Distinct desmocollinisoforms occurin the same desmosomes ... · Distinct desmocollinisoforms...

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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 of DNA 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. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7701 Downloaded by guest on June 29, 2020

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Page 1: Distinct desmocollinisoforms occurin the same desmosomes ... · Distinct desmocollinisoforms occurin the samedesmosomesand show reciprocally graded distributions in bovinenasalepidermis

Proc. Natl. Acad. Sci. USAVol. 93, pp. 7701-7705, July 1996Cell Biology

Distinct desmocollin isoforms occur in the same desmosomes andshow 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. GARRODEpithelial 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 expressedin a tissue-specific and developmentally regulated pattern. Inbovine nasal epidermis, the three desmocollin (Dsc) isoformsare expressed in overlapping domains; Dsc3 expression isstrongest in the basal layer, while Dsc2 and Dscl are stronglyexpressed in the suprabasal layers. Herein we have investi-gated whether different isoforms are assembled into the sameor distinct desmosomes by performing double immunogoldlabeling using isoform-specific antibodies directed againstDscl and Dsc3. The results show that individual desmosomesharbor both isoforms in regions where their expression ter-ritories overlap. Quantification showed that the ratio of theproteins in each desmosome altered gradually from basal toimmediately suprabasal and upper suprabasal layers, labelingfor Dscl increasing and Dsc3 decreasing. Thus desmosomesare constantly modified as cells move up the epidermis, withcontinuing turnover of the desmosomal glycoproteins. Statis-tical analysis of the quantitative data showed a possiblerelationship between the distributions of the two isoforms.This gradual change in desmosomal composition may consti-tute a vertical adhesive gradient within the epidermis, havingimportant consequences for cell positioning and differentiation.

The major desmosomal glycoproteins, termed the desmocol-lins and desmogleins, are members of the cadherin superfamilyof calcium-dependent adhesion molecules (1, 2). Each isrepresented by three isoforms, the products of different genes,so that they form distinct cadherin subfamilies (3-9). Theseisoforms show tissue-specific expression (6, 7, 10). They alsoshow distinct patterns of expression in epidermis and otherstratified epithelia (6, 9, 11-13), suggesting an important rolein epithelial differentiation.Our recent studies have focused on the expression of

desmocollins (Dsc) 1, 2, and 3 in bovine tissues (5, 6, 9). Wehave shown that Dscl (6, 14) is expressed in terminallydifferentiating cells of epidermis and tongue epithelium, whileDsc2 (6, 15) is ubiquitously expressed in desmosome-bearingtissues, 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), whileNorthern blot analysis suggests that the tissue distributions forall three human desmocollins resemble those found in bovinetissues (10). It has also been shown (17, 18) that the Dsc2message is upregulated at the 16-cell stage of murine devel-opment, apparently contributing to the regulation of glycoproteinsynthesis and initial desmosome assembly in the morula.

In bovine nasal epidermis, Dscl is expressed throughout thespinous 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 celllayers appear not to express Dscl. Dsc2 (in situ hybridizationonly) shows roughly the same expression territory as Dscl butis most strongly expressed at the bases of the rete ridges inthose cell layers lacking Dscl expression: we cannot be certainwhether it is expressed in the basal layer. Dsc3 is stronglyexpressed basally (in situ hybridization and immunofluores-cence), expression gradually fading toward the mid-spinouslayers (6, 9).

Desmocollin isoform expression clearly overlaps in theepidermis. 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 layerwhereas Ki and K10 are expressed only suprabasally.The expression patterns of desmocollins raises intriguing

questions regarding their role in epidermal differentiation andstratification (5, 9). Herein we ask how desmocollin isoformsare distributed between desmosomes (i) within the same celland (ii) at different levels in the epidermis. Three possibledistributions for two desmocollin isoforms between junctionswithin the same cell (Fig. 1 A-C) are restriction to distinctjunctions, regional restriction within the same junction, ormixing within the same junction, respectively.We raised a rabbit polyclonal antibody specific for Dscl that,

with our previously described mouse monoclonal antibody toDsc3 (9), permitted double immunogold labeling of ultrathinsections. We show that the two desmocollin isoforms are mixedwithin individual desmosomes. Further, the isoforms showreciprocal graded distributions with depth in the epidermis,Dscl increasing suprabasally and Dsc3 decreasing. Thesediscoveries have profound implications for epidermal stratifi-cation and differentiation.

