Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into 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 of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.

Amagai
M.
,
Klaus-Kovtun
V.
,
Stanley
J. R.
(
1991
).
Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.
Cell
67
,
869
877
Amagai
M.
(
1995
).
Adhesion molecules. I: Keratinocyte-keratinocyte interactions; cadherins and pemphigus.
J. Invest. Dermatol
104
,
146
152
Angst
B. D.
,
Nilles
L. A.
,
Green
K. J.
(
1990
).
Desmoplakin II expression 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.
,
Hughes
A. E.
(
1999
).
Haploinsufficiency of desmoplakin causes a striate subtype of palmoplantar keratoderma.
Human Mol. Genet
8
,
143
148
Arnemann
J.
,
Sullivan
K. H.
,
Magee
A. I.
,
King
I. A.
,
Buxton
R. S.
(
1993
).
Stratification-related expression of isoforms of the desmosomal cadherins in human epidermis.
J. Cell Sci
104
,
741
750
Bardsley
W. G.
,
Ackerman
R. A.
,
Bukhari
N. A.
,
Deeming
D. C.
,
Ferguson
M. W.
(
1995
).
Mathematical models for growth in alligator (Alligator mississippiensis) embryos developing at different incubation temperatures.
J. Anat
187
,
181
190
Bardsley
W. G.
,
Bukhari
N. A. J.
,
Ferguson
M. W. J.
,
Cachaza
J. A.
,
Burguillo
F. J.
(
1995
).
Evaluation of model discrimination, parameter estimation and goodness of fit in nonlinear regression problems by test statistics distributions.
Comput. Chem
19
,
75
84
Bonne
S.
,
van Hengel
J.
,
Nollet
F.
,
Kools
P.
,
van Roy
F.
(
1999
).
Plakophilin-3, a novel Armadillo-like protein present in nuclei and desmosomes of epithelial cells.
J. Cell Sci
112
,
2265
2276
Bornslaeger
E. A.
,
Corcoran
C. M.
,
Stappenbeck
T. S.
,
Green
K. J.
(
1996
).
Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.
J. Cell Biol
134
,
985
1001
Brandner
J. M.
,
Reidenbach
S.
,
Franke
W. W.
(
1997
).
Evidence that pinin, reportedly a differentiation-specific desmosomal protein, is actually a 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.
,
Magee
A. I.
(
1992
).
Structure and interactions of desmosomal and other cadherins.
Semin. Cell Biol
3
,
157
167
Chitaev
N. A.
,
Leube
R. E.
,
Troyanovsky
R. B.
,
Eshkind
L. G.
,
Franke
W. W.
,
Troyanovsky
S. M.
(
1996
).
The binding of plakoglobin todesmosomal cadherins: patterns of binding sites and topogenic potential.
J. Cell Biol
133
,
359
369
Chitaev
N. A.
,
Averbakh
A. Z.
,
Troyanovsky
R. B.
,
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.
,
Garrod
D. R.
(
1991
).
Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.
J. Cell Biol
113
,
381
391
Cowin
P.
,
Burke
B.
(
1996
).
Cytoskeleton-membrane interactions.
Curr. Opin. Cell Biol
8
,
56
65
Cowin
P.
,
Kapprell
H. P.
,
Franke
W. W.
,
Tamkun
J.
,
Hynes
R. O.
(
1986
).
Plakoglobin: a protein common to different kinds of intercellular adhering junctions.
Cell
46
,
1063
1073
Eger
A.
,
Stockinger
A.
,
Wiche
G.
,
Foisner
R.
(
1997
).
Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells.
J. Cell Sci
110
,
1307
1316
Garrod
D. R.
(
1993
).
Desmosomes and hemidesmosomes.
Curr. Opin. Cell Biol
5
,
30
40
Green
K. J.
,
Parry
D. A.
,
Steinert
P. M.
,
Virata
M. L.
,
Wagner
R. M.
,
Angst
B. D.
,
Nilles
L. A.
(
1990
).
Structure of the human desmoplakins. Implications for function in the desmosomal plaque [published erratum appears in J. Biol. Chem.265, 11406–11407].
J. Biol. Chem
265
,
2603
2612
Green
K. J.
,
Jones
J. C.
(
1996
).
Desmosomes and hemidesmosomes: structure and function of molecular components.
FASEB J
10
,
871
881
Hatzfeld
M.
,
Kristjansson
G. I.
,
Plessmann
U.
,
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.
,
Franke
W. W.
(
1994
).
Cell type-specific desmosomal plaque proteins of the plakoglobin family: plakophilin 1 (band 6 protein).
