INTRODUCTION

Desmosomes are symmetrical, disc-shaped intercellularjunctions found primarily in epithelial tissues. They performdual roles in mediating adhesion between cells and in linkingthe intermediate filaments (IF) of one cell to those of itsneighbour, thereby establishing an integrated scaffold acrossthe entire epithelium. The importance of this scaffold isreflected by the existence of severe skin blistering diseases,such as the autoimmune pemphigus disorders that target thedesmosomal cadherins (Amagai, 1995), and by recentlydescribed inherited disorders caused by mutations indesmosomal genes (Armstrong et al., 1999; McGrath et al.,1997). The ultrastructural organisation of desmosomes hasbeen described in detail (Burdett, 1998; McNutt and Weinstein,1973; Staehelin, 1974) and their principal molecularcomponents have been well characterised (Buxton and Magee,1992; Garrod, 1993; Green and Jones, 1996; Koch and Franke,

1994), enabling detailed investigation of the molecularinteractions that lead to IF attachment and adhesion.Biochemical analysis and in vitro binding or transfectionstudies employing mutant constructs have revealed much aboutinteractions between specific domains of the principaldesmosomal components (see Discussion and referencestherein). However, it is also crucial to determine the preciselocations of the different components within the nativestructure, in order to relate structure and function. This studyset out to provide such complementary data, and thereby to testspecific predictions that have emerged from molecular studies,by performing ultrastructural localisation using domain-specific antibodies against all of the principal desmosomalcomponents.

Desmosomes are readily identified in transmission electronmicrographs of conventional thin sections by theircharacteristic ultrastructural appearance (see Fig. 1 for aschematic diagram). They consist of two principal domains: (1)

4325Journal of Cell Science 112, 4325-4336 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0608

Recent biochemical and molecular approaches have begunto establish the protein interactions that lead to desmosomeassembly. To determine whether these associations occur innative desmosomes we have performed ultrastructurallocalisation of specific domains of the major desmosomalcomponents and have used the results to construct amolecular map of the desmosomal plaque. Antibodiesdirected against the amino- and carboxy-terminal domainsof desmoplakin, plakoglobin and plakophilin 1, and againstthe carboxy-terminal domains of desmoglein 3, desmocollin2a and desmocollin 2b, were used for immunogold labellingof ultrathin cryosections of bovine nasal epidermis. Foreach antibody, the mean distance of the gold particles, andthus the detected epitope, from the cytoplasmic surface ofthe plasma membrane was determined quantitatively.Results showed that: (i) plakophilin, although previouslyshown to bind intermediate filaments in vitro, is localisedextremely close to the plasma membrane, rather than in theregion where intermediate filaments are seen to insertinto the desmosomal plaque; (ii) while the ‘a’ form of

desmocollin overlaps with plakoglobin and desmoplakin,the shorter ‘b’ form may be spatially separated from them;(iii) desmoglein 3 extends across the entire outer plaque,beyond both desmocollins; (iv) the amino terminus ofdesmoplakin lies within the outer dense plaque and thecarboxy terminus some 40 nm distant in the zone ofintermediate filament attachment. This is consistent with aparallel arrangement of desmoplakin in dimers or higherorder aggregates and with the predicted length ofdesmoplakin II, indicating that desmoplakin I may befolded or coiled.

Thus several predictions from previous work were borneout by this study, but in other cases our observationsyielded unexpected results. These results have significantimplications relating to molecular interactions indesmosomes and emphasise the importance of applyingmultiple and complementary approaches to biologicalinvestigations.

Key words: Desmosome, Immunogold, Ultrastructure

SUMMARY

Molecular map of the desmosomal plaque

Alison J. North1,*, William G. Bardsley1, Janine Hyam1, Elayne A. Bornslaeger2, Hayley C. Cordingley3,Brian Trinnaman3, Mechthild Hatzfeld4, Kathleen J. Green2, Anthony I. Magee3 and David R. Garrod1

1University of Manchester, School of Biological Sciences, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, UK2Departments of Pathology, Dermatology and R. H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois60611, USA3Division of Membrane Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK4Molecular Biology Group of the Medical Faculty, University of Halle, Magdeburger Strasse 18, 06097 Halle, Germany*Author for correspondence

Accepted 24 September; published on WWW 17 November 1999

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the extracellular core domain (ECD) or ‘desmoglea’, ~30 nmwide and bisected by an electron-dense mid-line region; (2)symmetrical dense cytoplasmic plaques lying parallel to theplasma membrane and separated from it by a less dense zone.Each plaque is commonly described as consisting of tworegions, the outer dense plaque (ODP), 15-20 nm thick, andseparated by an 8 nm electron-lucent zone from a slightly lessdense inner plaque (inner dense plaque; IDP), into which theIF are seen to insert. The region between the inner face of theODP and the IF domain has also been referred to as the‘satellite region’ (Garrod et al., 1990; Miller et al., 1987).Differing values have been reported regarding the width ofthese domains, particularly the IDP (Garrod et al., 1990; Nilleset al., 1991; Steinberg et al., 1987). The orientation of IF atdesmosomes is also unclear, although in classical thin sectionsIF were reported to converge towards the plaque and then loopaway from it at a distance of 20-40 nm from the cell membrane(Fawcett, 1961; Kelly, 1966). Further ultrastructural detailshave been revealed by alternative methods of tissuepreparation. In the ECD, lanthanum infiltration exposedstaggered quadratic arrays of side-arms linking the dense mid-line to the plasma membrane (Rayns et al., 1969). In thecytoplasmic plaque, a population of 4-5 nm wide ‘traversingfilaments’ (TF) which intervene between the IF loops and thedesmosomal membranes were visualised by freeze-fractureEM (Kelly and Kuda, 1981; Leloup et al., 1979; McNutt andWeinstein, 1973).

The ECD of the desmosome is largely composed of theextracellular domains of desmocollins (Dsc) and desmogleins(Dsg), two families of transmembrane glycoproteins belongingto the cadherin superfamily of cell-cell adhesion molecules(Buxton and Magee, 1992; Koch and Franke, 1994). Both Dscand Dsg occur as three related proteins, products of distinctgenes, that show graded and overlapping distributions acrossthe different cell layers of epidermis (Arnemann et al., 1993;Legan et al., 1994; North et al., 1996; Shimizu et al., 1995).Each Dsc is additionally subject to alternative splicing,resulting in the ‘a’ and ‘b’ forms, which differ in the length oftheir cytoplasmic tail (Collins et al., 1991; Parker et al., 1991).

The cytoplasmic dense plaque is composed of thecytoplasmic tails of the glycoproteins and a number ofcytoplasmic components, including desmoplakins (DP) I and

II, plakoglobin (PG) and plakophilins (PP) (reviewed by Cowinand Burke, 1996). DPI and DPII are also produced byalternative splicing: both proteins consist of an α-helicalcoiled-coil rod domain between globular amino (N)- andcarboxy (C)-termini, DPII lacking most of the rod domain(Green et al., 1990). Desmoplakins are constitutivedesmosomal components, although DPII is absent from cardiacmuscle tissue (Angst et al., 1990), and their critical role inepidermal integrity is demonstrated by the striate subtype ofpalmoplantar keratoderma caused by DP haploinsufficiency(Armstrong et al., 1999). DPI is believed to occur ashomodimers or higher order filamentous structures indesmosomes, but whether by parallel or antiparallelaggregation remains unclear (Bornslaeger et al., 1994). PG andPP belong to the armadillo gene family of signalling proteins(Peifer et al., 1992), with distinct N- and C-termini flanking anumber of central arm repeats. PG contains 13 arm repeats,while PP comprise a separate subclass of armadillo proteins,each including 9 arm repeats preceded by an N-terminal headdomain of variable length (Hatzfeld et al., 1994; Heid et al.,1994; Riggleman et al., 1989). PG is notable as the onlycommon component of desmosomes and adherens junctions(Cowin et al., 1986). PP1 is localised in the desmosomalplaques of certain stratified and complex epithelia (Heid et al.,1994; Kapprell et al., 1988, 1990), PP2 in the desmosomes ofa wide range of cell types (Mertens et al., 1996) and PP3 inthe desmosomes of most simple and almost all stratifiedepithelia as well as cell lines derived from these tissues (Bonnéet al., 1999; Schmidt et al., 1999). Additional minorcomponents, such as IFAP 300 (Skalli et al., 1994), pinin(Ouyang and Sugrue, 1996; but see also Brandner et al., 1997),desmocalmin (Tsukita and Tsukita, 1985), plectin (Eger et al.,1997), envoplakin and periplakin (Ruhrberg and Watt, 1997)may also contribute to plaque structure.

