Proc. Natl. Acad. Sci. USA
Vol. 93, pp. 7701-7705, July 1996
Distinct desmocollin isoforms occur in the same desmosomes and
show reciprocally graded distributions in bovine nasal epidermis
(immunogold labeling/polyvinyl alcohol embedding/epidermal differentiation)
ALISON J. NORTH*, MARTYN A. J. CHIDGEY, JONATHAN P. CLARKE, WILLIAM G. BARDSLEY, AND DAVID R. GARROD
Epithelial Morphogenesis Research Group, School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Road,
Manchester M13 9PT, United Kingdom
Communicated by John Gurdon, Wellcome CRC Institute, Cambridge, United Kingdom, March 26, 1996 (received for review January 19, 1996)
ABSTRACT The adhesive core of the desmosome is com-
posed of cadherin-like glycoproteins of two families, desmo-
collins and desmogleins. Three isoforms of each are expressed
in a tissue-specific and developmentally regulated pattern. In
bovine nasal epidermis, the three desmocollin (Dsc) isoforms
are expressed in overlapping domains; Dsc3 expression is
strongest in the basal layer, while Dsc2 and Dscl are strongly
expressed in the suprabasal layers. Herein we have investi-
gated whether different isoforms are assembled into the same
or distinct desmosomes by performing double immunogold
labeling using isoform-specific antibodies directed against
Dscl and Dsc3. The results show that individual desmosomes
harbor both isoforms in regions where their expression ter-
ritories overlap. Quantification showed that the ratio of the
proteins in each desmosome altered gradually from basal to
immediately suprabasal and upper suprabasal layers, labeling
for Dscl increasing and Dsc3 decreasing. Thus desmosomes
are constantly modified as cells move up the epidermis, with
continuing turnover of the desmosomal glycoproteins. Statis-
tical analysis of the quantitative data showed a possible
relationship between the distributions of the two isoforms.
This gradual change in desmosomal composition may consti-
tute a vertical adhesive gradient within the epidermis, having
important consequences for cell positioning and differentiation.
The major desmosomal glycoproteins, termed the desmocol-
lins and desmogleins, are members of the cadherin superfamily
of calcium-dependent adhesion molecules (1, 2). Each is
represented by three isoforms, the products of different genes,
so that they form distinct cadherin subfamilies (3-9). These
isoforms show tissue-specific expression (6, 7, 10). They also
show distinct patterns of expression in epidermis and other
stratified epithelia (6, 9, 11-13), suggesting an important role
in epithelial differentiation.
Our recent studies have focused on the expression of
desmocollins (Dsc) 1, 2, and 3 in bovine tissues (5, 6, 9). We
have shown that Dscl (6, 14) is expressed in terminally
differentiating cells of epidermis and tongue epithelium, while
Dsc2 (6, 15) is ubiquitously expressed in desmosome-bearing
tissues, and Dsc3 (9) is expressed most strongly in the basal-
most layers of stratified epithelia. Reverse transcription-
coupled PCR studies suggest a similar ubiquitous tissue dis-
tribution for murine Dsc2 (ref. 16 and unpublished data), while
Northern blot analysis suggests that the tissue distributions for
all three human desmocollins resemble those found in bovine
tissues (10). It has also been shown (17, 18) that the Dsc2
message is upregulated at the 16-cell stage of murine devel-
opment, apparently contributing to the regulation of glycoprotein
synthesis and initial desmosome assembly in the morula.
In bovine nasal epidermis, Dscl is expressed throughout the
spinous layer (in situ hybridization and immunofluorescent
staining), expression ceasing in the granular layer. Dscl ex-
pression generally begins in the first layer of suprabasal cells.
However, at the bottoms of the deep rete ridges, 5-10 cell
layers appear not to express Dscl. Dsc2 (in situ hybridization
only) shows roughly the same expression territory as Dscl but
is most strongly expressed at the bases of the rete ridges in
those cell layers lacking Dscl expression: we cannot be certain
whether it is expressed in the basal layer. Dsc3 is strongly
expressed basally (in situ hybridization and immunofluores-
cence), expression gradually fading toward the mid-spinous
layers (6, 9).
Desmocollin isoform expression clearly overlaps in the
epidermis. By contrast the keratin intermediate filament pro-
teins that are linked to desmosomal plaques show nonover-
lapping distributions (19, 20). The basal keratins, keratin (K)
5 and K14, are strongly expressed only in the basal cell layer
whereas Ki and K10 are expressed only suprabasally.
The expression patterns of desmocollins raises intriguing
questions regarding their role in epidermal differentiation and
stratification (5, 9). Herein we ask how desmocollin isoforms
are distributed between desmosomes (i) within the same cell
and (ii) at different levels in the epidermis. Three possible
distributions for two desmocollin isoforms between junctions
within the same cell (Fig. 1 A-C) are restriction to distinct
junctions, regional restriction within the same junction, or
mixing within the same junction, respectively.