MATERIALS AND METHODS

Preparation of a Polyclonal Dscl-Specific Antiserum. Anexpression plasmid, pGEXDscEC, encoding a 1.1-kb fragmentof the bovine Dscl extracellular domain linked to the gluta-thione S-transferase (GST) gene was constructed using cloneCN35 (14), encoding full-length bovine Dsclb (Fig. 24), asstarting material. DNA encoding the transmembrane andcytoplasmic domains was deleted by site-directed mutagenesis.The resulting construct was cut with Aflll and BsmI, brieflydigested with Si nuclease, and ligated. A clone containing anin-frame fusion was cut with Narl, blunt-ended with theKlenow fragment ofDNA polymerase I, and cut with Sall. TheNarI(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.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 93 (1996)

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FIG. 1. Scheme depicting possible organization of desmocollinisoforms in desmosomes. (A) Isoforms are localized in distinct des-mosomes within the same cell. (B) Multiple isoforms are locatedwithin 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 coliXL1-blue. GST-Dscl fusion protein expression was inducedfor 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). TheDsc moiety (apparent Mr 50,000) was recovered by digestionwith bovine thrombin (Pharmacia; 10 units/ml of beads for16 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 beenreported (9). Fusion protein production and immunoblotanalysis 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 inneat supernatant of monoclonal antibody 07-4G (againstDsc3; ref. 9), followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Sigma) and Cy3-conjugateddonkey 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°Cand then 1 h at room temperature. After washes in 200 mMHepes buffer (pH 7.3), the tissue was infused with 20%aqueous polyvinyl alcohol (PVA; refs. 22 and 23) and left atroom temperature to harden (minimum 3 weeks). Ultrathinsections (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 plus5-nm gold-conjugated goat anti-mouse IgG (Biocell Labora-tories). Appropriate negative controls were undertaken. Afterextensive washes in PBS, sections were post-fixed for 10 min in2.5% glutaraldehyde/PBS, washed thoroughly with double-

1 2 3 4 5 6 7 8

FIG. 2. (A) Schematic representation of bovine Dsclb. Solid areasindicate the regions of the molecule used to generate antibody JCMC(see text for details). PRE, signal peptide; PRO, propeptide; EC,extracellular domain; TM, transmembrane domain; CYT, cytoplasmicdomain. (B) Polyclonal antibody JCMC is specific for Dscl. Immu-noblot analysis of bacterial cell lysates after isopropyl f-D-thiogalactoside induction of XL1-blue cells transformed with clonespGEX3X/Dsclb (lanes 1 and 5), pGEX4T/Dsc2b (lanes 2 and 6),pGEX2T/Dsc3b (lanes 3 and 7), and pGEX alone (lanes 4 and 8).Blots were probed with pan Dsc-specific monoclonal antibody 52-3D(ref. 21; lanes 1-4) and polyclonal antibody JCMC (serum diluted to1:250,000; lanes 5-8).

distilled H20, contrasted using 2% uranyl acetate oxalate,rinsed briefly with double-distilled H20, and embedded in 2%PVA/0.2% uranyl acetate (24).Gold Quantification and Statistical Analysis. Sections were

cut from eight horizontal levels of the epidermis, at verticalintervals of 60 ,tm from the basal surface. Thirty desmosomeswere selected for each level from nonoverlapping micrographs.The most basal sections were taken at the bottoms of reteridges. Above this level, desmosomes were selected as far as

possible from dermal papillae to avoid the basal layer. Goldparticles overlying the extracellular domain or plasma mem-

brane region were counted, and the length of the correspond-ing desmosome was measured. The results were expressed as

numbers of gold particles per ,um of desmosome length (linearparticle density or LPD; ref. 25).

