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Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Identification of an Epithelial Protein Related
to the Desmosome and Intermediate Filament Network
P. Ouyang and S. E Sugrue
Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115
Abstract. Using a mAb, referred to as 08L, we have
identified a protein, of Mr = 140,000, associated with
desmosomes of epithelial cells. The 08L antibody
stained the intracellular side of lateral cell margins of
monolayer epithelial cells but did not stain cell mar-
gins free of cell contact. Immunoelectron microscopy
revealed that the 08L antigen was localized to the cyto-
solic surface of the desmosomal plaque near points of
intermediate filament convergence with apparently little
staining of the desmosomal plaque proper. Western
blots revealed the 08L antigen to be a protein, of Mr
= 140,000, found in the Triton-X 100 insoluble pellet.
High salt-containing buffers extracted the 08L antigen
from the insoluble material. Examination of the as-
sembly of 08L to the desmosome complex, in cells
grown in low confluent culture or in calcium-switch
assays, by double immunofluorescence with 08L and
anti-desmoplakin antibody, revealed that 08L was re-
cruited to morphologically identifiable desmosomes.
08L antigen may exist in a cytosolic pool prior to as-
sembly to the cell surface. The solubility of 08L in low
calcium and normal calcium conditions, however, was
similar. 08L association to the desmosome was corre-
lated with increased organization of the intermediate
filament network. We suggest that the 08L antigen may
be involved in the organization and stabilization of the
desmosome-IF complexes of epithelia.
T HE desmosome (macula adherens) is a major compo-
nent of the epithelial intercellular junctional complex.
Desmosomes are intimately involved in the structural
and functional integration of adjacent epithelial cells (Fey et
al., 1984; Cowin et al., 1985a; Garrod et al., 1990). The
desmosome serves as a site of reinforcement of cell-cell
adhesion as well as an anchorage point for the intermediate
filament scaffold of the cell. Therefore, the desmosome is in-
tegral in epithelial cell and epithelial sheet organization. The
intercellular space at the desmosome is 20-30 nm and con-
tains an electron-dense midplate. A pair of dense cytoplas-
mic plaques are found beneath lateral cell membranes and
are associated with looping bundles of intermediate fila-
ments radiating from the cytoplasm.
Because of the availability of isolation procedures for des-
mosomes, detailed morphological, biochemical, and molec-
ular analyses of desmosomal components have led to the
identification of, and putative function for as many as eight
desmosomal proteins (Drochmans et al., 1978; Suhrbier and
Garrod, 1986; Schwartz et al., 1990). The constituent pro-
teins include desmoplakins (I and II) which are non-
glycosylated proteins (Mr = 280,000 and 250,000, respec-
tively) found within the desmosomal plaque (Mueller and
Franke, 1983; Green et al., 1990), plakoglobin (Mr =
83,000), a plaque protein which is apparently related to the
recently described catenins (Gorbsky et al., 1985; Cowin et
al., 1986; Franke et al., 1989; McCrea et al., 1991), des-
mocollin (I and II) (Mr ~- 115-130,000), and desmoglein
(Mr = 160,000) which are Ca 2+ sensitive cell adhesion
transmembrane glycoproteins with distinct homology to the
cadherins (Cowin et al., 1984; Schmelz et al., 1986a,b; Hol-
ton et al., 1990; Koch et al., 1990; Collins et al., 1991; Ma-
gee and Buxton, 1991; Mechanic et al., 1991). Other des-
mosomal components have been reported to be found in a
limited subset of desmosomes, such as desmoplakin IV or
band 6 polypeptide (Kapprell et al., 1988). In addition, still
other reports of minor or cell specific desmosomal-asso-
ciated proteins are plentiful (Tsukita and Tsukita, 1985;
Jones et al., 1986; Jones, 1988; Hieda et al., 1989). The mo-
lecular complexity of the desmosome may be indicative of
its functional diversity and tissue heterogeneity. Although
much is known about desmosome composition, many ques-
tions remain concerning the specific role of individual com-
ponents of the desmosomal complex, their regulation, and
means of assembly and disassembly.
As mentioned above, the desmosome serves as an an-
chorage point for intermediate filaments (IF) ~. However,
the specific molecular entities involved in IF binding at the
desmosome remain uncertain. Location on the cytoplasmic
portion of the desmosomal plaque (Jones and Green, 1991)
and the predicted molecular structure of desmoplakin iden-
1. Abbreviations used in this paper:
CSK, cytoskeleton; IF, intermediate
filament; IgM, immunoglobulin-M; LCM, low calcium medium; NCM,
normal calcium medium.
