adhesion sites became weaker and more indistinct in sMDCK-RacDN-2 cells
(Fig. 2 p). The changes in the actin cytoskeleton observed in
sMDCK-RhoDA-5, -RacDA-1, -RacDN-2, -Cdc42DA-2,
and -Cdc42DN-6 cells were similarly observed in all the
other clones stably expressing V14RhoA, V12Rac1, N17Rac1,
V12Cdc42, and N17Cdc42, respectively (data not shown). We have previously obtained an MDCK cell line transfected with the pSR
The shapes of sMDCK-RacDA-1 and -RacDN-2 cells
were different from those of the other cell lines. sMDCK-RacDA-1 cells were slightly thicker, and sMDCK-RacDN-2
cells were thinner than the other cell lines (Fig. 2, m-r).
Moreover, the diameter of sMDCK-RacDN-2 cells was
apparently larger than those of the other cell lines (Fig. 2,
m-r). The essentially same results were obtained in the other cell lines stably expressing V14RhoA, V12Rac1,
N17Rac1, V12Cdc42, or N17Cdc42 (data not shown). These
results indicate that the Rac subfamily furthermore affects
the cell shape of MDCK cells.
Localization of E-Cadherin, Because markedly different localization patterns of actin
filaments were observed at the cell-cell adhesion sites of
sMDCK-RacDA and -RacDN cells, compared with those
of the other cell lines, as described above, we next investigated the localization of E-cadherin and
Consistent with the results obtained by immunofluorescence microscopy, the detergent solubility assay indicated
that the amount of the actin cytoskeleton-associated
E-cadherin increased in sMDCK-RacDA-1 cells, whereas
it decreased in sMDCK-RacDN-2 cells. Wild-type MDCK,
sMDCK-RacDA-1, and -RacDN-2 cells were incubated
with various concentrations of NP-40, and the amounts of
E-cadherin in the detergent-soluble and -insoluble fractions were measured by immunoblotting. The amount of
E-cadherin remaining in the insoluble fraction increased in sMDCK-RacDA-1 cells and decreased in sMDCK-RacDN-2 cells, compared with that in wild-type MDCK
cells (Fig. 4). About 15, 50, and 7% of E-cadherin was insoluble in the presence of 0.5% NP-40 in wild-type MDCK,
sMDCK-RacDA-1, and -RacDN-2 cells, respectively.
We confirmed by immunoblotting that the expression
levels of E-cadherin and
ZO-1 is one of the structural proteins of tight junctions
(Woods and Bryant, 1993
Electron Microscopic Analyses on the
Cell-Cell Adhesion of sMDCK-RacDA and
sMDCK-RacDN Cells
To understand the detailed changes in the localization of
actin filaments, E-cadherin, and
Immunogold electron microscopic analysis showed that
the gold particles of E-cadherin tended to be concentrated
at the apical area from one fourth to one third of the lateral membranes, whereas they were sparsely observed at
the basal side of the lateral membranes in wild-type
MDCK and sMDCK-RacDN-2 cells (Fig. 8, a and e). The gold particles were observed densely throughout the lateral membranes in sMDCK-RacDA-1 cells (Fig. 8 c). The
localization patterns of
Inhibition of the Formation of the Adherens
and Tight Junctions by Microinjection of C3 into
Wild-type MDCK Cells
The stable expression of V14RhoA in MDCK cells did not
affect the cell-cell adhesion under the conditions where it
induced the formation of stress fibers and focal adhesions
as described above. We attempted to obtain MDCK cell
lines stably expressing a dominant negative mutant of
RhoA, but we did not succeed in obtaining this type of
transformant. Therefore, we investigated the effect of microinjection of C3, a Clostridium botulinum ADP-ribosyltransferase, into wild-type MDCK cells on the cell-cell adhesion. C3 ADP-ribosylates only the Rho subfamily of
>60 members of the small G protein superfamily (Aktories et al., 1988
We next investigated the effect of microinjection of C3
into wild-type MDCK cells on the Ca2+-induced formation
of both the adherens and tight junctions using Ca2+-switch
experiments. At 30 min before the Ca+-switch experiments, the cells were microinjected with C3. The cells were
then incubated in a low Ca2+ medium for 2 h, followed by
the 2-h incubation in a normal Ca2+ medium. The incubation in a low Ca2+ medium induced disappearance of the
staining of E-cadherin and ZO-1 at the cell-cell adhesion
sites and the disruption of the cell-cell adhesion (data not
shown), and the following incubation in a normal Ca2+
medium induced the localization of both E-cadherin and
ZO-1 at the cell-cell adhesion sites and the formation of
the cell-cell adhesion (Fig. 10, a, b, d, and e). The localization of both E-cadherin and ZO-1 at the cell-cell adhesion
sites between the microinjected cells was inhibited,
whereas that between the microinjected and unmicroinjected cells was not apparently inhibited (Fig. 10, a, b, d,
and e). These results indicate that the Rho subfamily is also necessary for the Ca2+-induced formation of both the
adherens and tight junctions.
The intercellular binding of cadherins triggers the formation of adherens junctions, followed by the formation
of tight junction in the Ca2+-induced formation of cell-cell
adhesion (Gonzalez-Mariscal et al., 1985 In contrast to the effect of C3 on wild-type MDCK cells,
microinjection of C3 showed little effect on the localization of actin filaments and E-cadherin at the cell-cell adhesion sites in sMDCK-RacDA-1 cells (Fig. 11). The actin
filaments at the cell-cell adhesion sites in sMDCK-RacDA-1 cells did not markedly change at 2 h after the
microinjection, although a part of the actin filaments at the
cell-cell adhesion sites was decreased, whereas the formation of stress fibers was completely inhibited (Fig. 11, a-d,
and f). The staining of E-cadherin at the cell-cell adhesion sites was not also apparently affected by the microinjection, although a part of E-cadherin at the cell-cell adhesion sites was decreased (Fig. 11, e and f). Even up to 4 h
after the microinjection of C3 into sMDCK-RacDA-1
cells, the increased stainings of the actin filaments and
E-cadherin were still observed at most of the cell-cell adhesion sites (data not shown). These results indicate that
the Rho subfamily is not necessary for the Rac-induced increase in the localization of both the actin filaments and
E-cadherin at the cell-cell adhesion sites.