Immunoelectron microscopy has been used previously toassign the major components to extracellular or intracellulardesmosomal domains (Miller et al., 1987; Steinberg et al.,1987). These studies revealed important information, butlargely employed polyclonal antisera raised against wholemolecules or monoclonal antibodies of unknown epitopespecificity. Many further immunoEM studies have indicatedthe approximate locations of components within the

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Intermediate filaments

Inner dense plaque

Outer dense plaque

Dense mid-linePlasma membrane

Satellitezone

Extracellularcore domain

PlakoglobinDesmogleinDesmocollins

Desmoplakins

Fig. 1. Schematic diagram of the desmosome showing the principal components and ultrastructural domains (reproduced with permission, andmodified from, Garrod, 1993).

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desmosomal ultrastructure, but precise localisations could notbe achieved without a quantitative approach. Here we useantibodies of known specificity directed against the ends ofthe major desmosomal components, in conjunction withquantitative immunogold EM, to construct a molecular map ofthe cytoplasmic plaque of desmosomes in bovine nasalepidermis. Important information concerning the relativelocations and the structural organisations of the componentproteins has been gleaned. The relevance of previous in vitroor transfection data to desmosome organisation has in somecases been confirmed and in others brought into question. Thismap will aid the interpretation of new results and allow futurestudies to be focussed on potentially important interactions.

MATERIALS AND METHODS

AntibodiesWhere possible, antibodies against the extreme termini of each proteinwere obtained. However the two anti-plakophilin antibodies werespecific only to one half of the molecule, rather than to the actualprotein termini (see Discussion). Desmoplakin N terminus (NW161):rabbit antiserum against bovine DP, residues 1-189, expressed as aHis-tag fusion protein (Bornslaeger et al., 1996) (used at 1:250 onsections and 1:2500 on blots). Desmoplakin C terminus (11-5F):mouse monoclonal IgG against bovine DP (Parrish et al., 1987). Theepitope was mapped using deletion constructs to between residues2019-2803 within the C terminus (T. Stappenbeck and K. Green,unpublished data) (used at 1:20 on sections and 1:500 on blots).Desmocollin ‘a’ form C terminus: rabbit antiserum against mouseDsc2a C-terminal residues 838-904, expressed as a GST fusionprotein (J. Hyam and D. R. Garrod, unpublished) (affinity purified IgGused at 650 µg/ml on sections and 40 µg/ml on blots). Desmocollin‘b’ form C terminus: rabbit antiserum against a peptide comprisingthe last 11 residues of human Dsc2b ESIRGHTLIKN (KLHconjugate) (A. I. Magee and B. Trinnaman, unpublished) (used at 1:40on sections and 1:500 on blots). Desmoglein C terminus (#10): rabbitantiserum against human Dsg3-specific C-terminal peptide residuesLCTEDPCSRLI (KLH conjugate) (A. I. Magee and B. Trinnaman,unpublished) (used at 1:100 on sections and 1:1000 on blots).Plakoglobin N terminus (#2008): rabbit antiserum against XenopusPG residues 1-106, expressed as a GST fusion protein (H. C.Cordingley and A. I. Magee; Cordingley, 1996) (used at 1:100 onsections and 1:1000 on blots). Plakoglobin C terminus (#1): rabbitantiserum against Xenopus PG residues 666-738, expressed as afusion protein (H. C. Cordingley and A. I. Magee; Cordingley, 1996)(used at 1:100 on sections and 1:1000 on blots). Plakophilin N-region:rabbit antiserum against human PP1, residues 1-285, expressed as aHis-tag fusion protein (Kowalczyk et al., 1999; M. Hatzfeld,manuscript in preparation) (used at 1:50 on sections and 1:500 onblots). Plakophilin C-region: rabbit antiserum against human PP1,residues 286 – 726, expressed as a His-tag fusion protein (Kowalczyket al., 1999; M. Hatzfeld, manuscript in preparation) (used at 1:50 onsections and 1:500 on blots). Both PP antibodies are known to bespecific for the PP1 isoform (M. Hatzfeld, unpublished data).

ImmunoblottingEpidermis was dissected from bovine nose, frozen in liquid nitrogenand fractured into powder using a pestle and mortar while still frozen.The powder was thawed into Laemmli sample buffer (Bio-Rad),homogenized, boiled for 5 minutes, and spun for 5 minutes at 10,000g in a bench centrifuge. SDS-PAGE was performed as described(Laemmli, 1970), using an 8% gel for subsequent immunoblottingwith PG and PP antibodies and a 6% gel for DP, Dsc and Dsgantibodies. Proteins were transferred onto a nitrocellulose membrane,

which was then blocked with 5% skimmed milk powder beforeprimary antibody incubation. Bound antibodies were detected with anappropriate peroxidase-conjugated secondary antibody and an ECLdetection system (Amersham).

Tissue preparation for electron microscopyPreparation of conventional and polyvinyl alcohol (PVA)-embedded samples for ultrastructural studiesFreshly obtained bovine nasal epidermis was dissected into smallpieces (approximately 1 × 1 × 0.5 mm) and fixed by immersion in 2%formaldehyde (FA) (freshly made from paraformaldehyde powder)plus 2% glutaraldehyde (GA) in 0.1 M sodium cacodylate buffer, pH7.3. Tissue was fixed for 2 hours at room temperature (RT), then takenthrough 4 washes of cacodylate buffer. Samples for resin embeddingwere post-fixed for 2 hours with 1% osmium tetroxide, dehydratedthrough an ethanol series and embedded in Spurr’s resin. Samples tobe embedded in PVA were immersed in 20% aqueous PVA, M. Wt.10,000 (Air Products and Chemicals Inc., Allentown, Pennsylvania)and hardened by drying overnight at 60°C. Sectioning of PVA blockswas performed as described (Small et al., 1986).

Resin sections were contrasted using uranyl acetate and lead citrate.PVA sections were incubated on aqueous buffer to extract the PVAfrom the section, rinsed with 40 µg/ml aqueous bacitracin (Sigma),and contrasted by negative staining using 2% aqueous uranyl acetate.

Preparation of ultrathin cryosections for immunoEMBovine nasal epidermis was dissected as above and fixed in 2% FAin 200 mM Hepes buffer, pH 7.4 (Hepes buffer) for 1 hour at RT.Samples were washed in 4 changes of Hepes buffer, 10 minutes ineach, and then infiltrated with a sucrose/polyvinyl pyrrolidone mixture(Tokuyasu, 1989) for a minimum of 2 hours at RT. Infiltratedspecimens were plunge-frozen and stored in liquid nitrogen.

Cryosectioning was performed using a Leica Ultracut S/FCScryoultramicrotome following the method of Tokuyasu (1980).Ultrathin sections were cut using tungsten-coated glass knives(Roberts, 1975) at temperatures around −100°C. Sections wereretrieved on a droplet of 2 M sucrose plus 0.75% gelatin (in Hepesbuffer) and transferred to Formvar-coated grids. Grids were invertedonto 2% gelatin/PBS (solidified) and stored overnight at 4°C.