Statistical analysis and curve-fitting were performed usingthe statistical package SIMFIT (26, 27).

RESULTS

Specificity ofAntibody JCMC. A rabbit antibody was raised,affinity-purified, and shown to be specific for Dscl by immu-noblot analysis (Fig. 2B).

General Distribution ofDscl and Dsc3. Immunofluorescentstaining of cryostat sections using antibodies specific to Dscland Dsc3 confirmed the contrasting distributions of the twoisoforms (Fig. 3). Dsc3 labeling (Fig. 3A) was strongest in thebasal layers of the deep rete ridges and around the dermalpapillae, gradually fading in intensity toward the upper epi-dermis. In contrast, Dscl labeling (Fig. 3B) began in the

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Proc. Natl. Acad. Sci. USA 93 (1996) 7703

FIG. 3. Immunofluorescent labeling of bovine nasal epidermisusing antibodies 07-4G (Dsc3) (A) and JCMC (Dscl) (B). dp,Dermal papilla; rr, rete ridges. (Bar = 100 ,um.)

suprabasal layers and increased in intensity upward throughthe spinous layer.

Ultrastructural Distribution of Dscl and Dsc3. Immuno-gold labeling was performed on methanol-fixed tissue since thereactivity of antibody 07-4G was highly sensitive to aldehydefixation. By combining this fixation with the PVA-embeddingmethod (22), adequate tissue preservation was achieved, whileantigenicity was retained using a modified procedure in whichthe resin was hardened at room temperature rather than at60°C. Similar gold distributions were obtained whichever sizeof IgG-gold was used to detect each isoform; thus, only theresults using 10 nm gold to detect Dscl and 5-nm gold to detectDsc3 are presented. For both antibodies, specific labeling wasfound only over desmosomes while the background level andlabeling of negative controls were negligible.From the first suprabasal layer of cells upward immunogold

labeling of both Dsc3 and Dscl was observed (Fig. 4 A-G).Colabeling of individual desmosomes was invariably seen,indicating that the two isoforms were present in each desmo-some. Moreover, the different-sized gold particles were mixedalong each desmosome, arguing against regional restriction ofisoforms (Fig. 1B). Desmosomes in the basal layer labeled forDsc3 only (Fig. 4H).

It was evident from longitudinal sections that the proportionof gold particles corresponding to a particular isoform variedwith the distance from the basal surface. Therefore, labelingwas quantified on horizontal sections of tissue collected atintervals from the basal surface up through the spinous layer(Fig. 4). The LPD was plotted against distance from the basalsurface (Fig. 5 A and B) and a best fit curve for each isoformdetermined (Fig. SC). It was found that the mean LPD for eachisoform changed gradually from one band of sections to thenext with a gradual increase in Dscl and decrease in Dsc3 withlinear distance from the basal surface (Fig. SC).

Such data suggest that there might be a relationship betweenthe distributions of the two isoforms. Statistical analysisshowed that the distributions could be described by exponen-tial curves, the rate constants of which were not significantlydifferent. Thus our experimental results could be consistentwith a process in which synthesis of the two glycoproteins iscoregulated, for example, by linked gene expression, generatedas follows. If the individual cells were expressing Dsc3 aproportion p of the time and Dscl a proportion 1 - p of thetime, then the amounts visualized would be proportional to pand 1 - p, respectively. Assuming an exponential decrease inp as a function of distance x would then lead to Dsc3 =

FIG. 4. Double immunogold labeling of desmosomes from eightdifferent levels of bovine nasal epidermis. Dsc3 (07-4G), 5-nm gold;Dsc1 (JCMC), 10-nm gold. Vertical distance from the basal layer (H)is shown in ,um. Note the reciprocal changes in the frequency of eachsize of gold particle. (Bar = 0.1 ,Am.)aexp(-kx) and Dscl = 13[1-exp(-kx)] for some parametersa, ,B, and k to account for the linked spatial dependence ofDsc3 and Dscl.