Localization of RhoA, Rac1, and Cdc42 Mutants in
MDCK Cell Lines Stably Expressing Various Rho, Rac,
and Cdc42 Mutants
In the last set of experiments, we analyzed the localization
of the myc-tagged proteins myc-V14RhoA, -V12Rac1,
-N17Rac1, -V12Cdc42, and -N17Cdc42 and actin filaments
in the stable transformant cell lines by double staining
with the anti-myc mAb and rhodamine-phalloidin, respectively. Confocal microscopic images at the junctional levels showed that the significant staining was not observed in
wild-type MDCK cells (Fig. 12, a and e). The weak and
diffuse staining of V14RhoA was observed in sMDCK-RhoDA-5 cells (Fig. 12, b and f). However, V12Rac1 and
N17Rac1 were highly concentrated at the cell-cell adhesion sites of sMDCK-RacDA-1 and -RacDN-2 cells, respectively, but the staining of V12Rac1 was stronger than
that of N17Rac1 (Fig. 12, c, d, g, and h). The weak and diffuse stainings of V12Rac1 and N17Rac1 were also observed in the cytosol of both cell lines. At both the basal
and apical levels, the weak and diffuse staining of V14RhoA
was observed in sMDCK-RhoDA-5 cells (data not shown). The specific stainings of V12Rac1 and N17Rac1, except
for the stainings at the cell-cell adhesion sites and the cytosol, were not observed at both the basal and apical levels
in sMDCK-RacDA-1 and -RacDN-2 cells (data not shown).
The essentially same results were obtained in the other cell
lines stably expressing V14RhoA, V12Rac1, and N17Rac1
(data not shown). These results indicate that both the
dominant active and negative mutants of Rac1 are localized mainly at the cell-cell adhesion sites, whereas the dominant active mutant of RhoA is localized mainly at the
cytosol. However, because vinculin at the focal adhesions
and the stress fibers markedly increased in sMDCK-RhoDA cells, compared with those of both wild-type MDCK,
sMDCK-RacDA, and -RacDN cells (data not shown), the
dominant active mutant of RhoA, which might be localized
at the focal adhesions, might be masked by the cytosolic diffuse staining.
As to the localization of V12Cdc42, V12Cdc42 was highly
concentrated at the cell-cell adhesion sites of sMDCK-Cdc42DA-2 cells as well as at the cytosol, although the
localization of both the actin filaments, E-cadherin, and
We have shown here by use of stable transformants of
MDCK cells expressing various RhoA, Rac1, and Cdc42
mutants that the Rac subfamily regulates the cadherin-based cell-cell adhesion, but not the formation of the tight
junction, that the Rho subfamily is necessary for both the
cadherin-based cell-cell adhesion and the formation of the
tight junction, and that the Cdc42 subfamily does not affect the cadherin-based cell-cell adhesion.
The expression of myc-V12Rac1 in sMDCK-RacDA
was undetectable by immunoblotting with the anti-myc
mAb using the cell lysates, whereas it was detected in the
immunoprecipitated samples from the cell lysates. However, the localization of the actin filaments, E-cadherin,
and Immunohistochemical analysis revealed more intense
staining of the actin filaments, E-cadherin, and The mechanism by which the Rac subfamily regulates
the E-cadherin-based cell-cell adhesion remains to be
clarified, but one possibility is that it primarily induces the
assembly of the actin filaments at the cell-cell adhesion
sites, eventually leading to the assembly of E-cadherin and
its related molecules, including The putative target molecules of the Rac subfamily were
identified. They were p65PAK, MLK2/3, MSE55, p60
S6kinase, POR-1, IQGAP1/2, WASP, and p67phox (for review see Tapon and Hall, 1997 We showed here by both confocal and electron microscopies that sMDCK-RacDA cells were thicker and
sMDCK-RacDN cells thinner than the other cell lines.
Moreover, the diameter of sMDCK-RacDN cells was apparently larger than those of the other cell lines. The morphological changes in the cell shapes of sMDCK-RacDA
and -RacDN cells may be due to alteration in the basolateral and/or the apical endo-exocytosis, because the Rac
subfamily regulates endo-exocytosis (Komuro et al., 1996 The Rac subfamily regulates the formation of lamellipodia and membrane ruffles in many types of cultured cells,
such as Swiss 3T3 and KB cells (Ridley et al., 1992 We previously showed that microinjection of the guanosine 5 We showed here that both the dominant active mutant
of Rac1, myc-V12Rac1, and the dominant negative mutant
of Rac1, myc-N17Rac1, were localized at the cell-cell adhesion sites. myc-V12Rac1 may interact with the downstream target molecule at the cell-cell adhesion sites and
exert its function there. Tiam1, a GDP/GTP exchange protein (GEP) for the Rac subfamily, was localized at the
plasma membrane in fibroblasts (Michiels et al., 1995 During the review process of this manuscript, one report
appeared, suggesting that both the Rho and Rac subfamilies are necessary for the E-cadherin-based cell-cell adhesion in human keratinocytes (Braga et al., 1997; Takai et al.,
1995
). The Rho subfamily, consisting of three members,
RhoA, -B, and -C, regulates a wide variety of cell functions, such as cell shape change (Rubin et al., 1988
; Chardin et al., 1989
; Paterson et al., 1990
; Miura et al., 1993
), formation of stress fibers and focal adhesions (Paterson et al., 1990
; Ridley and Hall, 1992
, 1994
; Self et al., 1993
; Ridley
et al., 1995
), cell motility (Stasia et al., 1991
; Takaishi et
al., 1993
, 1994
), platelet aggregation (Morii et al., 1992
),
smooth muscle contraction (Hirata et al., 1992
), lymphocyte toxicity (Lang et al., 1992
), cytokinesis (Kishi et al., 1993
;
Mabuchi et al., 1993
), lymphocyte aggregation (Tominaga
et al., 1993
), neurite retraction (Jalink et al., 1994
), formation of tight junction and perijunctional actin (Nusrat et al.,
1995
), endocytosis (Schmalzing et al., 1995
; Lamaze et al.,
1996
), and exocytosis (Komuro et al., 1996
). The Rac subfamily, consisting of two members, Rac1 and -2, regulates
formation of lamellipodia and membrane ruffles (Ridley
et al., 1992
), NADPH oxidase-catalyzed superoxide formation (Abo et al., 1991
; Knaus et al., 1991
; Ando et al.,
1992
; Mizuno et al., 1992
), endocytosis (Lamaze et al., 1996
),
and exocytosis (Komuro et al., 1996
; O'Sullivan et al.,
1996
). The Cdc42 subfamily, consisting of one member, regulates formation of filopodia (Kozma et al., 1995; Nobes
and Hall, 1995
) and adhesion of helper T cells towards antigen-presenting cells (Stowers et al., 1995
).