Immunogold labellingImmediately prior to immunolabelling, cryosections on 2% gelatinplates were incubated at 37°C to fluidify the gelatin and then for afurther 10 minutes to block non-specific labelling. All other steps ofthe labelling procedure were carried out at RT. Grids were transferredacross 3 droplets of 0.02 M glycine/PBS (10 minutes total), thenblocked for 15 minutes with 5% normal goat serum (NGS) plus 1%BSA. Sections were transferred to primary antibodies (diluted into 1%BSA/PBS to give optimal staining) for 1-2 hours, then washed 5 timesin 0.1% BSA/PBS (total 10 minutes). Goat anti-mouse or anti-rabbitIgG gold conjugates (BioCell Research Laboratories, Cardiff) werediluted 1 in 15 in 10% NGS, 1% BSA/PBS, and incubated for 30minutes on ice before use. Gold labelling was performed for 30minutes, the sections washed 5 times in PBS (total 20 minutes), andsections were then post-fixed using 2.5% GA/PBS for 10 minutes.After 4 washes in ddH2O (1 minute each), sections were contrastedfor 5 minutes on 2% uranyl acetate oxalate (Tokuyasu, 1980), washedbriefly 3 times in ddH2O, then incubated on 2% aqueous PVA plus0.2% uranyl acetate (Tokuyasu, 1989). Grids were looped out anddrawn across filter paper to remove excess PVA, air-dried, andexamined under a Phillips 400 transmission electron microscope. Allmicrographs were taken at a magnification of 46,000.

Gold quantification30 desmosomes, which had been sectioned in a plane normal to theplasma membrane, were selected for each antibody. In the case of theDsg3 C-terminal antibody, desmosomes were selected from the basal

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layers and lower spinous layers, as the labelling for this Dsg isstrongest in this region. Desmosomes from the mid-spinous regionwere selected for all other antibodies. Electron micrographs wereprinted at high magnifications and the perpendicular distance of eachgold particle from the cytoplasmic surface of the plasma membranewas measured to the nearest millimetre on print. For this purpose,desmosomes were divided into two symmetrical halves and thedistance of each gold particle measured from the nearer membrane.Particles lying over the extracellular region were assigned a negativevalue. To minimize variation and bias, all measurements wereperformed blind by the same author and the print magnification foreach set was calculated only after all measurements had beencompleted. The number of gold particles was plotted against distance(in whole mm on print) using the statistical package SIMFIT(Bardsley et al., 1995a,b). For all epitopes the distribution of goldparticles approximated to a normal distribution, and a best fit curvewas fitted using SIMFIT. Distances were converted back tonanometres (nm) on section (from total magnification) and the meandistance from the plasma membrane was obtained from the curveparameters.

Immunogold labelling was typically performed as double labelling,using the antibody of choice together with 11-5F (anti-DP Cterminus). This allowed the distance calculated for the DP C terminusto be compared between data sets, thereby assessing thereproducibility of the results. Three of the many data sets obtainedusing antibody 11-5F are presented below.

RESULTS

Antibody specificityImmunoblots (Fig. 2) demonstrated that the antibodies used inthis study detected proteins of the appropriate molecular massand antibodies against the N- and C-termini of the same proteinrecognized bands of the same size. The anti-Dsg 3 antibodystained a single band of around 135 kDa, confirming that it didnot react with the larger Dsg 1 or 2. The slight doubletsobtained with the Dsc antibodies probably reflected weakcross-reactivity with other Dsc isoforms, but there was clearlyno cross-reactivity between the two splice variants.

It should be noted that throughout the Results and Figures,DP refers to DP I and II, PP to PP1, and Dsg to Dsg 3.

Desmosomal ultrastructureThe ultrastructural appearance of desmosomes was dependenton the chosen method of tissue preparation and sectioncontrasting, yet all preparations revealed a highly orderedarrangement of components. Fig. 3A shows a typicaldesmosome from the spinous layers of bovine nasal epidermisprepared by a conventional method (see legend). The inner faceof the ODP was located 15 to 20 nm from the plasmamembrane, and was separated from the IDP by an electron-lucent zone less than 10 nm wide. The width of the morediffuse IDP was more difficult to determine but was in theregion of 15 to 20 nm. Thus the total width of the desmosomalplaque was up to 50 nm. In classical images from tissues suchas newt epidermis, the ODP, IDP and regions of less electrondensity on either side are clearly revealed, as are individualtonofilaments converging upon the plaque region at a distanceof 40-70 nm from the plasma membrane (Kelly, 1966).However, in bovine nasal epidermis a space between theplasma membrane and the plaque was hardly discernible andclearly defined IF could not be distinguished close to theplaque, consistent with previous studies (Leloup et al., 1979).

More detail was revealed within the negatively-stainedcytoplasmic plaque of PVA-embedded desmosomes. Thehighly ordered arrangement of desmosomal components wasobserved as lines of differential staining both parallel andperpendicular to the membrane (Fig. 3B). At highmagnifications perpendicular fine filaments could bedistinguished across the ODP, in some regions appearing astwo parallel arrays (Fig. 3C). These filaments may correspondto the 4-5 nm TF previously reported to extend from theintermediate filaments to the desmosomal membrane (Kellyand Kuda, 1981; Kelly and Shienvold, 1976; Leloup et al.,1979; McNutt and Weinstein, 1973).

The structure of desmosomes in ultrathin cryosections ofPFA-fixed bovine nasal epidermis has been describedpreviously (Miller et al., 1987). Consistent with this, fewstructural features could be discerned within the low contrastimages (Fig. 3D). The two plasma membranes were clearlyvisible as well as a cytoplasmic lamina parallel to each. Its innerface, situated around 20 nm from the membrane, was presumedto mark the extent of the ODP. Regular periodic striations acrossthis lamina (Miller et al., 1987) were occasionally seen inoptimally contrasted desmosomes (not shown).

Immunogold labellingOn ultrathin cryosections all antibodies labelled desmosomesstrongly, with negligible labelling across the rest of the tissue(Fig. 3F). Control sections labelled with gold conjugates aloneshowed minimal background labelling (Fig. 3D and E). Fig. 4shows representative desmosomes labelled with each antibody.Gold particles were largely confined to a band of label, whichvaried in distance from the plasma membrane for differentantibodies. To assess the reproducibility of the measurementsmost sections were double-labelled with the antibody underinvestigation together with antibody 11-5F against the DP Cterminus (Fig. 4A-F). The size of secondary gold conjugateused did not affect the particle distributions (not shown).Although all of the detected epitopes were cytoplasmic, goldparticles could also be seen in the extracellular domain usingcertain antibodies, in particular those directed against PP1 (seeDiscussion).

Quantification of gold particle distributionsThe distribution of gold particles obtained using each antibody

A. J. North and others

Fig. 2. Immunoblot analysis of bovine nasal epidermis extract toconfirm the specificity of each antibody used in this study. DPI andII, Dsg and Dsc were resolved on a 6% polyacrylamide gel (A), andPG and PP on an 8% gel (B). ‘N’ indicates N-terminal antibody and‘C’ indicates C-terminal antibody. Molecular mass markers areindicated in kilodaltons (kDa). The molecular masses of the majordesmosomal components determined by SDS-PAGE under reducingconditions are typically described as: DPI and II, a doublet around250 and 215 kDa; Dsg3, 135 kDa; Dsc ‘a’ and ‘b’ forms, 115 and107 kDa; PG, 83 kDa; PP (B6P), 75 kDa (Garrod, 1993).

4329Molecular map of desmosome

approximated a normal distribution curve. Fig. 5 depicts thedata for each antibody plotted together with a best fit curve.The mean gold particle distance from the plasma membranewas estimated for each antibody from the best fit curve (Table1), and the results were used to construct a preliminary map ofthe desmosomal plaque (Fig. 6).

The standard error of the mean and standard deviation werealso estimated using the best fit curve (Table 1). The standarddeviations, reflecting the spread of gold particles within anindividual data set (Table 1), were similar for all antibodies.The ‘tail’ which was apparent on the right hand side of eachgraph was probably contributed by non-specific labelling. Asymmetrical tail on the left side of each graph would not havebeen detected because the labelling at this position would have

been attributed to the other half of the desmosome and thusmeasured as a positive value from the other membrane leaflet.

In repeat experiments the same pattern of results (that is,order of increasing distance from the membrane) was obtained.However, some variability in the mean distances was foundbetween different tissue specimens. Therefore the resultspresented here were all obtained from the same specimen. Totest for variability between experiments, several sets of datawere obtained for the DP C terminus (Fig. 4A-C). The meanvalues and 99% confidence limits of the three data setspresented here were estimated to be 52.0±1.28 nm, 51.4±2.84nm and 49.5±1.60 nm. Thus no statistically significantdifferences were found between them at the 99% confidencelevel, since these confidence limits overlap.