9 The Rockefeller University Press, 0021-9525/92/09/1477/12 $2.00
The Journal of Cell Biology, Volume 118, Number 6, September 1992 1477-1488 1477
tify it as a putative IF linker (Green et al., 1990), yet, no
firm biochemical evidence exists for direct IF-desmoplakin
interaction (O'Keefe et al., 1989). Other desmosomal pro-
teins such as desmocalmin and desmoplakin IV have been
shown to bind IFs in vitro (Tsukita and Tsukita, 1985; Kap-
prell et al., 1988), however, these proteins exhibit a limited
tissue distribution and, therefore, would be unlikely candi-
dates for general mediators of IF-desmosomal interactions.
Recently, Cartaud et al. (1990) have reported a desmosomal
plaque protein with Mr = 140,000 which is related to lamin
B, the IF receptor of the nuclear lamina. This protein has
been shown to bind vimentin filaments.
Here, we report the identification of a desmosome-IF
complex-associated protein, Mr = 140,000, which is as-
sociated with most if not all desmosomes. This protein was
localized to the periphery of the desmosomal plaque and ad-
jacent amorphous material near the convergence of inter-
mediate filaments. This 140-kD protein assembles to
preformed desmosomes, and therefore, is not integral to des-
mosomal structure. However, the presence of 08L at the des-
mosome is correlated with the organization of keratin fila-
ment network. We speculate that this protein may be relevant
to desmosomal stability and involved in other functions of
the desmosome such as intermediate filament organization
and assembly.
Materials and Methods
Reagents
DME and suspension-MEM (S-MEM) were purchased from Gibco-BRL
(Grand Island, NY). RPMI medium, Hanks medium, and FCS, for growing
hybridoma ceils were purchased from Irvine Scientific (Santa Ana, CA).
FCS, used for culture of MDCK cells, was purchased from ICN Biomedi-
cals, Inc. (Costa Mesa, CA). PMSE leupeptin, chemostatin, and pepstatin
were purchased from Sigma Chemical Co. (St. Louis, MO). Unconjngated
and FITC-conjugated goat anti-mouse lgM were purchased from Chemicon
International, Inc. (Temecula, CA). mAb against pan-cytokeratin, FITC-
conjugated goat anti-mouse IgG, and IgM and peroxidase-conjugated goat
anti-mouse IgM were purchased from Boehringer-Mannheim Corp. (Indi-
anapolis, IN). Texas red-conjugated goat anti-rabbit IgG was purchased
from Cappel (Organon Teknika Corp., Durham, NC). Type I collagen was
purified from rat tail tendons as described by Dodson and Hay (1971). Rab-
bit polyclonal anti-desmoplakin antibody was kindly provided by Dr.
Manijeh Pasdar (University of Alberta in Edmonton, Canada).
mAb Production
MDCK cells grown in 150-ram 2 tissue culture flask were harvested with a
rubber policeman and lysed in 20 mM Tris, pH 7.4, 150 mM NaCI, 5 mM
EDTA containing 1% NP-40 for 60 rain at 4~ Detergent soluble fractions
were used as immunogen and mixed with complete Freunds adjuvant to in-
ject Balb/c mice. Subsequent to three or four boost injections, at 2-wk inter-
vals, mice were sacrificed and splenocytes fused with P3 myelorna cells,
similar to that described by K6hler and Milstein (1975). Hybridoma ceils
grown in HAT-containing RPMI medium were plated into 24-well dishes.
Superrmtants were screened by immunofluorescence microscopy of MDCK
cells cultured on collagen gels and dot-blot using whole cell protein extracts.
Cells of positive wells were selected and cloned by limited dilution. We thus
obtained a mAb, designated 08L, which was an IgM. For Western blot,
08L-IgM was concentrated with 40% ammonium sulfate followed by exten-
sive washing with 25 and 20% ammonium sulfate.
Purification of lgM
Goat anti-mouse IgM was coupled to CNBr activated Sepharose-4B ac-
cording to manufacturer's instructions (Pharmacia LKB Biotechnology,
Inc., Piscataway, NJ). The anti-immunoglobtdin-M (anti-IgM) Sepharose
affinity column was pre-equlibrated with 20 mM Tris, pH 8.0 (running
buffer), and washed sequentially with 0.1 M glycine, pH 2.6 (elution buffer),
20 mM Tris, pH 8.8, 0.1 M triethylamine, pH 11, and running buffer. 08L
hybridoma supernatant was filtered through a 0.2-~m filter before passing
to an anti-IgM column. The bound IgM was eluted from the column with
eltuion buffer, neutralized with 1 M Tris, pH 8.0, dialyzed extensively
against PBS, and stored at -200C.