; Tsukita et al., 1992
, 1993
). Cadherins, constituting a family of transmembrane proteins that interact with each other at the extracellular surface, are
localized at adherens junctions, and are responsible for
Ca2+-dependent cell-cell adhesion (for reviews see Tsukita et al., 1992
; Takeichi, 1995
). Cadherins are associated
with several cytoplasmic proteins, such as
-,
-, and
-catenin (plakoglobin) and p120 (Vestweber and Kemler,
1984
; Peyrieras et al., 1985
; Ozawa et al., 1989
; Shibamoto
et al., 1995
). As to the regulatory mechanism of cadherins,
the tyrosine phosphorylation of
-catenin is associated with dysfunction of cadherins (Matsuyoshi et al., 1992
; Behrens et al., 1993
; Hamaguchi et al., 1993
). APC competes
for the interaction of cadherins with
-catenin (Hülsken
et al., 1994
). However, the regulatory mechanism of the
cadherin-based cell-cell adhesion is not fully understood,
and the molecular mechanism of the linkage between cadherins and actin filaments also remains to be clarified. Occludins, transmembrane proteins that interact with each
other at the extracellular surface, are localized at tight junctions (Furuse et al., 1993
). The occludin cytoplasmic tail
interacts with the cytoplasmic plaque proteins, ZO-1 and -2 (Woods and Bryant, 1993
; Furuse et al., 1994
; Jesaitis and
Goodenough, 1994
). In addition, several proteins, including cingulin (Citi et al., 1988
), the 7H6 antigen (Zhong et
al., 1993
), Rab13 small G protein (Zahraoui et al., 1994
), and
symplekin (Keon et al., 1996
) are localized at tight junctions. As to the regulatory mechanism of the formation of
tight junctions, the intercellular binding of cadherins triggers the formation of adherens junctions, followed by the
formation of tight junctions in the Ca2+-induced formation
of cell-cell adhesion (Gonzalez-Mariscal et al., 1985
, 1990
;
Contreras et al., 1992
). Activation of protein kinase C
(PKC)1 induces the formation of tight junctions without
the formation of adherens junctions in epithelial cells cultured in a low Ca2+ medium (Balda et al., 1993
). However,
the regulatory mechanism of the formation of tight junctions is not fully understood, and the molecular mechanism of the linkage between occludin and actin filaments
also remains to be clarified.
). Moreover, the Drosophila Rac subfamily regulates
actin assembly at the adherens junctions of the wing disc
epithelium (Luo et al., 1994
; Eaton et al., 1995
). However,
it has not been studied whether the mammalian Rho family regulates the formation of the cadherin-based cell-cell adhesion. Here we attempted to address this issue using
MDCK cell lines stably expressing various mutants of the
Rho family members.
Materials and Methods
neo
expression plasmid was donated from A. Miyajima (Tokyo University,
Tokyo, Japan). C3 was supplied by S. Narumiya (Kyoto University, Kyoto, Japan). The C3 sample for the microinjection experiments was prepared as described (Kotani et al., 1997
). Hybridoma cells expressing the
anti-myc mouse mAb (9E10) were purchased from American Type Culture Collection (Rockville, MD). The anti-E-cadherin rat mAb (ECCD-2)
and mouse mAb (C20820) were obtained from TAKARA Shuzo, Inc.
(Shiga, Japan) and Transduction Laboratories, Inc. (Lexington, KY), respectively. The anti-
-catenin mouse mAb (5H10) and the anti-ZO-1
mouse mAb were supplied by M.J. Wheelock (University of Toledo, Toledo, Ohio) and Sh. Tsukita (Kyoto University, Kyoto, Japan), respectively. The anti-IQGAP rabbit polyclonal antibody was supplied by K. Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan).
Rhodamine-phalloidin was purchased from Molecular Probes, Inc. (Eugene, OR). Secondary antibodies for immunofluorescence microscopy
were obtained from Chemicon International, Inc. (Temecula, CA). Other
materials and chemicals were obtained from commercial sources.
-myc-tagged V14RhoA (myc-V14RhoA), pSR
-myc-tagged
V12Rac1 (myc-V12Rac1), pSR
-myc-tagged N17Rac1 (myc-N17Rac1),
pSR
-myc-tagged V12Cdc42 (myc-V12Cdc42), and pSR
-myc-tagged
N17Cdc42 (myc-N17Cdc42) expression plasmids were constructed as described (Takaishi et al., 1995
). Briefly, the pSR
neo plasmid was cut at the
XhoI and BamHI sites and ligated to a double-stranded oligonucleotide
encoding the peptide sequence, MEQKLISEEDL, which is the epitope of
the 9E10 anti-myc mAb to generate the pSR
-myc plasmid. The 0.6-kb fragments containing full length V14RhoA, V12Rac1, N17Rac1, V12Cdc42, and N17Cdc42 coding sequences with the BamHI site upstream of the initiation methionine codon and downstream of the termination codon were
synthesized by PCR. These fragments were digested by BamHI and ligated into the BamHI site of the pSR
-myc plasmid.
-myc-V14RhoA, -V12Rac1, -N17Rac1, -V12Cdc42,
or -N17Cdc42 was carried out using the calcium phosphate coprecipitation
method, and cell clones were isolated by resistance to G418. For the Ca2+-switch experiments, the cells were cultured in serum-free DME and 5 mM
EGTA (low Ca2+ DME) for 2 h. They were then transferred to serum-free DME (normal Ca2+ DME). MDCK cells for the microinjection experiments were seeded at a density of 3 x 104 cells/dish onto 35-mm grid
dishes. 2 d after seeding, C3 was comicroinjected with 5 mg/ml of rabbit
IgG into the cells as described (Kotani et al., 1997
).
-catenin in MDCK cells were
detected by immunoblotting with the 9E10, C20820, and 5H10 mAbs, respectively. For detection of myc-tagged proteins, subconfluent monolayers of MDCK cells were lysed in Lysis Buffer A (20 mM Tris/HCl, pH 7.4, containing 150 mM NaCl, 10 mM MgCl2, 1% NP-40, and 100 µM p-amidinophenyl methanesulfonyl fluoride) and sonicated. For detection of
E-cadherin and
-catenin, the cells were lysed in Lysis Buffer B (Hepes-buffered saline [HCMF; Takeichi, 1977
] containing 1 mM CaCl2, 1% NP-40,
100 µM p-amidinophenyl methanesulfonyl fluoride, and 20 µg/ml leupeptin) and sonicated. 50 µg of each protein sample from the homogenates
was subjected to SDS-PAGE, and the separated proteins were electrophoretically transferred to a nitrocellulose membrane sheet. The sheet
was processed to detect myc-tagged proteins with the 9E10 mAb, E-cadherin with the C20820 mAb, and
-catenin with the 5H10 mAb as a primary antibody, using the ECL detection kit (Amersham Corp., Arlington
Heights, IL). For immunoprecipitation of myc-tagged proteins with the
9E10 mAb, the homogenates lysed in Lysis Buffer A were clarified by
centrifugation at 12,000 g for 10 min, and the supernatants were immunoprecipitated with the 9E10 mAb crosslinked to protein A-Sepharose 4B
using dimethyl pimelimidate. The immunoprecipitates were boiled with
SDS-sample buffer. myc-Tagged proteins in the immunoprecipitates were
detected by immunoblotting with the 9E10 mAb as described above.