Fig. 3. Transmission electron micrographs of desmosomes in bovine nasal epidermis prepared by different embedding methods. ECD,extracellular core domain. PM, plasma membrane; ODP, outer dense plaque; IDP, inner dense plaque; CL, cytoplasmic lamina. (A) fixed with2% FA/2%GA, followed by osmium tetroxide, and embedded in epoxy resin. Sections were stained with uranyl acetate and lead citrate. Notethe heavily stained ODP (marked by a white bar) and IDP, separated by an electron-lucent zone, and the electron-dense mid-line in the ECD(marked by a white bar) linked by periodic cross-bridges to each leaflet of the PM. (B) Fixed with 2% FA/2% GA, embedded in PVA andcontrasted by negative staining using uranyl acetate. By this method the dense, proteinaceous cytoplasmic plaques appear lighter than thesurrounding structures, and the ECD and the so-called electron-lucent zones on either side of the ODP appear darker. (C) A highermagnification image of the preparation shown in B, revealing periodic filamentous structures, perpendicular to the PM, crossing thecytoplasmic plaques (large arrows). A thin white line (marked by small arrows), apparently divides the ODP into two parallel rows of filaments.This line indicates a region of particularly high protein density. (D) Ultrathin cryosection of FA-fixed tissue. The most notable features of thislower contrast image are the PM and the tripartite CL. (E) Lower magnification image of the preparation shown in D. D and E both showsections of tissue labelled using the immunogold secondary antibody alone as a negative control. Note the low background labelling.(F) Ultrathin cryosection labelled using anti-DP N. Desmosomes are labelled strongly, with negligible background labelling. Bars, 50 nm.

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The length of DP was calculated from the distance betweenthe C terminus (52 nm; first data set) and the N terminus (10.3nm; measured on the same desmosomes as the C terminususing double labelling). This gave a length of ~42 nm,measured perpendicular to the plasma membrane.

The N terminus of each of the desmosomal glycoproteins issituated in the extracellular core domain: therefore only the Cterminus was considered here.

DISCUSSION

Localisation of the terminal domains of all major desmosomalcomponents has enabled construction of a rudimentarymolecular map of the cytoplasmic plaque of desmosomes inbovine nasal epidermis (Fig. 6). The results have importantimplications for future investigations into molecularinteractions between desmosomal components. A number ofspecific predictions based on previous biochemical andmolecular studies have been confirmed or broughtinto question. Before expanding on these majoradvances in our understanding of structure-functionrelationships in desmosomes, several issues, whichmight impact the interpretation of our results, shouldbe considered.

First, in localising the terminal domains of themolecules, we have shown that no majordesmosomal component is aligned parallel to theplasma membrane along its entire length. This is inkeeping with ultrastructural features such as the TF(Kelly and Kuda, 1981; Leloup et al., 1979; McNuttand Weinstein, 1973), which have been visualisedperpendicular to the membrane, and with theperpendicular filamentous structures seen here inPVA-embedded tissue (Fig. 3C). However this studycould not provide information concerning theposition of internal regions of each protein. Thusother domains of certain components may bepositioned closer to or further from the membranethan the N- and C-termini.

Second, this study has demonstrated the benefit ofthe immunogold technique in resolving the positionof epitopes located only a few nm apart,perpendicular to the membrane. However, higherresolution methods will be required to investigatethe lateral organisation of the plaque.

Third, the calculated distances varied betweendifferent tissue specimens, and therefore cannot betaken as absolute. This variability might be causedby different degrees of fixation-induced tissue

A. J. North and others

Table 1. Statistical analysis of gold particle distributionsMean particle Standard Standard error ofdistance (nm) deviation (nm) the mean (nm)

DP N 10.3 14.3 0.98DP C 1st 52.0 16.0 0.44DP C 2nd 51.34 16.4 1DP C 3rd 49.5 13.9 0.56Dsg3 C 20.3 15.0 0.77Dsca C 15.4 13.6 0.87Dscb C 7.2 12.9 0.76PG N 22.9 11.5 0.45PG C 10.8 13.8 0.9PP N 15.8 14.3 1.1PP C 4.2 14.7 1.1

Mean distance of the gold particles from the plasma membrane for eachantibody together with the standard deviation of the gold particle distributionsand the standard error of the means.

Fig. 4. Representative desmosomes in ultrathincryosections of bovine nasal epidermis labelled usingeach of the antibodies indicated. (A to F) Doublelabelling with the antibody of choice (indicated in bold;5 nm gold) together with the DP C-terminal antibody(10 nm gold) as a comparison. (G and H) Singlelabelling. Note the subtle differences in the overalldistribution of gold particles relative to the plasmamembrane. Bar, 100 nm.

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shrinkage brought about by slight changes in the durationor temperature of fixation. However, the important pointsto emphasise are that the differences between repeatedexperiments on one specimen were not statisticallysignificant, and that the relative order of the differentprotein domains and thus the potential overlap in spatialdistribution of particular components remained constant.

The close fit of each gold particle distribution to a singlenormal distribution curve suggested that each of thedetected epitopes was localised in a single discrete bandparallel to the plasma membrane. The standard deviationswere consistent with the use of an indirect labellingprotocol, after which a gold particle could lie up to thelength of two antibodies (around 20 nm) in either directionfrom its bound epitope (see Griffiths, 1993). Thislocalisation of gold particles far from their bound epitopealso seems to be the likely explanation for the apparentextracellular signal observed for antigens close to theplasma membrane, such as PP1. Although a directlabelling method would have resulted in tighterdistributions, the reduced number of bound gold particleswould have rendered statistical analysis very difficult forthe weaker antibodies. Moreover it is probable that themean position of the gold particles would be unaltered.

The ultrastructural features of the desmosomalplaque reflect the organisation of its molecularcomponentsFig. 6 shows a rudimentary molecular map of thedesmosomal plaque, on which the approximate positionof each protein terminal domain has been marked relativeto the plasma membrane. This detailed analysis of thedesmosomal component locations has led to a greaterunderstanding of plaque ultrastructure.

The electron density of the ODP (Fig. 3A) can beexplained by the high concentration of proteins in thisregion. The positions of the PG N terminus and the Dsg3C terminus (around 20 nm from the membrane) werecoincident with the electron-opaque lamina observed oncryosections that appears to mark the distal limit of theplaque. In contrast, the PP1 C terminus was located nearto the membrane proximal face of the ODP. Thus the ODPcorresponds to the region where PG and PP1 mightinteract with the cytoplasmic tails of the desmosomalglycoproteins. DP spans both the IDP and most of theODP, the region harbouring its C-terminal domainmarking the innermost face of the IDP. The TF, previouslyvisualised between the ODP and the IF attachment zone(Kelly and Kuda, 1981; Leloup et al., 1979; McNutt andWeinstein, 1973), are most probably comprised of DP,possibly together with the most C-terminal domains of

A

B

Fig. 5. Best fit curves for each of the antibodies plotted againstthe gold particle distribution (circles). (A) Graphs showing DPN, three data sets for DP C, Dsg C, Dsc’a’ C and Dsc’b’ C;(B) graphs of PG and PP. A broken line at zero distanceindicates the position of the cytoplasmic surface of the plasmamembrane. Note the contrasting distances of the peak of eachparticle distribution from the membrane.

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Dsg1 and Dsg2, and/or with unravelled keratin protofilaments(see below).

The orientation of the desmoplakin N- and C-terminal domains is consistent with functions inplaque targeting and intermediate filamentanchorage, respectivelyThe DP N terminus was localised in the outer plaque around10 nm from the plasma membrane, in keeping with itsproposed role in attachment to the plaque (Bornslaeger et al.,1996; Smith and Fuchs, 1998; Stappenbeck et al., 1993). Incontrast, the C terminus lay much further from the membrane,consistent with its function in IF attachment (Bornslaeger etal., 1996; Kouklis et al., 1994; Meng et al., 1997; Stappenbecket al., 1993; Stappenbeck and Green, 1992). Contrastingreports from previous immunogold labelling studies reportedDP to extend from the cytoplasmic face of the plasmamembrane to the inner boundary of the satellite region(Steinberg et al., 1987) or to be a component of the satelliteregion only (Miller et al., 1987). Our results demonstrate thatDP extends across most of the outer plaque and the whole ofthe inner plaque and thus is in a position to interact with mostdesmosomal components.