DISCUSSIONThis study has two major findings (i) that individual desmo-somes can harbor more than one desmocollin isoform and (ii)that the distributions of Dscl and Dsc3 are reciprocally gradedwithin the epidermis.The distribution of two desmocollin isoforms, Dscl and

Dsc3, between individual desmosomes supports the modeldepicted in Fig. 1C. Since Dsc2 is expressed throughout thespinous layers (6) and all observed desmosomes were found tobe labeled for at least one of Dscl and Dsc3, then Dsc2 mustalso be codistributed with the other isoforms. Currently, wehave no Dsc2-specific antibody to prove this. Desmosomes inthe spinous layer are larger than those in the basal layer (25).Junctional size might be increased by addition of new materialto each end of the existing junction, giving rise to the situation

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Proc. Natl. Acad. Sci. USA 93 (1996)

(

FIG. 5. Stlabeling. (A a

basal surface.and Dscl (B)limits. The beto be Dsc3 =

but the 95%estimated as

4.65 x 10 -3different.

depicted inwould thenfor Dscl. Sinew constit

A. Data for Dsc3 and Best Fit Curve junction. Hence desmosomes, although mechanically resistantstructures, are also dynamic and subject to continuing turnoverof constituent proteins. This might be achieved by turnover of

o components within existing desmosomes: alternatively, entire0o o desmosomes may be disassembled and new ones assembled0° with random insertion of different glycoprotein isoforms.

Our second aim was to examine the change in desmocollinr O isoform distribution with relative position in the epidermis.

Previous results using immunofluorescence and in situ hybrid-0 \ J o ization have demonstrated the different expression territories

a 0 0a of the three isoforms (6). Moreover, a graded intensity ofo00'-o ~~Q ^^ staining was indicated for Dscl and 3 (refs. 6 and 9; Fig. 3).

Given the close correspondence between protein and mRNAo o 8distributions (6, 9), it is unlikely that the graded immunola-,1 ~I-- - 1 .~I---i--- Ibeling seen here was due to masking of epitopes in different

0 60 120 180 240 300 360 420 cell layers. Therefore, we extended these results by quantifyingDistance from Basal Surface (gm) the simultaneous immunogold labeling of both isoforms. It is

important to point out that the data presented in Fig. 5 are onlysemi-quantitative: the number of gold particles binding to each

B. Data for Dscl and Best Fit Curve isoform may not be regarded as a direct measure of theabsolute abundance of each isoform, given the different natureof the antibodies (polyclonal versus monoclonal) and the use

A A of different sized colloidal gold particles. The binding in eachcase may also have been influenced by factors such as the

A\ ^ i degree of fixation and steric hindrance. However, the data2AA ^ .--'. clearly indicate that the distributions of both isoforms changeA _- ,__--^''R lin a reciprocally graded manner.

A i --^S'iH SlThis gradual change contrasts strikingly with the distribu-~̂~~~~~A--~ a Itions reported for many other epidermal proteins for which the

zA- (^ cut-off points of expression appear to be fairly sharp andA ,"1

.

Acoincident with an alteration in cell phenotype (28). Thus basal

A' ^5 ^cells synthesize K5 and K14 (29), hemidesmosomal compo-

,^ R 5 A nents, integrins, and regulatory proteins including basonuclin(30). Upon entering the first suprabasal layer, the cells switch

0 60 120 180 240 300 360 420 from K5 and K14 to Kl and K10 synthesis, lose hemidesmo-Distance from Basal Surface (gm) somes and integrins, and begin to express proteins character-

istic of terminal differentiation including involucrin (31, 32).Strong Dsc3 expression is clearly associated with the prolifer-

:. Curves with 95% Confidence Limits ative layer but unlike other proteins so expressed, loss of Dsc3expression is graded. An inversely graded binding pattern of