) with slight modifications. Briefly, subconfluent monolayers of MDCK cells grown on 6-cm dishes were washed with HCMF containing 1 mM CaCl2 (HMF; Takeichi, 1977
), collected by scraping with a rubber policeman, and then centrifuged at
5,000 g for 1 min. After the supernatant was removed, 200 µl of various
concentrations (0-0.5%) of NP-40 in HMF were added. The samples were
incubated for 10 min with mild pipetting and then centrifuged at 100,000 g
for 30 min. To the supernatant, 100 µl of 3x SDS-sample buffer was
added and used as the detergent-soluble fraction. The pellet fraction was
dissolved in 300 µl of 1x SDS-sample buffer and used as the detergent-
insoluble fraction. E-Cadherin was detected by immunoblotting with the
C20820 mAb.
). Briefly, cells were fixed in 3.7% paraformaldehyde in PBS for 20 min. The fixed cells were incubated with 50 mM NH4Cl in PBS for 10 min
and permeabilized with PBS containing 0.2% Triton X-100 for 10 min. After being soaked in 10% FCS/PBS for 30 min, the cells were treated with
the first antibody in 10% FCS/PBS for 1 h. The cells were then washed
with PBS three times, followed by incubation with the second antibody in
10% FCS/PBS for 1 h. For the detection of actin filaments, rhodamine-phalloidin was mixed with the second antibody solution. After being
washed with PBS three times, the cells were examined using a confocal laser scanning microscope (LSM 410; Carl Zeiss, Oberkochen, Germany). For localization of E-cadherin and
-catenin, cells were fixed in 3.7%
paraformaldehyde in HMF for 20 min, followed by the same procedures
as described above.
). Briefly, cells cultured on
the poly-L-lysine-coated celldesk LF1 were fixed with 4% paraformaldehyde in PBS. The samples were incubated with the ECCD-2 anti-E-cadherin mAb or the 5H10 anti-
-catenin mAb followed by incubation with
an anti-rat IgG antibody or an anti-mouse IgG antibody, respectively,
coupled with 1.4-nm gold particles (Nanoprobes Inc., Stony Brook, NY).
After being washed, the samples were fixed with 1% glutaraldehyde in
PBS for 10 min, and the sample-bound gold particles were silver enhanced
by the HQ-silver kit (Nanoprobes Inc.) at 18°C for 8 min. The samples
were again washed and postfixed with 0.8% OsO4 in 0.1 M cacodylate-HCl, pH 7.4, for 1 h. The samples were dehydrated in a graded series of
ethanol, embedded in Epon812, and examined using an electron microscope (JEM-1200EX; JEOL, Tokyo, Japan).
Results
neo expression plasmid, containing each of the
cDNAs of myc-V14RhoA, a dominant active mutant of RhoA,
myc-V12Rac1, a dominant active mutant of Rac1, myc-N17Rac1, a dominant negative mutant of Rac1, myc-V12Cdc42,
a dominant active mutant of Cdc42, and myc-N17Cdc42,
a dominant negative mutant of Cdc42, was transfected into MDCK cells, and cell clones were isolated by resistance to G418. Three clones expressing myc-V14RhoA
(sMDCK-RhoDA), four clones expressing myc-V12Rac1
(sMDCK-RacDA), two clones expressing myc-N17Rac1
(sMDCK-RacDN), two clones expressing myc-V12Cdc42 (sMDCK-Cdc42DA), and four clones expressing myc-N17Cdc42 (sMDCK-Cdc42DN) were obtained. myc-V14RhoA in sMDCK-RhoDA cell line clone 5 (sMDCK-RhoDA-5) and myc-V12Rac1 in sMDCK-RacDA cell line
clone 1 (sMDCK-RacDA-1) were undetectable by immunoblotting with the anti-myc Ab using the cell lysates (Fig.
1 a, lanes 2 and 3), but the proteins were detected in the
immunoprecipitated samples from the cell lysates (Fig. 1
b, lanes 2 and 3). myc-N17Rac1 in the sMDCK-RacDN
cell line clone 2 (sMDCK-RacDN-2), myc-V12Cdc42 in the
sMDCK-Cdc42DA cell line clone 2 (sMDCK-Cdc42DA-2), and myc-N17Cdc42 in the sMDCK-Cdc42DN cell line
clone 6 (sMDCK-Cdc42DN-6) were detected in both the cell lysates and the immunoprecipitated samples (Fig. 1, a
and b, lanes 4-6). The expression level of myc-V14RhoA
was calculated to be less than one tenth of endogenous
RhoA in sMDCK-RhoDA-5 when the expression level of
endogenous RhoA was estimated by immunoblotting with
the anti-RhoA polyclonal antibody (data not shown). Because any anti-Rac1 Ab or anti-Cdc42 Ab is not currently available, we could not compare the expression levels of
Rac1 and Cdc42 mutants with those of endogenous Rac1
and Cdc42 in sMDCK-RacDA-1, -RacDN-2, -Cdc42DA-2,
and -Cdc42DN-6 cells, respectively.
Fig. 1.
Expression of various Rho, Rac, and Cdc42 mutants in
stable transformants of MDCK cell lines. Subconfluent MDCK
cells were lysed and protein samples were directly subjected to
SDS-PAGE (13% polyacrylamide). The separated proteins were
processed for immunoblotting with the 9E10 anti-myc mAb (a).
Subconfluent MDCK cells were lysed, and protein samples were
immunoprecipitated with the 9E10 anti-myc mAb crosslinked to
protein A-Sepharose 4B. The immunoprecipitates were subjected to SDS-PAGE (13% polyacrylamide) and the separated
proteins were processed for immunoblotting with the 9E10 mAb
(b). lane 1, Wild-type MDCK cell line; lane 2, sMDCK-RhoDA-5 cell line expressing myc-V14RhoA; lane 3, sMDCK-RacDA-1 cell
line expressing myc-V12Rac1; lane 4, sMDCK-RacDN-2 cell line expressing myc-N17Rac1; lane 5, sMDCK-Cdc42DA-2 cell line expressing myc-V12Cdc42; lane 6, sMDCK-Cdc42DN-6 cell line
expressing myc-N17Cdc42. Arrows indicate myc-tagged proteins.
The mobility of myc-V14RhoA was slightly slower than that of
myc-V12Rac1, -N17Rac1, -V12Cdc42, and -N17Cdc42. An arrowhead indicates the light chain of the 9E10 anti-myc mAb,
which was detected because a part of the light chain of the 9E10
anti-myc mAb crosslinking to protein A-sepharose 4B was detached from the beads by boiling in SDS-sample buffer. The prestained protein markers used were soybean trypsin inhibitor (molecular weight, 28,300) and lysozyme (molecular weight,
19,800).
[View Larger Version of this Image (28K GIF file)]
-myc vector alone (cMDCK; Takaishi et al., 1995
), and the staining pattern of the actin filaments at the cell-cell adhesion sites in cMDCK cells was
similar to that in wild-type MDCK cells (data not shown).
These results indicate that the Rac subfamily regulates the
formation of the actin filaments at the cell-cell adhesion
sites, whereas the Rho subfamily regulates the formation of stress fibers.
Fig. 2.