Localisation of the desmoplakin N- and C-terminaldomains demonstrates that higher order oligomersmust interact in a parallel, unstaggered fashionA single peak of labelling was obtained with both anti-DPantibodies. Thus all the N-termini of DP molecules are locatedat the same distance from the plasma membrane, as are allthe C-termini. This indicates that if DP self-aggregates toform homodimers, tetramers or higher-ordered filamentousstructures, it must do so in a parallel fashion. This result isconsistent with the demonstration that DP can self-associate

through head-head interactions (Smith and Fuchs, 1998).However, this observation differs from the situation recentlydescribed for the related plakin family member plectin, whichhas been proposed to exist in the form of both parallel andantiparallel oligomers within hemidesmosomes (Rezniczek etal., 1998).

Desmoplakin I may be coiled or folded in situ whilethe width of the desmosomal plaque may be definedby desmoplakin IIDPI and DPII are predicted to be of very different lengths. Thelength of the rod domain was predicted to be 130 nm for DPIand 43 nm for DPII, both from sequence data (Green et al.,1990) and from rotary shadowing of isolated molecules(O’Keefe et al., 1989). Both DPI and DPII are present in thistissue in approximately equal amounts, and the antibodies wereshown to detect both isoforms (Fig. 2). Therefore a double peakof labelling might be expected using either antibody. Yet wefound that the C-termini and N-termini of both DPI and DPIIwere located in a single region respectively and the total lengthof DP, calculated perpendicular to the plasma membrane, wasaround 42 nm. This figure is thus consistent with the predictedlength of DPII, but not of DPI, suggesting that DPI is eitheroriented at an angle to the membrane or is somehow folded orcoiled in native tissue. Several possible break points andstutters along the length of the rod domain, together with anextended region of predicted flexibility (Green et al., 1990),may allow the molecule to bend. Fig. 7 shows three of the manypossible configurations of DPI that would fit our data andreconcile our results with those of O’Keefe et al. (1989).However we consider model A to be unlikely as it would beinconsistent with the reported perpendicular appearance of theTF.

The functional significance of the different lengths of DP Iand II is unknown. Here we found that the distance of the DPC terminus from the membrane (around 51 nm) was entirelyconsistent with the total width of the entire desmosomal plaquemeasured on frozen sections by Miller et al. (1987), that is a17 nm outer plaque plus a 34 nm satellite zone. Theseobservations are consistent with the idea that the width of thedesmosomal plaque is defined by the extent of DP. It istherefore conceivable that DPII determines the extent of theinner plaque, while lateral associations between the longer roddomains of DPI could contribute to the structure of bothplaques (model B) or the IDP alone (model C). Since cardiacmuscle desmosomes contain only the DPI isoform we wereinterested to determine whether the C-terminal antibody wouldbe localised further from the membrane in this tissue. However,insufficient gold labelling could be achieved on theseconsiderably smaller desmosomes to permit statistical analysis.

The positions of the C-termini of the desmosomalglycoproteins are consistent with their relative sizesand their predicted interactions with other plaquecomponents The C terminus of Dsg3 was located further from themembrane than that of Dsca or Dscb, consistent with theirmolecular sizes and the predictions of Miller et al. (1987). Wefound the Dsg3 C terminus to be located at the inner face ofthe ODP, suggesting that the entire Dsg3 cytoplasmic tail maybe folded within the ODP. Thus Dsg3 is unlikely to participate

A. J. North and others

0 20 40-10 10 30 50 nm

PM ECD PM ODP IDP

Dsc"a" C

Dsc"b" C

DP N DP C

Dsg3 C

PG NPG C

PP NPP C

Fig. 6. A map of the desmosomal plaque relating the positions of themajor molecular components to the principal ultrastructural domains.The apex of each pentagon marks the mean gold particle position forthe detected epitope. The protein domains linking the termini of eachprotein are depicted as a straight line for the sake of simplicity. Notethe high concentration of proteins located within the ODP. ECD,extracellular core domain; PM, plasma membrane; ODP, outer denseplaque; IDP, inner dense plaque.

4333Molecular map of desmosome

significantly in direct IF binding, unless keratins penetrate intothe ODP in the form of unravelled protofilaments (see below).However, Dsg3 is the smallest of the three Dsg isoforms: its CIII domain comprises only 2 of the unique 29-amino acidrepeats, compared to 5 in Dsg1 and 6 in Dsg2, and it lacks theglycine-rich C-terminal cytoplasmic domain (Amagai et al.,1991). Thus it is still possible that the longer cytoplasmicdomains of Dsg1 and 2 could span the region between the ODPand IDP and even extend through the IDP to interact with IF,as proposed from sequence analysis (Nilles et al., 1991) androtary shadowing studies (Rutman et al., 1994). Since noantibodies against the extreme C terminus of Dsg1 or Dsg2have been raised successfully, we were unable to address thispoint.

Our results concerning the Dsc ‘a’ and ‘b’ forms areconsistent with previous studies using transfected cells and invitro binding assays. The ‘a’ form of Dsc1 has been shown tobind both PG and DP and to recruit them in transfectedcells to form a plaque (Chitaev et al., 1996; Smith and Fuchs,1998; Troyanovsky et al., 1993, 1994; Witcher et al., 1996).

Consistent with these results we found that Dsca, PG and DPall overlap spatially. Conversely, we found that the ‘b’ form ofDsc overlapped with neither PG nor DP (unless with internaldomains of these proteins: see following section), consistentwith the demonstrated inability of Dsc1b to nucleate plaqueformation in transfected cells (Troyanovsky et al., 1993). Atpresent the function of the ‘b’ form of Dsc remains unclear.PP1, for which strong in vitro association with the ‘a’ form ofDsc1 has been reported (Smith and Fuchs, 1998), was foundto overlap also with the ‘b’ form. The possibility that Dscbcontains a PP1-binding domain should therefore beinvestigated. Alternatively the clue to the function of theshorter ‘b’ form may lie in its apparent inability to recruitcytoplasmic plaque proteins. The extremely high concentrationof proteins localised within the outer plaque is reflected by themarked electron density of this region under the EM. It ispossible that the density of Dsc extracellular domains requiredfor adhesion is greater than the density of Dsc plaquecomponent-binding domains that is optimal for plaqueassembly.

Plakoglobin is located in the ODP with the potentialto interact with desmoplakin, desmoglein anddesmocollin a The localisation of PG is in keeping with its orientation andinteractions predicted from biochemical and transfectionstudies (reviewed by Cowin and Burke, 1996). Thus the Cterminus was localised nearer to the plasma membrane than theN terminus and the protein overlapped with its known bindingpartners Dsca, Dsg and DP, as well as with PP1. PG is believedto bind to the C-domains of the desmosomal glycoproteins viaa binding site localised to the first 3 arm repeats (Chitaev etal., 1998; Troyanovsky et al., 1996; Wahl et al., 1996; Witcheret al., 1996). Our results would not be compatible with theseinteractions if PG were an elongated molecule orientatedperpendicular to the membrane. However, it is more probablethat PG is globular (Kapprell et al., 1987; Ruediger et al.,1994), folded such that the internal arm repeats are positionedcloser to the plasma membrane than either its C- or N terminus(as depicted by Cowin and Burke, 1996). This orientation isalso consistent with the binding of central PG domains to theDP N terminus (Kowalczyk et al., 1997, 1998). Thus we cannotexclude the possibility that the arm repeat domain would alsobe available for binding to Dscb.