Cell Culture
Cell cultures of the MDCK cell line of passage 10-60 (provided by Dr. Karl
Matlin, Harvard Medical School, Boston, MA), were maintained in DME
supplemented with 10% FCS, 2 mM glutamine, and 200 U/ml each of
streptomycin and penicillin G (normal calcium medium, NCM). Ceils were
passed with 0.1% trypsin and 0.04% EDTA in Hanks medium. For MDCK
cells cultured on collagen gel, a Lab-Tek culture chamber (Nunc Inc.,
Naperville, IL) precoated with type I collagen was used. For calcium switch
assays, confluent MDCK cells grown on coverslips in NCM were changed
to low calcium medium (LCM) for 2 d and then shifted back to NCM for
varied time points before immunostaining (see below). LCM had the same
formulation as NCM with the exceptions that calcium salts were omitted
by replacing DME with S-MEM and the FCS was depleted of divalent cat-
ion by using Chelex membrane (Bio-Rad, Richmond, CA).
Immunofluorescence Microscopy
MDCK cells grown to confluence on collagen gels were scraped from the
culture chamber with a razor blade. Samples were frozen and mounted in
Tissue Tek R OCT embedding medium (Miles Scientific, Naperviile, IL)
and 8-/~m sections were cut on a cryostat. Ceils on coverslips were permea-
bilized with acetone for 3 rain at -20~ Sectioned material and eoverslips
were washed briefly in PBS and incubated with 08L supernatant for 1 h at
room temperature. After three 5-rain washes in PBS, the samples were in-
cubated with FITC-conjugated goat anti-mouse IgM diluted 1:100 in PBS
for 1 h at room temperature. The samples were then washed extensively in
PBS, mounted with 50% glycerol containing 0.4% n-propylgallate and ex-
amined with a photo microscope (Carl Zeiss, Oberkoehen, Germany)
equipped with epifluorescence. Double immunofluorescence microscopy
used primary antibody mixtures of 08L supernatantJanti-desmoplakin (di-
luted 1:200), 08L/anti-cytokeratin (diluted 1:10) and anti-desmoplakin/anti-
cytokeratin.
Immunoelectron Microscopy
Purified 08L IgM was dialyzed extensively against 20 mM sodium borate,
pH 9.0, and coupled to 10 nm colloidal gold as described by Larsson (1988).
08L-conjugated colloidal gold was concentrated by centrifugation at 45,000
g for 1 h using a rotor (SW60; Beckman Instruments, Inc., Palo Alto, CA)
and resuspended in 20 rhM Tris, pH 7.6, containing 150 mM NaCI, 1%
BSA, and 0.02% sodium azide. For immunogold labeling, 17-d-old em-
bryonic chick corneal epithelia were detached from underlying stroma with
0.4 mg/ml EDTA, permeabilized with buffer containing 0.5 % Triton-X 100
for 30 rain at 4~ and incubated with 08L-conjugated colloidal gold for
1 h at 4~ Alternatively, MDCK cells cultured on coverslips were fixed
briefly in 1.5% formaldehyde, washed with PBS, permeabilized, and finally
incubated with 08L-gold. Subsequent to the 08L-gold incubation, the
epithelia and MDCK ceils were washed extensively with 20 mM Tris, 150
mM NaC1 containing 1% BSA and fixed with 2.5% glutaraldehyde, 2%
formaldehyde plus 1% picric acid buffered in 0.1 M eacodylate for 1 h. After
washing in 0.1 M caeodylate buffer, the samples were postfixed in 1% os-
mium tetroxide, en bloc stained with uranyl acetate, and prepared by routine
methods for EM. Thin sections were viewed on an electron microscope
(1200; JEOL USA, Peabody, MA).
Protein Extracts
MDCK cells in culture dishes were scraped with a rubber policeman and
homogenized briefly in either cytoskeleton (CSK) buffer (Fey et al., 1984;
10 mM Pipes, 300 mM sucrose, 150 mM NaC1, 3 mM MgCI2, 1 mM
EDTA, 0.1 mM DTT and 0.5% Triton-X 100, pH 6.8, 1 mM PMSF, and
1 t~g/mi each of pepstatin, leupeptin, and chemostatin) or CSK butter con-
taining 1.5 M KCI. Cells were lysed on ice for 30 min followed by centrifu-
gation for 30 rain at 10,000 g at 4oc. The supernatants and pellets were sub-
jected to SDS-PAGE and Western blotting.
SDS-PAGE and Immunoblot
Protein extracts as described above were resuspended in sample buffer and
The Journal of Cell Biology, Volume 118, 1992 1478
electrophoresed on 6% polyacrylamide gels according to the system of
Laemmli (1970). Immunoblot analysis was performed as described by Tow-
bin ct ai. (1979) with a few modifications. Proteins were transferred from
the gel to nitrocellulose paper (Sehleicher & Schuell, Keene, Nil). The pa-
per was blocked with 5% non-fat dried milk in PBS followed by washing
in PBS. Primary antibody incubations were carried out with 08L concen-
trated by ammonium sulfate for 1 h at room temperature. After extensive
washing in PBS, the paper was incubated for 1 h at room temperature with
HRP conjugated goat anti-mouse IgM (1:1000 in PBS). The peroxidase-
labeled blots were reacted with 0.5 mg/ml diaminobenzidine in PBS and
color reactions were developed using 0.01% hydrogen peroxide.