Localization of the actin cytoskeleton and cell shape in
MDCK cells stably expressing various Rho, Rac, and Cdc42 mutants. Wild-type MDCK cells (a, g, and m), sMDCK-RhoDA-5
cells (b, h, and n), sMDCK-RacDA-1 cells (c, i, and o), sMDCK-RacDN-2 cells (d, j, and p), sMDCK-Cdc42DA-2 cells (e, k, and
q), and sMDCK-Cdc42DN-6 cells (f, l, and r) were stained with
rhodamine-phalloidin and analyzed by confocal microscopy. (a-
f) Basal levels; (g-l) apical, junctional levels; (m-r) vertical sections. White arrowheads indicate the cell-cell adhesion sites.
Black arrowheads and arrows indicate the apical and basal levels
of the cells, respectively. Note that the scale in RacDN panels (d,
j, and p) is different from those in the other panels. The results
shown are representative of three independent experiments.
Bars, 10 µm.
[View Larger Version of this Image (107K GIF file)]
-Catenin, and ZO-1 in
MDCK Cell Lines Stably Expressing Various Rho, Rac,
and Cdc42 Mutants
-catenin, both of
which are the structural proteins of adherens junction (for
reviews see Tsukita et al., 1992
; Takeichi, 1995
), in these
cell lines. The staining of E-cadherin at the junctional levels
in sMDCK-RhoDA-5 cells was similar to that in wild-type MDCK cells (Fig. 3, a and b). The staining of E-cadherin
was apparently denser and thicker in sMDCK-RacDA-1
cells but weaker in sMDCK-RacDN-2 cells (Fig. 3, c and
d). The staining of E-cadherin at the area of about apical
two thirds of the lateral membranes was relatively stronger
than that at the area of about basal one third of the lateral
membranes in wild-type MDCK and sMDCK-RhoDA-5
cells (Fig. 3, e and f). However, the staining of E-cadherin
increased at all of the lateral membranes in sMDCK-RacDA-1 cells (Fig. 3 g), whereas it was weak throughout
the lateral membranes in sMDCK-RacDN-2 cells (Fig. 3
h). The staining patterns of
-catenin were essentially similar to those of E-cadherin in wild-type MDCK, sMDCK-RhoDA-5, -RacDA-1, and -RacDN-2 cells (Fig. 3, i-p).
These staining patterns of both E-cadherin and
-catenin
were essentially similar to those of the actin filaments at
the cell-cell adhesion sites. The essentially same results were
obtained when E-cadherin and
-catenin were stained in
the other MDCK cell lines stably expressing V14RhoA, V12Rac1, or N17Rac1 (data not shown). The stainings of
both E-cadherin and
-catenin at the junctional levels in
all the clones of both sMDCK-Cdc42DA and -Cdc42DN
were similar to those in wild-type MDCK cells (data not
shown). These results indicate that the Rac subfamily regulates not only the formation of the actin filaments but
also the localization of E-cadherin and
-catenin at the
cell-cell adhesion sites in MDCK cells.
Fig. 3.
Localization of E-cadherin and -catenin in sMDCK-RhoDA-5, -RacDA-1, and
-RacDN-2 cells. Wild-type
MDCK cells (a, e, i, and m),
sMDCK-RhoDA-5 cells (b, f, j,
and n), sMDCK-RacDA-1 cells (c, g, k, and o), and sMDCK-RacDN-2 cells (d, h, l, and p)
were stained with the ECCD-2
anti-E-cadherin mAb (a-h) or
the 5H10 anti-
-catenin mAb
(i-p) and analyzed by confocal
microscopy. (a-d and i-l) Junctional levels; (e-h and m-p) vertical sections. White arrowheads
indicate the cell-cell adhesion
sites. Black arrowheads and arrows indicate the apical and basal
levels of the cells, respectively.
Note that the scales in RacDN
panels (d, h, l, and p) are different
from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 µm.
[View Larger Version of this Image (101K GIF file)]
Fig. 4.
Detergent solubility of E-cadherin from wild-type
MDCK, sMDCK-RacDA-1, and -RacDN-2 cells. Wild-type
MDCK (a), sMDCK-RacDA-1 (b), and sMDCK-RacDN-2 cells
(c) were incubated with various concentrations of NP-40, and the
amounts of E-cadherin in the detergent-soluble (S) and -insoluble
(I) fractions were measured by immunoblotting with the C20820
anti-E-cadherin mAb. d shows the amount of the insoluble
E-cadherin as a percentage of the amount of total E-cadherin, i.e., S plus I, by estimation using densitometer.
[View Larger Versions of these Images (34 + 26K GIF file)]
-catenin were not markedly different among wild-type, sMDCK-RhoDA-5, -RacDA-1,
and -RacDN-2 cells (Fig. 5). The expression levels of
E-cadherin and
-catenin in sMDCK-Cdc42DA-2 and
-Cdc42DN-6 cells were also similar to those in wild-type
MDCK cells (data not shown). These results indicate that
the changes in the staining intensity and the distribution of
both E-cadherin and
-catenin are not due to the changes
in the expression levels of both E-cadherin and
-catenin
in sMDCK-RacDA-1 and -RacDN-2 cells.
Fig. 5.
Expression levels of
E-cadherin and -catenin in
sMDCK-RhoDA-5, -RacDA-1,
and -RacDN-2 cells. Sub-confluent MDCK cells were lysed, and protein samples
were directly subjected to
SDS-PAGE (10% polyacrylamide). The separated proteins were processed for immunoblotting with the
C20820 anti-E-cadherin mAb (a) and the 5H10 anti-
-catenin
mAb (b). Lane 1, wild-type MDCK cells; lane 2, sMDCK-RhoDA-5 cells; lane 3, sMDCK-RacDA-1 cells; lane 4, sMDCK-RacDN-2 cells. The prestained protein markers used were
myosin (molecular weight, 201,000),
-galactosidase (molecular
weight, 117,000), and bovine serum albumin (molecular weight,
82,000).
[View Larger Version of this Image (50K GIF file)]
) but is also partly colocalized
with E-cadherin in MDCK cells (Howarth et al., 1994
).
We analyzed the localization of ZO-1 in sMDCK-RhoDA,
-RacDA, -RacDN, -Cdc42DA, and -Cdc42DN cells. The
staining patterns of ZO-1 in sMDCK-RhoDA-5, -RacDA-1, and -RacDN-2 cells were apparently indistinguishable
from those in wild-type MDCK cells (Fig. 6). The staining
patterns of ZO-1 in the other MDCK cell lines stably expressing V14RhoA, V12Rac1, or N17Rac1 were also similar to that in wild-type MDCK cells (data not shown). The
staining patterns of ZO-1 in all the clones of sMDCK-Cdc42DA and -Cdc42DN were also similar to that in wild-type MDCK cells (data not shown). These results indicate
that neither the Rho, the Rac, nor the Cdc42 subfamily affects the localization of ZO-1.
Fig. 6.