We raised a rabbit polyclonal antibody specific for Dscl that,
with our previously described mouse monoclonal antibody to
Dsc3 (9), permitted double immunogold labeling of ultrathin
sections. We show that the two desmocollin isoforms are mixed
within individual desmosomes. Further, the isoforms show
reciprocal graded distributions with depth in the epidermis,
Dscl increasing suprabasally and Dsc3 decreasing. These
discoveries have profound implications for epidermal stratifi-
cation and differentiation.
MATERIALS AND METHODS
Preparation of a Polyclonal Dscl-Specific Antiserum. An
expression plasmid, pGEXDscEC, encoding a 1.1-kb fragment
of the bovine Dscl extracellular domain linked to the gluta-
thione S-transferase (GST) gene was constructed using clone
CN35 (14), encoding full-length bovine Dsclb (Fig. 24), as
starting material. DNA encoding the transmembrane and
cytoplasmic domains was deleted by site-directed mutagenesis.
The resulting construct was cut with Aflll and BsmI, briefly
digested with Si nuclease, and ligated. A clone containing an
in-frame fusion was cut with Narl, blunt-ended with the
Klenow fragment ofDNA polymerase I, and cut with Sall. The
NarI(blunt-end)-SalI fragment (solid boxes; Fig. 2A) was then
Abbreviations: Dsc, desmocollin; LPD, linear particle density; K,
keratin; GST, glutathione S-transferase; PVA, polyvinyl alcohol.
*To whom reprint requests should be addressed.
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Proc. Natl. Acad. Sci. USA 93 (1996)
0 060 p.....000
*@000@ * 000
(B) Distinct territories
*00 0 0 0--
Nar I Af
PRE PRO EC
III Bsm I
FIG. 1. Scheme depicting possible organization of desmocollin
isoforms in desmosomes. (A) Isoforms are localized in distinct des-
mosomes within the same cell. (B) Multiple isoforms are located
within the same desmosomes but restricted to distinct domains. (C)
Multiple isoforms are mixed along each desmosome.
cloned into the SmaI and Sall sites ofpGEX-4T-3 (Pharmacia)
in-frame with the GST gene to produce construct pGEXDscEC.
Plasmid pGEXDscEC was transformed into Escherichia coli
XL1-blue. GST-Dscl fusion protein expression was induced
for 3 h at 28°C with 1 mM isopropyl ,B-D-thiogalactoside.
Bacteria were sonicated, and the fusion protein was affinity-
purified using glutathione-Sepharose 4B (Pharmacia). The
Dsc moiety (apparent Mr 50,000) was recovered by digestion
with bovine thrombin (Pharmacia; 10 units/ml of beads for
16 h at 22°C) and used to generate rabbit antiserum JCMC.
Antibody JCMC was affinity-purified using the purified 50-
kDa protein immobilized on CNBr-activated Sepharose 4B
Immunoblot Analysis. Construction of plasmids which en-
code full-length Dsclb (pGEX3X/Dsclb), Dsc2b (pGEX4T/
Dsc2b), and Dsc3b (pGEX2T/Dsc3b) linked to GST has been
reported (9). Fusion protein production and immunoblot
analysis were carried out as described (9).
Immunofluorescence. Cryostat sections (7 ,um) of bovine
nasal epidermis were stained as described (9), using JCMC
(against Dscl), affinity-purified, and applied at 2.3 ,tg/ml in
neat supernatant of monoclonal antibody 07-4G (against
Dsc3; ref. 9), followed by fluorescein isothiocyanate-
conjugated goat anti-mouse IgG (Sigma) and Cy3-conjugated
donkey anti-rabbit IgG (The Jackson Laboratory).
Immunoelectron Microscopy. Small pieces ( 1 mm3) of
bovine nasal epidermis were fixed in methanol for 1 h at 4°C
and then 1 h at room temperature. After washes in 200 mM
Hepes buffer (pH 7.3), the tissue was infused with 20%
aqueous polyvinyl alcohol (PVA; refs. 22 and 23) and left at
room temperature to harden (minimum 3 weeks). Ultrathin
sections (80-110 nm) were floated onto a boat of87% glycerol,
retrieved on Formvar-coated nickel grids, and incubated on
PBS at 4°C overnight to extract the PVA.
Immunolabeling was performed as described (23), using the
same primary antibodies used for immunofluorescence fol-
lowed by 10-nm gold-conjugated goat anti-rabbit IgG plus
5-nm gold-conjugated goat anti-mouse IgG (Biocell La