Plakophilin 1 is located near to the plasmamembrane and distant from desmosome-associatedclassical intermediate filamentsAn important point to emerge from this study is the localisationof PP1. This plaque component (Kapprell et al., 1988, 1990)was reported to bind keratin IF in vitro (Hatzfeld et al., 1994;Heid et al., 1994; Kapprell et al., 1988), and henceforth hascommonly been assumed to be involved in mediatingattachment of IF to the desmosome. Moreover, PP2 has beenlocalised near the cytoplasmic face of the desmosomal plaquein bovine nasal epidermis (Mertens et al., 1996), consistentwith such a role, although its localisation in cardiacdesmosomes appeared closer to the membrane and evenenriched over the desmoglea. We have demonstrated PP1 to liecloser to the plasma membrane than either of the other majorcytoplasmic plaque components, PG or DP. This may reflect a

A

C

IDP

ODP

PM

IDP

ODP

PM

DPII DPI

N

C

B

IDP

ODP

PM

DPII DPI

N

C

DPII DPI

N

C

DPII DPI

N

C

DPII DPI

N

C

C'

B'

Fig. 7. Some possible arrangements of DPI and II within thedesmosomal plaque to explain the coincidence of their N- and C-termini. (A) DP I is orientated to subtend an angle of approximately17° to the plasma membrane, so that the distance between its N- andC-termini perpendicular to the membrane is the same as for DPII. (Band B′) DP bends at more than one position along the rod domain, sothat this domain crosses the plaque more than once, possibly forming(by itself or together with other components) the TF. (C and C′) DPbends at one point along the rod domain, with the result that most ofthe rod domain is located within the IDP and oriented parallel to theplasma membrane. Note that model B′ differs from B, and model C′from C, in the portion of the rod domain of DPI that would beavailable for interaction with the rod domain of DPII.

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true functional difference between PP1 and PP2, as the latterhas a significantly longer N-terminal domain that could extendfurther out from the ODP. A recent study suggests that PP3,like PP1, may be localised deep within the dense plaque ofdesmosomes in cultured epithelial cells, near the plasmamembrane (Schmidt et al., 1999).

It is important to reiterate that the epitopes of the two PP1antibodies are known with less precision than are those of theother antibodies used in this study. The ‘N-region’ antibodywas raised against the whole PP1 head domain and the ‘C-region’ antibody against the whole PP1 repeat domain, andreactivity with deletion constructs suggests that both PP1antibodies recognise numerous epitopes localised along thelength of the fragments (M. Hatzfeld, unpublished data).Therefore broader gold particle distribution curves might havebeen expected using these two antibodies. In fact, the standarddeviations of the distributions were similar to those of the otherantibodies. The calculated distance between the mean positionof the N- and C-terminal regions of PP1 was very similar tothe distance between the termini of PG, which is of a similarsize and closely related to PP1. It is thus unlikely that the actualtermini of PP1 were located more than a few nm from thepositions measured above.

Our results were inconsistent with a function for PP1 indirect binding of full diameter 10 nm IF in native desmosomes.However, the association of PP1 with keratins observed in vitro(Hatzfeld et al., 1994; Heid et al., 1994; Kapprell et al., 1988;Smith and Fuchs, 1998) could also occur in vivo if otheroligomeric forms of keratins penetrate further into thedesmosome than full diameter IF. It has been postulatedpreviously that keratins may penetrate into the plaque in theform of unravelled protofilaments (Leloup et al., 1979).Moreover, filamentous structures resembling the 4-5 nm TFhave been formed from aggregates of DP construct proteinscontaining both the rod and C-terminal domains together withIF proteins. This meshwork was not seen with DP alone,suggesting that keratin IF may be present in the plaque as ananastomosing network of fine protofilaments associated withDP (Stappenbeck and Green, 1992).

Plakophilin could associate with desmoplakin andthe desmosomal glycoproteins The finding that PP1 resides close to the plasma membrane isalso interesting given the binding of its close relative, p120ctn,to the juxtamembrane region of the E-cadherin cytoplasmic tail(Yap et al., 1998). Moreover our results are in line with datashowing that PP1 binds to the desmosomal glycoproteinsDsc1a and Dsg1 (Mathur et al., 1994; Smith and Fuchs, 1998;but see also Kowalczyk et al., 1999). However our results areequally consistent with the reported in vitro binding of PP1 tothe DP head domain (Smith and Fuchs, 1998) and in particularwith more recent data demonstrating that the DP binding sitein PP1 resides in its N-terminal non-armadillo head domain(Kowalczyk et al., 1999). Importantly, desmosomes of a patientwith no PP1, due to mutations in the PP1 gene, displayedalterations both in adhesion and in DP and keratin IForganization (McGrath et al., 1997), suggesting a key linkingrole for PP1 in desmosome structure. Recent resultsdemonstrating that PP1 enhances recruitment of DP to cell-cellborders and promotes lateral interactions among plaquecomponents suggest that its loss in patients could compromise

epidermal integrity by decreasing the number of binding sitesfor IF at the desmosome (Kowalczyk et al., 1999).

ConclusionOur results demonstrate the value of the immunogold labellingtechnique for low resolution mapping of multimolecularstructures. Such data provide crucial in situ information againstwhich to interpret the results of molecular studies. Thistechnique, when used in conjunction with additional domain-specific antibodies, has the potential to yield further valuableinformation on structure-function relationships withindesmosomes and other cellular structures.

A.J.N. and D.R.G. are grateful for the generous support of theWellcome Trust, the University of Manchester and the CancerResearch Campaign during these studies. The production ofantibodies was also supported by the Medical Research Council(A.I.M), the NIH (grants RO1 AR43380 and AR41836 to K.J.G.) andthe DFG (grant Ha 1791/3-1 to M.H.). We also thank members of theGarrod and Green laboratories for insightful discussions, the staff ofNewton Heath Abattoir for providing bovine nose tissue and the staffof the Biological Sciences E.M. Unit in Manchester for their support.

REFERENCES

Amagai, M., Klaus-Kovtun, V. and Stanley, J. R. (1991). Autoantibodiesagainst a novel epithelial cadherin in pemphigus vulgaris, a disease of celladhesion. Cell 67, 869-877.

Amagai, M. (1995). Adhesion molecules. I: Keratinocyte-keratinocyteinteractions; cadherins and pemphigus. J. Invest. Dermatol. 104, 146-152.

Angst, B. D., Nilles, L. A. and Green, K. J. (1990). Desmoplakin IIexpression is not restricted to stratified epithelia. J. Cell Sci. 97, 247-257.

Armstrong, D. K., McKenna, K. E., Purkis, P. E., Green, K. J., Eady, R.A., Leigh, I. M. and Hughes, A. E. (1999). Haploinsufficiency ofdesmoplakin causes a striate subtype of palmoplantar keratoderma. HumanMol. Genet. 8, 143-148.

Arnemann, J., Sullivan, K. H., Magee, A. I., King, I. A. and Buxton, R. S.(1993). Stratification-related expression of isoforms of the desmosomalcadherins in human epidermis. J. Cell Sci. 104, 741-750.

Bardsley, W. G., Ackerman, R. A., Bukhari, N. A., Deeming, D. C. andFerguson, M. W. (1995a). Mathematical models for growth in alligator(Alligator mississippiensis) embryos developing at different incubationtemperatures. J. Anat. 187, 181-190.

Bardsley, W. G., Bukhari, N. A. J., Ferguson, M. W. J., Cachaza, J. A. andBurguillo, F. J. (1995b). Evaluation of model discrimination, parameterestimation and goodness of fit in nonlinear regression problems by teststatistics distributions. Comput. Chem. 19, 75-84.

Bonné, S., van Hengel, J., Nollet, F., Kools, P. and van Roy, F. (1999).Plakophilin-3, a novel Armadillo-like protein present in nuclei anddesmosomes of epithelial cells. J. Cell Sci. 112, 2265-2276.

Bornslaeger, E. A., Stappenbeck, T. S., Kowalczyk, A. P., Palka, H. L. andGreen, K. J. (1994). Molecular genetic analysis of desmosomal proteins.In Molecular Biology of Desmosomes and Hemidesmosomes (ed. J. E.Collins and D. R. Garrod), pp. 35-52. Austin: RG Landes Company.

Bornslaeger, E. A., Corcoran, C. M., Stappenbeck, T. S. and Green, K. J.(1996). Breaking the connection: displacement of the desmosomal plaqueprotein desmoplakin from cell-cell interfaces disrupts anchorage ofintermediate filament bundles and alters intercellular junction assembly. J.Cell Biol. 134, 985-1001.

Brandner, J. M., Reidenbach, S. and Franke, W. W. (1997). Evidence thatpinin, reportedly a differentiation-specific desmosomal protein, is actuallya widespread nuclear protein. Differentiation 62, 119-127.

Burdett, I. D. (1998). Aspects of the structure and assembly of desmosomes.Micron 29, 309-328.