Differential KC1 Extraction
A series of confluent MDCK cells, cultured in 60-ram cultured dishes, were
extracted in situ with CSK buffer containing different concentrations of KCl
(0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 M) at 4 *C for 30 rain. After KCI extrac-
tion, cellular remnants remaining in the culture dishes were boiled in sam-
ple buffer and collected. Both the KCI extracts and salt-insoluble material
were subjected to SDS-PAGE and immunoblot as described above. In paral-
lel, MDCK cells, cultured on covet'slips coated with either polyornithine
or ConA, were extracted with KC1, as above, and processed for im-
munofluorescence with 08L.
Results
The mAb, 08L, which was derived from ongoing studies on
epithelial adhesion, presented an intriguing lateral epithelial
boundary staining pattern. Immunofluorescence microscopy
of permeabilized monolayer cultures of densely grown
MDCK cells revealed the distribution of 08L antigen along
cell boundaries corresponding to cell-cell contact regions
(Fig. 1 a). The epitope appeared to reside on or near the cy-
toplasmic face of the lateral membrane. Free cell boundaries
along the margins of epithelial colonies exhibited no stain-
ing. No staining of nuclear envelope, Golgi complexes, or
other distinct cytoplasmic structures was seen (Fig. 1, a and
b). Sections of MDCK cells grown on collagen gels demon-
strated the 08L antigen to be localized to the lateral borders
of the cells with no apical or basal staining (Fig. 1 b).
The mAb, 08L, stained stratified epithelium as well as
simple epithelium of numerous species. Immunostaining of
rat stomach revealed that 08L was primarily localized to the
lateral surface of epithelial cells, especially evident at lateral
cell boundaries near the junctional region (Fig. 1 c). Sec-
tions of rat liver (Fig. 1 d) stained with 08L demonstrated
staining along the lateral plasmalemma of hepatocytes and
presented a pattern parallel to the course of bile canaliculi.
Strong immunofluorescence was also observed along epithe-
lial lateral boundaries in the bile duct (Fig. 1 d). Immuno-
fluorescence of rat corneal epithelium clearly demonstrated
08L antigen at the lateral cell boundaries of basal ceils and
lateral surfaces of the stratifying wing cells (Fig. 1 e).
Epidermis stained with 08L demonstrated staining of the
lateral surface of all cell layers except the stratum corneum
(Fig. 1 f). Interestingly, 08L did not immunostaln the basal
epithelial surfaces of corneal epithelium or epidermal cells,
both of which contained well formed hemidesmosomes. In
all tissues surveyed, we found no 08L staining in connective
tissues, such as lamina propria of the gut, corneal stroma,
or dermis. In addition, endothelium, smooth muscle, and
nervous tissue appeared negative for 08L staining.
Immunoelectron microscopy revealed the 08L antigen to
be associated with the cytosolic face of desmosomes. Chick
corneal epithelia incubated with colloidal gold-conjugated
08L subsequent to detergent permeabilization (Fig. 2, a, b,
and d) and MDCK cells which were fixed and then detergent
permeabilized and incubated in 08L-gold (Fig. 2 c) exhibited
similar immunostalning patterns. The 08L antigen was lo-
calized prim~'fly to the periphery of desmosomes and to in-
termediate filaments immediately adjacent to the desmo-
some (Fig. 2). Gold particles did not appear to be associated
with desmosome plaque proper, even in areas where the
plaque appeared clearly accessible, rather they were as-
sociated with amorphous material adjacent to the plaque and
with IFs in the immediate vicinity of desmosomes. Samples,
fixed before immunogold labeling, demonstrated a better
preserved cytoarchitecture and a more dense IF meshwork.
The gold labeling was still restricted to the areas immedi-
ately adjacent to the desmosome, indicating that the immu-
noloeations in the extensively extracted epithelia did not re-
sult from a collapsing of 08L onto the desmosome. This EM
distribution of 08L antigen suggests that 08L may recognize
a novel component of the IF-desmosome complex.
The 08L antibody, when used to immunostaln Western
blots of extracts from confluent MDCK cells, revealed a
prominent immunoreactive band with a relative molecular
mass of 140,000 (Fig. 3, lanes 9-1/). The 08L antigen dis-
played the same mobility on SDS-PAGE under reducing and
non-reducing conditions. This 140-kD protein was not pres-
ent in the Triton-X 100 soluble fraction from MDCK cells
(Fig. 3, lane 8), but was observed in the detergent-resistant
fraction (Fig. 3, lane 9). However, the 140-kD detergent-
resistant protein could be extracted from the CSK-insoluble
pellet (Fig. 3, lane
10)
or directly from lysed cells (Fig. 3,
lane 1/) with buffers containing KC1 (Fig. 3, lane
10 and 11),
leaving a small residual amount of 08L protein in the KC1
insoluble pellet (Fig. 3, lane
12).