-35 pemphigus vulgaris and pemphigus foliaceus autoantisera to

Dsc3 desmosomes in the basal, squamous, and granular layers of30\ I T 30 human epidermis has been reported (33). These antibodies

Dscl i -,, 25 recognize desmoglein 3 and desmoglein 1, respectively; hence,I\--"' r - 25the different desmoglein isoforms also appear to be differen-

\ i I-20 § tially distributed across the epidermis. By only sampling three" T -

1 positions in the epidermis, however, the authors did notobserve the smooth distribution curve we report for desmo-

10 ^ collins. The distribution of desmocollin isoforms in humanepidermis is controversial. In one study Dscl expression was

-5 detected only high in the epidermis and not in the rete ridges_L'0 (13). In another, a desmocollin antibody, now believed to be

Dscl specific, stained from the first suprabasal layer (34). This0 60 120 180 240 300 360 420 may be explained by primary antibodies of different affinities

Distance from Basal Surface (jm) or by the contrasting sensitivities of different detection sys-tems, as seen by our detection of Dsc3 in higher epidermal

tatistical analysis of the data derived from immunogold layers by immunofluorescence but not by the less sensitiveind B) Plots of LPD against distance of section from the immunogold technique.These graphs depict the total data collected for Dsc3 (A) When plotted against distance from the basal surface, the

). (C) Means of experimental data with 95% confidence LPDs for both isoforms appeared approximately exponential.st fit exponential curves shown in this figure were found We therefore investigated the possibility of fitting exponential22.5exp(-.0047) and Dscl = 35.5[-exp(-0.0022)], curves to the data. We found that a satisfactory fit to the twoconfidence limits on the two decay constants were

4.14 x i10- to 5.25 x 10-3 (Dsc3) and 1.80 x 10 -3 to curves could be achieved, by a single exponential term. This(Dscl). Thus, the rate constants were not significantly result merely demonstrates that this is one particular family of

curves that is consistent with the experimental data. We do,however, observe that if a critical event occurs at a limiting

Fig. 1B. The central portion of each desmosome boundary at one end of a column of cells and this effect isbe labeled only for Dsc3 and the peripheral region propagated in a geometric progression from cell to cell, theince this was clearly not the case, we suggest that resulting spatial dependence of the effect will be approxi-uents are inserted along the entire length of the mately an exponential function of the distance from the

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Proc. Natl. Acad. Sci. USA 93 (1996) 7705

boundary. Moreover, the description of both curves by a singleexponential term is consistent with a process of linked geneexpression. It is therefore interesting to note that the desmo-collin genes are located on the same chromosome (35, 36).What is the significance of the graded distributions? Perhaps

the desmosomal cadherin isoforms are characterized by dif-ferential adhesiveness. Their distribution patterns would thenestablish an adhesive gradient through the epidermis thatcould provide information for cell positioning in differentstrata. Additionally, the desmocollins may signal positionalinformation that regulates cell differentiation. They are asso-ciated cytoplasmically with plakoglobin and plakophilin, mem-bers of the armadillo/03-catenin family of signaling proteins(37-39). Differential binding of these by the cytoplasmicdomains of the different desmocollin isoforms might generategraded signals within the tissue.

We thank Dr. C. Byrne for valuable discussion. This work wassupported by the Wellcome Trust and the Cancer Research Campaign.

1. Holton, J. L., Kenny, T. P., Legan, P. K., Collins, J. E., Keen,J. N., Sharma, R. & Garrod, D. R. (1990) J. Cell Sci. 97, 239-246.

2. Koch, P. J., Walsh, M. J., Schmelz, M., Goldschmidt, M. D.,Zimbelmann, R. & Franke, W. W. (1990) Eur. J. Cell Biol. 53,1-12.

3. Buxton, R. S., Cowin, P., Franke, W. W., Garrod, D. R., Green,K. J., King, I. A., Koch, P. J., Magee, A. I., Rees, D. A., Stanley,J. R. & Steinberg, M. S. (1993) J. Cell Biol. 121, 481-483.

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