Buxton, R. S. and Magee, A. I. (1992). Structure and interactions ofdesmosomal and other cadherins. Semin. Cell Biol. 3, 157-167.

Chitaev, N. A., Leube, R. E., Troyanovsky, R. B., Eshkind, L. G., Franke,W. W. and Troyanovsky, S. M. (1996). The binding of plakoglobin to

A. J. North and others

4335Molecular map of desmosome

desmosomal cadherins: patterns of binding sites and topogenic potential. J.Cell Biol. 133, 359-369.

Chitaev, N. A., Averbakh, A. Z., Troyanovsky, R. B. and Troyanovsky, S.M. (1998). Molecular organization of the desmoglein-plakoglobin complex.J. Cell Sci. 111, 1941-1949.

Collins, J. E., Legan, P. K., Kenny, T. P., MacGarvie, J., Holton, J. L. andGarrod, D. R. (1991). Cloning and sequence analysis of desmosomalglycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesionmolecules with heterogeneous cytoplasmic domains. J. Cell Biol. 113, 381-391.

Cordingley, H. C. (1996). Investigation into the role of plakoglobin inXenopus development. Ph.D. thesis, University of London.

Cowin, P. and Burke, B. (1996). Cytoskeleton-membrane interactions. Curr.Opin. Cell Biol. 8, 56-65.

Cowin, P., Kapprell, H. P., Franke, W. W., Tamkun, J. and Hynes, R. O.(1986). Plakoglobin: a protein common to different kinds of intercellularadhering junctions. Cell 46, 1063-1073.

Eger, A., Stockinger, A., Wiche, G. and Foisner, R. (1997). Polarisation-dependent association of plectin with desmoplakin and the lateralsubmembrane skeleton in MDCK cells. J. Cell Sci. 110, 1307-1316.

Fawcett, D. W. (1961). Intercellular bridges. Exp. Cell Res. (suppl.) 8, 174-187.

Garrod, D. R., Parrish, E. P., Mattey, D. L., Marston, J. E., Measures, H.R. and Vilela, M. J. (1990). Desmosomes. In Morphoregulatory Molecules(ed. G. M. Edelman, B. A. Cunningham and J. P. Thiery), pp. 315-339. NewYork: John Wiley and Sons.

Garrod, D. R. (1993). Desmosomes and hemidesmosomes. Curr. Opin. CellBiol. 5, 30-40.

Green, K. J., Parry, D. A., Steinert, P. M., Virata, M. L., Wagner, R. M.,Angst, B. D. and Nilles, L. A. (1990). Structure of the human desmoplakins.Implications for function in the desmosomal plaque [published erratumappears in J. Biol. Chem. 265, 11406-11407]. J. Biol. Chem. 265, 2603-2612.

Green, K. J. and Jones, J. C. (1996). Desmosomes and hemidesmosomes:structure and function of molecular components. FASEB J. 10, 871-881.

Griffiths, G. (editor) (1993). Fine Structure Immunocytochemistry. SpringerVerlag, Heidelberg.

Hatzfeld, M., Kristjansson, G. I., Plessmann, U. and Weber, K. (1994).Band 6 protein, a major constituent of desmosomes from stratified epithelia,is a novel member of the armadillo multigene family. J. Cell Sci 107, 2259-2270.

Heid, H. W., Schmidt, A., Zimbelmann, R., Schafer, S., Winter-Simanowski, S., Stumpp, S., Keith, M., Figge, U., Schnolzer, M. andFranke, W. W. (1994). Cell type-specific desmosomal plaque proteins ofthe plakoglobin family: plakophilin 1 (band 6 protein). Differentiation 58,113-131.

Kapprell, H. P., Cowin, P. and Franke, W. W. (1987). Biochemicalcharacterization of the soluble form of the junctional plaque protein,plakoglobin, from different cell types. Eur. J. Biochem. 166, 505-517.

Kapprell, H. P., Owaribe, K. and Franke, W. W. (1988). Identification of abasic protein of Mr 75, 000 as an accessory desmosomal plaque protein instratified and complex epithelia. J. Cell Biol. 106, 1679-1691.

Kapprell, H. P., Duden, R., Owaribe, K., Schmelz, M. and Franke, W. W.(1990). Subplasmalemmal plaques of intercellular junctions: common anddistinguishing proteins. In Morphoregulatory Molecules (ed. G. M.Edelman, B. A. Cunningham and J. P. Thiery), pp. 285-314. New York: JohnWiley and Sons.

Kelly, D. E. (1966). Fine structure of desmosomes, hemidesmosomes, and anadepidermal globular layer in developing newt epidermis. J. Cell Biol. 28,51-72.

Kelly, D. E. and Shienvold, F. L. (1976). The desmosome: fine structuralstudies with freeze-fracture replication and tannic acid staining of sectionedepidermis. Cell Tissue Res. 172, 309-323.

Kelly, D. E. and Kuda, A. M. (1981). Traversing filaments in desmosomaland hemidesmosomal attachments: freeze-fracture approaches toward theircharacterization. Anat. Rec. 199, 1-14.

Koch, P. J. and Franke, W. W. (1994). Desmosomal cadherins: anothergrowing multigene family of adhesion molecules. Curr. Opin. Cell Biol. 6,682-687.

Kouklis, P. D., Hutton, E. and Fuchs, E. (1994). Making a connection: directbinding between keratin intermediate filaments and desmosomal proteins. J.Cell Biol. 127, 1049-1060.

Kowalczyk, A. P., Bornslaeger, E. A., Borgwardt, J. E., Palka, H. L.,Dhaliwal, A. S., Corcoran, C. M., Denning, M. F. and Green, K. J.(1997). The amino-terminal domain of desmoplakin binds to plakoglobin

and clusters desmosomal cadherin-plakoglobin complexes. J. Cell Biol. 139,773-784.

Kowalczyk, A. P., Navarro, P., Dejana, E., Bornslaeger, E. A., Green, K.J., Kopp, D. S. and Borgwardt, J. E. (1998). VE-cadherin and desmoplakinare assembled into dermal microvascular endothelial intercellular junctions:a pivotal role for plakoglobin in the recruitment of desmoplakin tointercellular junctions. J. Cell Sci. 111, 3045-3057.

Kowalczyk, A. P., Hatzfeld, M., Bornslaeger, E. A., Kopp, D. S.,Borgwardt, J. E., Corcoran, C. M., Settler, A. and Green, K. J. (1999).The head domain of plakophilin-1 binds to desmoplakin and enhances itsrecruitment to desmosomes: implications for cutaneous disease. J. Biol.Chem. 274, 18145-18148.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227, 680-685.

Legan, P. K., Yue, K. K., Chidgey, M. A., Holton, J. L., Wilkinson, R. W.and Garrod, D. R. (1994). The bovine desmocollin family: a new gene andexpression patterns reflecting epithelial cell proliferation and differentiation.J. Cell Biol. 126, 507-518.

Leloup, R., Laurent, L., Ronveaux, M. F., Drochmans, P. and Wanson, J.C. (1979). Desmosomes and desmogenesis in the epidermis of calf muzzle.Biol. Cellulaire 34, 137-152.

Mathur, M., Goodwin, L. and Cowin, P. (1994). Interactions of thecytoplasmic domain of the desmosomal cadherin Dsg1 with plakoglobin. J.Biol. Chem. 269, 14075-14080.

McGrath, J. A., McMillan, J. R., Shemanko, C. S., Runswick, S. K., Leigh,I. M., Lane, E. B., Garrod, D. R. and Eady, R. A. (1997). Mutations inthe plakophilin 1 gene result in ectodermal dysplasia/skin fragilitysyndrome. Nature Genet. 17, 240-244.

McNutt, N. S. and Weinstein, R. S. (1973). Membrane ultrastructure atmammalian intercellular junctions. Prog. Biophys. Mol. Biol. 26, 45-101.

Meng, J. J., Bornslaeger, E. A., Green, K. J., Steinert, P. M. and Ip, W.(1997). Two-hybrid analysis reveals fundamental differences in directinteractions between desmoplakin and cell type-specific intermediatefilaments. J. Biol. Chem. 272, 21495-21503.

