Distinct desmocollinisoforms occurin the same desmosomes ... Distinct desmocollinisoforms occurin...
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7701-7705, July 1996 Cell Biology
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)
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(A) Distinct desmosomes
(B) Distinct territories within desmosomes
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Nar I Af
PRE PRO EC
III Bsm I
zI1I TMIY-TM CYT
-116 - 97
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 (Pharmacia). 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