Localization of ZO-1 in sMDCK-RhoDA-5, -RacDA-1,
and RacDN-2 cells. Wild-type MDCK cells (a), sMDCK-RhoDA-5
cells (b), sMDCK-RacDA-1 cells (c), and sMDCK-RacDN-2
cells (d) were stained with the anti-ZO-1 mAb and analyzed by
confocal microscopy. Confocal images are shown at the junctional levels. Note that the scale in RacDN panels (d) is different
from those in the other panels. The results shown are representative of three independent experiments. Bars, 10 µm.
[View Larger Version of this Image (45K GIF file)]
-catenin in the MDCK
cells stably expressing V12Rac1 or N17Rac1, we performed
electron microscopic analyses on the morphology of the
cell-cell adhesion and the localization of E-cadherin and
-catenin at the cell-cell adhesion sites in these cell lines.
Wild-type MDCK cells tightly and linearly made contact
with each other at the apical area of the lateral membranes, whereas the cells loosely made contact at the other
areas of the lateral membranes (Fig. 7 a). In contrast,
sMDCK-RacDA-1 cells tightly made contact with each
other throughout the lateral membranes (Fig. 7 c). No intercellular-free spaces were observed in these cells. Moreover, sMDCK-RacDA-1 cells were thicker and showed
more markedly intermingled lateral membranes than wild-type MDCK cells. The morphology of the cell-cell adhesion in sMDCK-RacDN-2 cells resembled that in wild-type
MDCK cells. sMDCK-RacDN-2 cells tightly made contact
with each other at the apical area of the lateral membranes, whereas the cells loosely made contact at the basal side of the lateral membranes, although the cells were
thinner than wild-type MDCK cells (Fig. 7 e). The desmosomes were apparently indistinguishable in all the cell
lines (Fig. 7, b, d, and f). These results indicate that the activation of the Rac subfamily strengthens the cell-cell adhesion without affecting the formation of the desmosomes.
Fig. 7.
Electron microscopic analysis on the morphology of the
cell-cell adhesion sites in wild-type MDCK, sMDCK-RacDA-1,
and -RacDN-2 cells. Wild-type MDCK cells (a and b), sMDCK-RacDA-1 cells (c and d), and sMDCK-RacDN-2 cells (e and f)
were fixed and processed for electron microscopy. b, d, and f
show the cell-cell adhesion sites at higher magnifications of a, c,
and e, respectively. Arrowheads indicate desmosomes. The results shown are representative of three independent experiments.
Bars: (a, c, and e) 1 µm; (b, d, and f) 0.5 µm.
[View Larger Version of this Image (106K GIF file)]
-catenin were similar to those of
E-cadherin (Fig. 8, b, d, and f). These results were consistent with those obtained by immunofluorescence microscopy and the detergent solubility assay.
Fig. 8.
Immunoelectron microscopy analysis of the localization of E-cadherin and -catenin at the cell-cell adhesion sites by
immunoelectron microscopy in wild-type MDCK, sMDCK-RacDA-1, and -RacDN-2 cells. Wild-type MDCK cells (a and b),
sMDCK-RacDA-1 cells (c and d), and sMDCK-RacDN-2 cells (e
and f) were fixed and processed for immunoelectron microscopy
using the ECCD-2 anti-E-cadherin mAb (a, c, and e) or the 5H10
anti-
-catenin mAb (b, d, and f). The results shown are representative of three independent experiments. Bars, 1 µm.
[View Larger Version of this Image (137K GIF file)]
; Kikuchi et al., 1988
; Narumiya et al., 1988
;
Braun et al., 1989
). C3 ADP-ribosylates Asn41 of the Rho
subfamily, which is located at the putative effector domain, and the ADP-ribosylation impairs the functions of
the Rho subfamily, presumably preventing the Rho subfamily from interacting with its effector protein (Sekine et
al., 1989
). We have previously shown that the most prominent and earliest effects of microinjection of C3 into wild-type MDCK cells were the disappearance of stress fibers
and peripheral bundles and the inhibition of the localization of the ERM (Ezrin, Radixin, Moesin) family at peripheral bundles and vinculin at both focal adhesions and
basal edges (Kotani et al., 1997
). These effects of C3 were
observed within 15 min after the microinjection. At 1 h after the microinjection, the actin filaments at the cell-cell
adhesion sites started to disappear, and the microinjected
cells separated with each other, although the actin filaments at the cell-cell adhesion sites between the microinjected and unmicroinjected cells were not apparently affected (Fig. 9, a and d). We then investigated the effect of
microinjection of C3 into the cells on the localization of
E-cadherin and ZO-1 at the cell-cell adhesion sites. At 30 min after the microinjection, the localization of both
E-cadherin and ZO-1 at the cell-cell adhesion sites was
not affected (data not shown), but at 1 h after the microinjection, the staining of both E-cadherin and ZO-1 at the
cell-cell adhesion sites between the microinjected cells
disappeared, whereas that between the microinjected and
unmicroinjected cells was not apparently affected (Fig. 9,
b, c, e, and f). These results indicate that the Rho subfamily is necessary for the maintenance of both the adherens
and tight junctions.
Fig. 9.
Disruption of the cell-cell adhesion in wild-type
MDCK cells by microinjection of C3. Wild-type MDCK cells
were fixed at 1 h after the microinjection with 40 µg/ml of C3 plus
5 mg/ml of rabbit IgG and stained with rhodamine-phalloidin (a),
the ECCD-2 anti-E-cadherin mAb (b), or the anti-ZO-1 mAb
(c), and analyzed by confocal microscopy. The microinjected cells
are shown by the staining of microinjected rabbit IgG (d-f). Confocal images are shown at the junctional levels. The localization
of the actin filaments, E-cadherin, and ZO-1 at the cell-cell adhesion sites was inhibited between the microinjected cells (arrowheads), whereas it was not inhibited between the microinjected
and unmicroinjected cells (arrows). The results shown are representative of three independent experiments. Bars, 10 µm.
[View Larger Version of this Image (88K GIF file)]
Fig. 10.
Inhibition of the formation of the cell-cell adhesion in
wild-type MDCK cells by microinjection of C3. Wild-type
MDCK cells cultured in normal Ca2+ DME were microinjected
with 40 µg/ml of C3 plus 5 mg/ml of rabbit IgG. At 30 min after
the microinjection the cells were transferred to low Ca2+ DME
and further incubated for 2 h. The cells were then transferred to
normal Ca2+ DME for 2 h (a, b, d, and e) or stimulated with 1 x 107 M TPA for 1 h (c and f). The cells were stained with the
ECCD-2 anti-E-cadherin mAb (a) or the anti-ZO-1 mAb (b and
c) and analyzed by confocal microscopy. The microinjected cells
are shown by the staining of microinjected rabbit IgG (d-f). Confocal images are shown at the junctional levels. The localization
of E-cadherin and ZO-1 at the cell-cell adhesion sites was inhibited between the microinjected cells (arrowheads), whereas it was
not inhibited between the microinjected and unmicroinjected
cells (arrows). The results shown are representative of three independent experiments. Bars, 10 µm.