Mertens, C., Kuhn, C. and Franke, W. W. (1996). Plakophilins 2a and 2b:constitutive proteins of dual location in the karyoplasm and the desmosomalplaque. J. Cell Biol. 135, 1009-2105.

Miller, K., Mattey, D., Measures, H., Hopkins, C. and Garrod, D. (1987).Localisation of the protein and glycoprotein components of bovine nasalepithelial desmosomes by immunoelectron microscopy. EMBO J. 6, 885-889.

Nilles, L. A., Parry, D. A., Powers, E. E., Angst, B. D., Wagner, R. M. andGreen, K. J. (1991). Structural analysis and expression of humandesmoglein: a cadherin-like component of the desmosome. J. Cell Sci. 99,809-821.

North, A. J., Chidgey, M. A., Clarke, J. P., Bardsley, W. G. and Garrod,D. R. (1996). Distinct desmocollin isoforms occur in the same desmosomesand show reciprocally graded distributions in bovine nasal epidermis. Proc.Nat. Acad. Sci. USA 93, 7701-7705.

O’Keefe, E. J., Erickson, H. P. and Bennett, V. (1989). Desmoplakin I anddesmoplakin II. Purification and characterization. J. Biol. Chem. 264, 8310-8318.

Ouyang, P. and Sugrue, S. P. (1996). Characterization of pinin, a novelprotein associated with the desmosome-intermediate filament complex. J.Cell Biol. 135, 1027-1042.

Parker, A. E., Wheeler, G. N., Arnemann, J., Pidsley, S. C., Ataliotis, P.,Thomas, C. L., Rees, D. A., Magee, A. I. and Buxton, R. S. (1991).Desmosomal glycoproteins II and III. Cadherin-like junctional moleculesgenerated by alternative splicing. J. Biol. Chem. 266, 10438-10445.

Parrish, E. P., Steart, P. V., Garrod, D. R. and Weller, R. O. (1987).Antidesmosomal monoclonal antibody in the diagnosis of intracranialtumours. J. Pathol. 153, 265-273.

Peifer, M., McCrea, P. D., Green, K. J., Wieschaus, E. and Gumbiner, B.M. (1992). The vertebrate adhesive junction proteins beta-catenin andplakoglobin and the segment polarity gene armadillo form a multigenefamily with similar properties. J. Cell Biol. 118, 681-691.

Rayns, D. G., Simpson, F. O. and Ledingham, J. M. (1969). Ultrastructureof desmosomes in mammalian intercalated disc; appearances afterlanthanum treatment. J. Cell Biol. 42, 322-326.

Rezniczek, G. A., de Pereda, J. M., Reipert, S. and Wiche, G. (1998).Linking integrin alpha6beta4-based cell adhesion to the intermediatefilament cytoskeleton: direct interaction between the beta4 subunit andplectin at multiple molecular sites. J. Cell Biol. 141, 209-225.

Riggleman, B., Wieschaus, E. and Schedl, P. (1989). Molecular analysis of

4336

the armadillo locus: uniformly distributed transcripts and a protein withnovel internal repeats are associated with a Drosophila segment polaritygene. Genes Dev. 3, 96-113.

Roberts, I. M. (1975). Tungsten coating–a method of improving glassmicrotome knives for cutting ultrathin sections. J. Microsc. 103, 113-119.

Ruediger, R., Hentz, M., Fait, J., Mumby, M. and Walter, G. (1994).Molecular model of the A subunit of protein phosphatase 2A: interactionwith other subunits and tumor antigens. J. Virol. 68, 123-129.

Ruhrberg, C. and Watt, F. M. (1997). The plakin family: versatile organizersof cytoskeletal architecture. Curr. Opin. Genet. Dev. 7, 392-397.

Rutman, A. J., Buxton, R. S. and Burdett, I. D. (1994). Visualisation byelectron microscopy of the unique part of the cytoplasmic domain of adesmoglein, a cadherin-like protein of the desmosome type of cell junction.FEBS Lett. 353, 194-196.

Schmidt, A., Langbein, L., Prätzel, S., Rode, M., Rackwitz, H. R. andFranke, W. W. (1999). Plakophilin 3 – a novel cell-type-specificdesmosomal plaque protein. Differentiation 64, 291-306.

Shimizu, H., Masunaga, T., Ishiko, A., Kikuchi, A., Hashimoto, T. andNishikawa, T. (1995). Pemphigus vulgaris and pemphigus foliaceus serashow an inversely graded binding pattern to extracellular regions ofdesmosomes in different layers of human epidermis. J. Invest. Dermatol.105, 153-159.

Skalli, O., Jones, J. C., Gagescu, R. and Goldman, R. D. (1994). IFAP 300is common to desmosomes and hemidesmosomes and is a possible linkerof intermediate filaments to these junctions. J. Cell Biol. 125, 159-170.

Small, J. V., Furst, D. O. and De Mey, J. (1986). Localization of filamin insmooth muscle. J. Cell Biol. 102, 210-220.

Smith, E. A. and Fuchs, E. (1998). Defining the interactions betweenintermediate filaments and desmosomes. J. Cell Biol. 141, 1229-12241.

Staehelin, L. A. (1974). Structure and function of intercellular junctions. Int.Rev. Cytol. 39, 191-283.

Stappenbeck, T. S. and Green, K. J. (1992). The desmoplakin carboxylterminus coaligns with and specifically disrupts intermediate filamentnetworks when expressed in cultured cells. J. Cell Biol. 116, 1197-1209.

Stappenbeck, T. S., Bornslaeger, E. A., Corcoran, C. M., Luu, H. H.,

Virata, M. L. and Green, K. J. (1993). Functional analysis of desmoplakindomains: specification of the interaction with keratin versus vimentinintermediate filament networks. J. Cell Biol. 123, 691-705.

Steinberg, M. S., Shida, H., Giudice, G. J., Shida, M., Patel, N. H. andBlaschuk, O. W. (1987). On the molecular organization, diversity andfunctions of desmosomal proteins. Ciba Found Symp. 125, 3-25.

Tokuyasu, K. T. (1980). Immunochemistry on ultrathin frozen sections.Histochem. J. 12, 381-403.

Tokuyasu, K. T. (1989). Use of poly(vinylpyrrolidone) and poly(vinylalcohol) for cryoultramicrotomy. Histochem. J. 21, 163-171.

Troyanovsky, R. B., Chitaev, N. A. and Troyanovsky, S. M. (1996).Cadherin binding sites of plakoglobin: localization, specificity and role intargeting to adhering junctions. J. Cell Sci. 109, 3069-3078.

Troyanovsky, S. M., Eshkind, L. G., Troyanovsky, R. B., Leube, R. E. andFranke, W. W. (1993). Contributions of cytoplasmic domains ofdesmosomal cadherins to desmosome assembly and intermediate filamentanchorage. Cell 72, 561-574.

Troyanovsky, S. M., Troyanovsky, R. B., Eshkind, L. G., Krutovskikh, V.A., Leube, R. E. and Franke, W. W. (1994). Identification of theplakoglobin-binding domain in desmoglein and its role in plaque assemblyand intermediate filament anchorage. J. Cell Biol. 127, 151-160.

Tsukita, S. and Tsukita, S. (1985). Desmocalmin: a calmodulin-binding highmolecular weight protein isolated from desmosomes. J. Cell Biol. 101,2070-2080.

Wahl, J. K., Sacco, P. A., McGranahan-Sadler, T. M., Sauppe, L. M.,Wheelock, M. J. and Johnson, K. R. (1996). Plakoglobin domains thatdefine its association with the desmosomal cadherins and the classicalcadherins: identification of unique and shared domains. J. Cell Sci. 109,1143-1154.

Witcher, L. L., Collins, R., Puttagunta, S., Mechanic, S. E., Munson, M.,Gumbiner, B. and Cowin, P. (1996). Desmosomal cadherin bindingdomains of plakoglobin. J. Biol. Chem. 271, 10904-10909.

Yap, A. S., Niessen, C. M. and Gumbiner, B. M. (1998). The juxtamembraneregion of the cadherin cytoplasmic tail supports lateral clustering, adhesivestrengthening, and interaction with p120ctn. J. Cell Biol. 141, 779-789.

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