The solubility of 08L antigen in KCI was examined in
more detail by differential salt extraction combined with im-
munoblot and immunofluorescence analyses. 08L antigen
was soluble at concentrations greater than 0.4 M KC1 (Fig.
4 A). A small, but significant, portion remained insoluble
even after 1.5 M KC1 extraction. The immunofluorescence
analyses supported the immunoblot profile with the dimin-
ishing of immunostalning with 08L following 0.4 M KC1 ex-
traction and the appearance of weak residual staining on the
MDCK lateral cell surfaces even subsequent to 1.5 M KC1
extraction (Fig. 4 B). MDCK cell cultures extracted with
KC1 also revealed weaker 160-170-kD immunoreactive
bands. At this time, it is not clear how these larger polypep-
tides are related to the 140-kD 08L polypeptide.
Under normal physiological conditions, 08L antigen was
seen to co-localize with desmoplakin on densely grown
MDCK cells. Immunostaining of low confluent cultures of
MDCK cells with 08L and anti-desmoplakin suggested that
08L assembled onto the lateral margins subsequent to des-
moplakin. Immunostaining of single cells demonstrated 08L
and desmoplakin to be intracellular with little or no co-
distribution (Fig. 5, a and a'). Desmoplakin staining was
concentrated to the paranuclear area. Staining of small cell
clusters revealed that desmoplakin assembled to the cell-ceU
contact surfaces before 08L (Fig. 5, c and c'). The larger
clusters exhibited extensive 08L-desmoplaldn co-aligument,
similar to that seen on confluent cultures (Fig. 5, d and d').
Taking advantage of the observation that the structural in-
tegrity of desmosomes is affected by extracellular calcium
(Hennings et al., 1980), we cultured MDCK cells at high
density in low calcium medium (LCM) for 2 d. Subse-
Ouyang and Sugrue
Identification of a New Desmosomal Protein
1479
Figure L
Confluent culture of MDCK cells stained with 08L, using routine indirect immunostaining procedures (a) revealed strong staining
for 08L along the cell boundaries, corresponding to cell-ceU contact sites, with a characteristic pattern of double parallel interrupted lines.
Note the apparent negative staining of 08L at cell margins with no cell-cell contact. MDCK cells cultured on collagen grown to confluence,
cut perpendicular to the plane of substratum (b) showed 08L staining restricted to a linear pattern at the lateral cell boundary. Little or
no staining was seen intracellularly or associated with apical or basal surfaces. Sectioned rat stomach (c) showed that 08L antigen was
localized to the lateral surface of epithelium, while the cells of connective tissues displayed little or no reactivity. Rat liver stained with
08L (d) demonstrated staining along the lateral surface of hepatocytes and presented a pattern representative of the course of bile canaliculi
(small arrows).
Bile duct epithelium also stained with 08L antibody (/arge arrow), whereas, the hepatic endothelium did not stain
(trian-
gles).
Rat cornea (e) and rabbit skin (f) showed epithelial staining for 08L, however, underlying connective tissue is not stained. Note
lateral epithelial surfaces stained with 08L whereas the basal surfaces of basal epithelial cells which contain numerous hemidesmosome
showed no immunoreactivity for 08L.
The Journal of Cell Biology, Volume 118, 1992 1480
Figure 2. Chick corneal epithelia (17 d, a, b, and d) and MDCK cells (c) fixed with 1.5% formaldehyde, were permeabilized with 0.5%
Triton-X 100. Subsequent to incubation with 10 nm gold-08L conjugates, the samples were processed for routine EM. Gold particles
can readily be seen in the region of desmosomes. Gold particles can be seen to decorate amorphous material on the cytoplasmic face of
desmosomes and the intermediate filaments immediately adjacent to the desmosomes (arrows), however, the desmosomal plaque proper
exhibited little or no staining. In addition, intermediate filaments distant from the desmosome complex bound little or no 08L-gold (d).
The survey micrograph shown in d demonstrates that 08L was localized only to areas in the vicinity of desmosomes, not to other cytosolic
structures. Inset in d demonstrates higher magnification of a desmosome decorated with 08L-gold.
Ouyang and Sugrue Identification of a New Desmosomal Protein 14.81
Figure 3.
Confluent MDCK cells were extracted with Triton-X 100
(lanes 2, 3, 8, and 9), Triton-X 100 and 1.5 M KCI (lanes
5, 6, 11,
and
12),
and successively with Triton-X 100 then 1.5 M KCI (lanes
4 and
10).