[View Larger Version of this Image (82K GIF file)]
, 1990
; Contreras
et al., 1992
). However, activation of PKC induces the formation of the tight junction without the formation of the
adherens junction in MDCK cells cultured in a low Ca+
medium (Balda et al., 1993
). We investigated the effect of
microinjection of C3 on the PKC-induced formation of the
tight junction. At 30 min before the Ca2+ switch, the cells
were microinjected with C3. The cells were then incubated
in a low Ca2+ medium for 2 h, followed by the 1-h incubation
in a low Ca2+ medium with 12-O-tetradecanoylphorbol-13-acetate (TPA). As shown previously (Balda et al.,
1993
), addition of TPA induced the localization of ZO-1 at
the cell-cell adhesion sites (Fig. 10, c and f), whereas it did
not induce the localization of E-cadherin at the cell-cell adhesion sites (data not shown). The TPA-induced localization of ZO-1 at the cell-cell adhesion sites between the
microinjected cells was inhibited (Fig. 10, c and f), whereas
that between the microinjected and unmicroinjected cells
was not apparently inhibited (data not shown). These results indicate that the Rho subfamily is also necessary for
the PKC-induced formation of the tight junction.
Fig. 11.
Inability of microinjection of C3 to disrupt the cell-
cell adhesion in sMDCK-RacDA-1 cells. (a-c); sMDCK-RacDA-1
cells were fixed at 2 h after the microinjection with 40 µg/ml of
C3 plus 5 mg/ml of rabbit IgG and stained with rhodamine-phalloidin (a and b) and analyzed by confocal microscopy. The microinjected cells are shown by the staining of microinjected rabbit
IgG (c). (a) Basal level; (b and c) junctional levels. Stress fibers
completely disappeared in the microinjected cells (stars), whereas
the unmicroinjected cells possessed weak stress fibers (arrow).
(d-f) sMDCK-RacDA-1 cells were fixed at 2 h after the microinjection with 40 µg/ml of C3 plus 5 mg/ml of rabbit IgG and stained
with rhodamine-phalloidin (d) or the ECCD-2 anti-E-cadherin
mAb (e) and analyzed by confocal microscopy. The microinjected cells are shown by the staining of microinjected rabbit IgG
(f). Confocal images are shown at the junctional levels. The results shown are representative of three independent experiments.
Bars, 10 µm.
[View Larger Version of this Image (124K GIF file)]
Fig. 12.
Localization of myc-tagged proteins in sMDCK-RhoDA-5, -RacDA-1, and -RacDN-2 cells. Wild-type MDCK
cells (a and e), sMDCK-RhoDA-5 cells expressing myc-V14RhoA
(b and f), sMDCK-RacDA-1 cells expressing myc-V12Rac1 (c
and g), and sMDCK-RacDN-2 cells expressing myc-N17Rac1 (d
and h) were double stained with rhodamine-phalloidin (a-d) and
the 9E10 anti-myc mAb (e-h) and analyzed by confocal microscopy. Confocal images are shown at the junctional levels. The results shown are representative of three independent experiments.
Bars, 10 µm.
[View Larger Version of this Image (88K GIF file)]
-catenin was not apparently affected in sMDCK-Cdc42DA-2
cells as described above (data not shown). N17Cdc42 was
localized mainly at the cytosol (data not shown).
Discussion
-catenin at the cell-cell adhesion sites markedly increased, suggesting that the dominant active mutant of
Rac1 expressed to a small extent is able to induce the
change in the actin cytoskeleton and adherens junctional
proteins. The expression of myc-V14RhoA in sMDCK-RhoDA was also undetectable by immunoblotting with
the anti-myc mAb using the cell lysates, whereas it was detected in the immunoprecipitated samples from the cell lysates, and the expression level of myc-V14RhoA was calculated to be less than one tenth of endogenous RhoA in
sMDCK-RhoDA, when the expression level of endogenous RhoA was estimated by immunoblotting with the
anti-RhoA polyclonal Ab. However, the formation of the
stress fibers and the focal adhesions apparently increased in sMDCK-RhoDA, suggesting that the dominant active mutant of RhoA expressed to a small extent is
enough to induce the change in the actin cytoskeleton. These results are consistent with the yeast results that activation of only a part of Rho1p is sufficient for the bud formation (Yamochi et al., 1994
).
-catenin
at the cell-cell adhesion sites and wider distribution of
these proteins in sMDCK-RacDA cells than in wild-type
MDCK cells, and conversely less intense staining and narrower distribution of these proteins in sMDCK-RacDN cells than in wild-type MDCK cells. The detergent solubility assay showed that the amount of detergent-insoluble
E-cadherin increased in sMDCK-RacDA cells, whereas it
decreased in sMDCK-RacDN cells. Electron microscopic
analyses revealed that sMDCK-RacDA cells made tight
contact with each other throughout the lateral membranes, whereas wild-type MDCK cells made contact tightly and
linearly only at the apical side of the lateral membranes
and that E-cadherin and
-catenin were localized densely
throughout the lateral membranes of sMDCK-RacDA
cells, whereas they were localized sparsely at the basal side
of the lateral membranes of wild-type MDCK cells. These
results clearly indicate that the E-cadherin-based cell-cell adhesion is markedly strengthened in sMDCK-RacDA
cells. We furthermore attempted to perform cell aggregation assay using wild-type MDCK, sMDCK-RacDA, and
-RacDN cells as described previously (Nagafuchi and
Takeichi, 1988
). However, we have not observed the significant difference in the activity of cell-cell adhesion in
this assay (data not shown), suggesting that a dominant active or negative mutant of the Rac subfamily does not up
regulate or down regulate, respectively, the activity of the
E-cadherin-based cell-cell adhesion to the levels detectable by this cell aggregation assay. However, because it is
generally difficult to measure correctly the activity of cell-
cell adhesion of epithelial cells, which have not only cadherin-based cell-cell adhesion but also tight junction, by this cell aggregation assay, we reserve the conclusion on
the effect of the Rac subfamily on this activity.
-,
-, and
-catenins, p120
(Vestweber and Kemler, 1984
; Peyrieras et al., 1985
;
Ozawa et al., 1989
; Shibamoto et al., 1995
), and unidentified molecules that link E-cadherin to actin filaments. Another possible mechanism is that the Rac subfamily directly regulates the function of E-cadherin or these related
molecules through its downstream target molecule, eventually leading to the assembly of the actin filaments at the
cell-cell adhesion sites.
). The target molecule of the Rac subfamily that regulates the E-cadherin-based
cell-cell adhesion is not known. Among the identified
putative target molecules, IQGAP1/2 was localized at the
cell-cell adhesion sites in MDCK cells (Hart et al., 1996
;
Kuroda et al., 1996
). However, the localization of IQGAP1/2
was not apparently affected in sMDCK-RacDA and -RacDN cells, compared with that in wild-type MDCK cells (data
not shown). Further studies are necessary to clarify the
mode of action of the Rac subfamily.
;
Lamaze et al., 1996
; O'Sullivan et al., 1996
). Either the inhibition of endocytosis, the stimulation of exocytosis, or
both may induce the enlargement of the plasma membrane, whereas either the stimulation of endocytosis, the
inhibition of exocytosis, or both may induce the reduction
of the plasma membrane.
; Nishiyama et al., 1994
). Microinjection of the dominant active
mutant of Rac1 induced membrane ruffling, whereas microinjection of the dominant negative mutant of Rac1 inhibited the growth factor-induced membrane rufflings in
Swiss 3T3 cells (Ridley et al., 1992
). However, in MDCK
cells, microinjection of the dominant active mutant of
Rac1 into this cell line did not induce membrane ruffling,
whereas microinjection of the dominant negative mutant
of Rac1 inhibited the hepatocyte growth factor-induced
membrane ruffling (Ridley et al., 1995
). In sMDCK-RacDA
cells, the formation of lamellipodia or membrane ruffling
did not increase, compared with that in wild-type MDCK cells, whereas the formation of lamellipodia and membrane ruffles apparently decreased in sMDCK-RacDN
cells (data not shown). These results indicate that the Rac
subfamily is necessary, but not sufficient, for the formation
of lamellipodia and membrane ruffles in MDCK cells,
whereas the Rac subfamily is sufficient for the formation
of lamellipodia and membrane ruffles in Swiss 3T3 cells. These different actions of the Rac subfamily may be due
to the difference of the cell types.
-(3-O-thio) triphosphate-bound form of Rho into
wild-type MDCK cells induced the formation of stress fibers and the localization of vinculin at the focal adhesions
but did not induce the increased localization of the ERM
family at the plasma membrane (Kotani et al., 1997
). We
showed here that the formation of stress fibers and focal
adhesions markedly increased in sMDCK-RhoDA cells.
However, the localization of the ERM family at the
plasma membrane did not apparently increase in sMDCK-RhoDA cells, compared with that in wild-type MDCK
cells (data not shown). These results are consistent with
our previous results (Kotani et al., 1997
). Moreover, we previously showed that microinjection of C3 into wild-type
MDCK cells inhibited both the localization of the ERM
family at the peripheral bundles and of vinculin at the
basal edges of the colonies of the cells within 15 min after
the microinjection (Kotani et al., 1997
). We showed here
that the later effect of microinjection of C3 into wild-type
MDCK cells was disruption of the E-cadherin-based cell-
cell adhesion and the tight junction. Furthermore, we
showed here that microinjection of C3 also inhibited the
Ca2+-induced cell-cell adhesion and the TPA-induced formation of the tight junction. However, these effects of C3
were observed at the cell-cell adhesion between the microinjected cells, but not between the microinjected and
unmicroinjected cells. Moreover, microinjection of C3
showed little effect on the increased localization of the actin filaments and E-cadherin at the cell-cell adhesion sites
in sMDCK-RacDA cells. It is likely that the C3-induced
loss of stress fibers, focal adhesions, and normal cell shape
secondarily disrupts the cell-cell adhesion.
).
N17Rac1 had higher affinity for GDP than for GTP and
acted as a dominant negative mutant by binding to GEP
(Ridley et al., 1992
). Therefore, the localization of myc-N17Rac1 at the cell-cell adhesion sites may be due to its
binding to GEP localized at the plasma membrane. In contrast to the localization of the Rac1 mutants, the dominant active mutant of RhoA, myc-V14RhoA, was localized
mainly at the cytosol in sMDCK-RhoDA cells. The localization of myc-V14RhoA might be masked by the cytosolic diffuse staining, even if activated RhoA was translocated to the focal adhesions and induced the formation of
the stress fibers and the focal adhesions through interaction with and activation of its downstream target molecule
localized there. It is also possible that activated RhoA induced the formation of the stress fibers and the focal adhesions through interaction with and activation of its downstream target molecule localized at the cytosol.
). Microinjection of C3, RacDN, or RacDA plus C3 inhibited the
localization of E-cadherin at the cell-cell adhesion, whereas
microinjection of neither RhoDA nor RacDA affected it. The effects of C3 and RacDN to inhibit the localization of
E-cadherin at the cell-cell adhesion were observed at 25 min
after the microinjection, and the inhibition of the E-cadherin-based cell-cell adhesion was observed between the
microinjected cells and between the microinjected cells and
unmicroinjected cells, suggesting that the Rho subfamily primarily regulates the E-cadherin-based cell-cell adhesion. As to the effect of RacDN and RhoDA, these results
are consistent with our results, but as to the effect of C3
and RacDA, there are apparent discrepancies between
these and our results. In our results, the time courses of C3
to inhibit the E-cadherin-based cell-cell adhesion in MDCK
cells were apparently delayed, and the effect of C3 to inhibit the E-cadherin-based cell-cell adhesion in MDCK
cells was observed only between the microinjected cells. Moreover, microinjection of C3 did not apparently inhibit
the increased localization of E-cadherin in sMDCK-RacDA
cells. Microinjection of RacDA did not affect the E-cadherin-based cell-cell adhesion in human keratinocytes,
whereas sMDCK-RacDA cells showed increased localization of E-cadherin at cell-cell adhesion sites. In another previous report, the Rho subfamily regulated the formation of the tight junction and the perijunctional actin but
did not affect the localization of E-cadherin or the actin
filaments at the cell-cell adhesion sites in polarized intestinal epithelial cells using DC3B, a chimeric toxin consisting
of C3 and diphtheria toxin (Nusrat et al., 1995
), whereas
microinjection of C3 into wild-type MDCK cells inhibited
the localization of both the actin filaments, E-cadherin,
and ZO-1 at the cell-cell adhesion sites. The reasons for
these discrepancies between these and our results are not known but may be due to the difference in assay conditions and/or difference of cell types.
Received for publication 3 April 1997 and in revised form 4 September 1997.
This investigation was supported by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan (1995-1997), by Grants-in-Aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1995-1997), and by grants from the Human Frontier Science Program (1995-1997) and the Uehara Memorial Foundation (1995-1996).We thank Dr. M. Takeichi (Kyoto University, Kyoto, Japan) for valuable
discussions, and Dr. Sh. Tsukita for providing the anti-ZO-1 mAbs and
valuable discussions. We thank Dr. W. Birchmeier for providing MDCK
cells, Dr. A. Hall for the cDNAs of V12Rac1 and N17Rac1, Dr. K. Kaibuchi for anti-IQGAP pAb, Dr. P. Madaule for the cDNA of RhoA, Dr. A. Miyajima for the pSRneo expression plasmid, Dr. S. Narumiya for C3,
Dr. P. Polakis for the cDNA of Cdc42, and Dr. M.J. Wheelock for the
anti-
-catenin mAb.
ERM, Ezrin, Radixin, Moesin; GEP, GDP/GTP exchange protein; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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