Lanes 2-6 contain Coomassie blue-stained SDS-PAGE
and lanes
8-12
demonstrate corresponding immunoblots. Subse-
quent to the extractions, supematants and insoluble materials were
resolved on SDS-PAGE and immunoblotted. The 08L antigen pre-
sented as a 140-kD protein. 08L antigen was found in the Triton-
insoluble fractions (lanes 3 and 9), little or no 08L antigen was rec-
ognized in Triton-soluble fractions (lanes 2 and 8). KCI extraction
of the Triton insoluble material released the 08L antigen (lanes 4
and 10).
The 08L antigen was detected in the soluble fractions of
MDCK extracted simultaneously with Triton-X 100 and KC1 (lanes
5 and
11).
The corresponding reduction of insoluble 08L can be
seen in lanes 6 and
12.
quently, cultures were switched to NCM and the distribu-
tions of 08L antigen and desmoplakin were examined at var-
ied time points. Double immunofluorescence microscopy
showed that in LCM, both 08L antigen and desmoplakin
were distributed intracellularly. The 08L staining appeared
more punctate than that of desmoplakin (Fig. 6, a and a'),
but they did not appear to co-localize. Upon shift to NCM,
desmoplakin staining appeared at the lateral cell boundary
as early as 15 min, exhibiting strong staining within 2 h (Fig.
6 b'). Significant levels of staining for 08L was not observed
until after 5 h (Fig. 6 c). With longer incubation times, the
cytoplasmic staining of 08L gradually diminished with a
corresponding increase in peripheral staining. 4-d cultures
in NCM showed that 08L staining on MDCK cells was re-
stricted to lateral boundary with little or no cytoplasmic
staining (as shown in Fig. 1). These data are suggestive that
08L may assemble to the vicinity of the desmosome after
desmoplakin can be detected and after morphologically
recognizable desmosomes can be observed (Mattey and Gar-
rod, 1986a). Whether or not these early junctions, which
lack 08L, represent mature and stable desmosome structures
still remains to be determined.
The calcium switch assays also suggest that 08L may exist
either complexed to desmosomes or in punctate locations
within the cytoplasm. We next examined the solubility state
of 08L during the transition from LCM to NCM conditions.
There was no change in the relative abundance of accumu-
lated 08L protein in soluble versus insoluble pools, with the
majority of 08L found in the Triton-insoluble material (Fig.
7). While these data do not rule out the possibility that 08L
Figure 4.
MDCK cells cultured in dishes
(A) or on coverslips (B) were extracted
with CSK with different concentrations
of KCI for 30 min at 4~ KCI extracts
(A, top)
or KCI insoluble material (A,
bottom)
were boiled with sample buffer
and subjected to SDS-PAGE and immu-
noblotted. Ceils on coverslips after ex-
traction as described above were fixed
and permeabilized with acetone and
analyzed by immunofluorescence mi-
croscopy as described previously (B).
The 08L antigen was extracted in buffers
containing KCI > 0.4 M, and the amount
of 08L antigen remaining in the KCI in-
soluble pellet decreased accordingly.
Immtmofluorescence microscopy showed
that 08L intensity decreased dramat-
ically upon extraction with 0.4 M KCI,
compared with those extracted with
0.2 M KC1.
The Journal of Cell Biology, Volume 118, 1992 1482
Figure 5. Double immunostaln-
ing of low confluence cultures of
MDCK cells with 08L antibodies
(a, b, c, and d) and anti-desmo-
plakin (a', b', c', and d') demon-
strated that, in isolated cells, both
08L (a) and desmoplakin (a')
were distributed intracellularly,
however, they exhibited little or
no co-distribution. Desmoplakin
was seen within aggregates in a
paranuclear location. In two cell
aggregates, 08L (b) appeared pre-
dominantly as intracellular punc-
tate staining with limited staining
along cell-cell contact surfaces,
whereas, desmoplakin (b') was
mostly seen assembled along the
lateral contacts. In the larger is-
lands of cells, 08L was seen to co-
distribute with desmoplakin at
the lateral surfaces with dimin-
ished cytosolic staining (c, c', d,
and d').
may exist in the soluble pool with a very short half-life, these
data do suggest that 08L may exist intracellularly in an in-
soluble state. Further in depth biochemical analyses, such as
gel filtration and/or sucrose gradient centrifugation of pulse
chased material are required to address the solubility state
of 08L and associated molecules during desmosome as-
sembly.
Further investigation into the timing of assembly of 08L,
desmoplakin, and cytokeratin was carried out. In low cal-
cium conditions the staining pattern of 08L did not seem to
co-align with that of cytokeratin (Fig. 8, a and a'). However,
at this resolution it is difficult to absolutely exclude the possi-
bility of 08L cytokeratin co-distribution. Desmoplakin,
however, showed some, albeit limited, co-distribution with
Ouyang and Sugrue
Identification of a New Desmosomal Protein
1483
The Journal of Cell Biology, Volume 118, 1992 1484
Figure 7.
MDCK cells cultured in low calcium medium then trans-
ferred to NCM for 0, 2, 6, and 20 h were extracted with Triton-X
100 containing buffers. Subsequent to extraction, Triton-X 100
soluble material (s) and insoluble pellet (p) were analyzed by West-
ern blots with 08L antibody. 08L was found predominantly in the
Triton insoluble material.
perinuclear keratin whorls (Fig. 8, d and d',
arrows).
Within
2 h after the switch to normal calcium levels, desmoplakin
was seen along cell-cell contact surfaces, whereas 08L
staining of lateral surface was limited (Fig. 8, b and e). Kera-
tin filament immunostaining at 2 h post-switch revealed rela-
tively little organization to the cell boundaries. However,
when cytokeratin filaments were seen to be organized per-
pendicularly to lateral surfaces, immunostaining for 08L
could be detected at the cell margin (Fig. 8, b and b'). The
cell margin immunostaining pattern for desmoplakin, how-
ever, did not seem to correlate with perpendicularly ar-
ranged cytokeratin filaments (Fig. 8, e and e'). While most
sites of cell-cell contact decorate with desmoplakin staining,
the majority of the desmoplakin staining did not appear to
correlate with anchorage for perpendicularly arranged inter-
mediate filaments. Cells immunostained 5 h after switching
revealed more 08L assembled to lateral surface and exten-
sive perpendicularly organized keratin filament bundles
(Fig. 8, c and c'). While these data tempt one to suggest a
correlation between the presence of 08L to the lateral cell
boundary and the higher organization of the keratin cytoar-
chitecture, more in depth studies including immunogold EM
will be necessary to confirm the role of 08L in the organiza-
tion of the epithelial cytokeratin scaffold.
Discussion
Cell-cell adhesion is widely believed to be a fundamental
process in the development of multicellular organisms. Junc-
tions of the adherens type are of particular interest and inte-
gral to our understanding of cell and tissue biology, because
they play a role in not only adhesion of epithelial cells but
also in the organization of the epithelial cytoskeleton (Cowin
et al., 1985a; Geiger and Ginsberg, 1991; Bologna et al.,
1986). In this work, we present data regarding the
identification of a novel protein Mr = 140,000, which is
found within most, if not all, epithelial ceils and is related
to the macula adherens junction (desmosome). Double stain-
ing with desmoplakin antibodies localized 08L to, or near,
the desmosome. Electron microscopic analyses of MDCK
cells and corneal epithelia confirmed the 08L protein loca-
tion to the cytoplasmic face of desmosomes.
The known molecular components of the desmosome
proper have been examined in much detail (for reviews see
Cowin et al. 1985a; Schwartz et al., 1990; Garrod et al.,
1990; Jones and Green, 1991). The 08L protein does not
seem to be identical to any known desmosomal proteins.
However, there have indeed been reports of desmosome as-
sociated proteins with relative molecular masses around
140,000. These include Mr = 125-140,000 cell surface gly-
coproteins which may be cell adhesion molecules (Jones et
al., 1986; Jones, 1988). In addition, Cartaud and co-workers
(1990) have described a 140-kD desmosomal protein which
binds intermediate filaments and is antigenically related to
lamin B. Although the 08L protein described here may be
similar or related to the protein reported by Cartaud et al.
(1990), the 08L protein, unlike the lamin B-related protein,
does not localize to the desmosomal plaque proper. Also, the
140-kD lamin B-related protein has a distinctly acidic PI of
5.4 with no indication of isoforms. The 08L protein displays
isoforms from PI 5.9-6.2 (data not shown). While our
studies suggest that 08L protein and the 140-kD protein de-
scribed by Cartaud and colleagues (1990) may be distinct,
definitive resolution of this point awaits more direct compari-
sons and/or primary sequence analysis.
The location of the 08L protein at the interface between
the desmosome proper and IFs may be suggestive that 08L
is involved in the interaction between IFs and the desmo-
Figure 6.
MDCK cells cultured in LCM, after 2 d, were shifted to NCM. Cells were double immuno-stained with 08L (a, b, c, and d)
and anti-desmoplakin (a', b', c', and d') after 0, 2, 5, 20 h of incubation in NCM. MDCK cells in LCM exhibited no obvious cell-cell
contacts and 08L and desmoplakin were distributed intracellularly (a and a', respectively), with little or no co-distribution. After shifting
to NCM, staining for desmoplakin can be seen at the lateral cell surface within 2 h (b'). The 08L antigen, showed limited cell margin
staining
(b, arrows)
until around 5 h (c). After 20 h in NCM both 08L and desmoplakin can be located along the lateral cell margins
with decreased cytosolic staining (d and d').
Ouyang and Sugrue
Identification of a New Desmosomal Protein
1485
The Journal of Cell Biology, Volume 118, 1992 1486
some. Thus far, a number of desmosomal components have
been described as putative linker molecules connecting IF to
the desmosome. For example, desmoplakin I and II are com-
ponents of the desmosomal plaque and have been shown to
associate with IF during desmosome formation and assem-
bly (Mueller and Franke, 1983; Jones and Goldman, 1985).
In addition, sequence information of desmoplakin predicts
that the carboxyl terminus consists of a globular domain with
basic and acidic residue periodicity complementary to the
rod domain of IFs (Green et al., 1990). However, studies
using a variety of in vitro assays have been unable to demon-
strate direct binding of desmoplakin to IF (O'Keefe et al.,
1989). It has been suggested that desmoplakin-IF binding
may require the involvement of additional accessory proteins
(Jones and Green, 1991; Stappenbeck and Green, 1992).
To gain insight into the 08L-antigen association with the
desmosome, we chose to examine desmosome-IF complex
assembly. In low calcium conditions both desmoplakin and
08L antigen were localized to the cytosol with little or no co-
alignment. After changing cells from low calcium to nor-
mal calcium medium, desmoplakins were seen to assemble
to cell-cell boundaries significantly before the 08L protein.
The timing of desmosome reformation after Ca 2§ switch in
our system, was consistent with that reported in previous
studies (Mattey and Garrod, 1986a). We have also found that
as MDCK cells advanced from low density to higher density,
the temporal sequence of assembly of 08L protein and des-
moplakin to the cell-cell contact boundaries was the same
as seen in the Ca 2+ switch assays. These data are suggestive
that the assembly of 08L protein to the lateral cell boundary
occurs subsequent to desmosome formation. Therefore, we
suggest that the 08L antigen most likely does not function as
the primary IF-desmosome linker but may be involved in the
assembly and/or organization of the intermediate filament
network.
The assembly of desmosomes by cultured epithelial cells
has been extensively studied (Overton, 1962; Lentz and
Trinkhaus, 1971; Jones and Goldman, 1985; Mattey and
Garrod, 1986a; Duden and Franke, 1988; Pasdar and Nel-
son, 1988a,b, 1989; Pasdar et al., 1991). It is clear that cer-
tain epithelial systems, such as the MDCK cell line, cells in
low calcium conditions (LCM) possess all the requisite com-
ponents for desmosome formation (Mattey and Garrod,
1986a; Pasdar and Nelson, 1988a, 1989). So that, when the
calcium level is restored there is a rapid assembly of well
formed desmosomes. We have shown that in low calcium
conditions, 08L and desmoplakin may be sequestered in dis-
tinct locations within the cytosol similar to the previous ob-
servations on desmoglein and desmoplakin (Pasdar et al.,
1991).
Mattey and Garrod (1986b) have described the formation
of stable desmosomal linkages in MDCK cells, They showed
that although morphologically "mature" desmosomes may be
established within the first hour in normal calcium concen-
trations, these desmosomes remain sensitive to low Ca 2+
conditions for some time. They suggest that additional acces-
sory desmosomal components reinforce the desmosome and
result in a stable, low Ca 2+ insensitive, epithelial adhesion.
The recruitment of 08L protein to the lateral membrane was
correlated with a more ordered array of the intermediate fila-
ment associating with desmosomes. It is tempting to specu-
late that the 08L protein may somehow contribute to the sta-
bility of the desmosomal junction.
Cycloheximide treatment does not interfere with 08L as-
sembly to the desmosome-IF complex, indicating de novo
protein synthesis is not required and that functional 08L anti-
gen may exist in a cytosolic pool in low calcium condition
(data not shown). The gross solubility of 08L protein does
not appear to change in cells under low or normal calcium
conditions, suggesting that 08L antigen is aggregated or
complexed with other cytosolic proteins. However, we have
shown that 08L does not co-localize with desmoplakin nor
cytokeratin under these conditions. Therefore, although 08L
is eventually recruited to the desmosome, it is not located in
preformed association with desmoplakin.
In this paper, we identify a novel protein localized to the
region of the IF-desmosome complex on the lateral epithe-
liai surface. This protein is not identical to any desmosomal
proteins nor to known IFs associated proteins. Based on its
location between desmosomal plaque and associated bundles
of IFs, we postulate that this molecule may be involved in the
association of IFs to the desmosome structure. Identification
of such molecules will contribute to our current understand-
ing of the functions of the IF-desmosome complex in epithe-
lial adhesion, and the role of specific cell surface domains
such as desmosomes in the integration and organization of
epithelial cytoskeleton.
This work was supported by grant R01 EY07883 from the National Insti-
rates of Health and March of Dimes Birth Defect Foundation award
1-1228.
Received for publication 28 January 1992.
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