Differentiation
58
,
113
131
Kapprell
H. P.
,
Cowin
P.
,
Franke
W. W.
(
1987
).
Biochemical characterization 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.
,
Franke
W. W.
(
1988
).
Identification of a basic protein of Mr 75, 000 as an accessory desmosomal plaque protein in stratified and complex epithelia.
J. Cell Biol
106
,
1679
1691
Kelly
D. E.
(
1966
).
Fine structure of desmosomes, hemidesmosomes, and an adepidermal globular layer in developing newt epidermis.
J. Cell Biol
28
,
51
72
Kelly
D. E.
,
Shienvold
F. L.
(
1976
).
The desmosome: fine structural studies with freeze-fracture replication and tannic acid staining of sectioned epidermis.
Cell Tissue Res
172
,
309
323
Kelly
D. E.
,
Kuda
A. M.
(
1981
).
Traversing filaments in desmosomal and hemidesmosomal attachments: freeze-fracture approaches toward their characterization.
Anat. Rec
199
,
1
14
Koch
P. J.
,
Franke
W. W.
(
1994
).
Desmosomal cadherins: another growing multigene family of adhesion molecules.
Curr. Opin. Cell Biol
6
,
682
687
Kouklis
P. D.
,
Hutton
E.
,
Fuchs
E.
(
1994
).
Making a connection: direct binding 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.
,
Green
K. J.
(
1997
).
The amino-terminal domain of desmoplakin binds to plakoglobinand 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.
,
Borgwardt
J. E.
(
1998
).
VE-cadherin and desmoplakin are assembled into dermal microvascular endothelial intercellular junctions: a pivotal role for plakoglobin in the recruitment of desmoplakin to intercellular 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.
,
Green
K. J.
(
1999
).
The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes: implications for cutaneous disease.
J. Biol. Chem
274
,
18145
18148
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Legan
P. K.
,
Yue
K. K.
,
Chidgey
M. A.
,
Holton
J. L.
,
Wilkinson
R. W.
,
Garrod
D. R.
(
1994
).
The bovine desmocollin family: a new gene and expression patterns reflecting epithelial cell proliferation and differentiation.
J. Cell Biol
126
,
507
518
Leloup
R.
,
Laurent
L.
,
Ronveaux
M. F.
,
Drochmans
P.
,
Wanson
J. C.
(
1979
).
Desmosomes and desmogenesis in the epidermis of calf muzzle.
Biol. Cellulaire
34
,
137
152
Mathur
M.
,
Goodwin
L.
,
Cowin
P.
(
1994
).
Interactions of the cytoplasmic 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.
,
Eady
R. A.
(
1997
).
Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome.
Nature Genet
17
,
240
244
McNutt
N. S.
,
Weinstein
R. S.
(
1973
).
Membrane ultrastructure at mammalian intercellular junctions.
Prog. Biophys. Mol. Biol
26
,
45
101
Meng
J. J.
,
Bornslaeger
E. A.
,
Green
K. J.
,
Steinert
P. M.
,
Ip
W.
(
1997
).
Two-hybrid analysis reveals fundamental differences in direct interactions between desmoplakin and cell type-specific intermediate filaments.
J. Biol. Chem
272
,
21495
21503
Mertens
C.
,
Kuhn
C.
,
Franke
W. W.
(
1996
).
Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque.
J. Cell Biol
135
,
1009
2105
Miller
K.
,
Mattey
D.
,
Measures
H.
,
Hopkins
C.
,
Garrod
D.
(
1987
).
Localisation of the protein and glycoprotein components of bovine nasal epithelial desmosomes by immunoelectron microscopy.
EMBO J
6
,
885
889
Nilles
L. A.
,
Parry
D. A.
,
Powers
E. E.
,
Angst
B. D.
,
Wagner
R. M.
,
Green
K. J.
(
1991
).
Structural analysis and expression of human desmoglein: 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.
,
Garrod
D. R.
(
1996
).
Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.
Proc. Nat. Acad. Sci. USA
93
,
7701
7705
O'Keefe
E. J.
,
Erickson
H. P.
,
Bennett
V.
(
1989
).
Desmoplakin I and desmoplakin II. Purification and characterization.
J. Biol. Chem
264
,
8310
8318
Ouyang
P.
,
Sugrue
S. P.
(
1996
).
Characterization of pinin, a novel protein 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.
,
Buxton
R. S.
(
1991
).
Desmosomal glycoproteins II and III. Cadherin-like junctional molecules generated by alternative splicing.
J. Biol. Chem
266
,
10438
10445
Parrish
E. P.
,
Steart
P. V.
,
Garrod
D. R.
,
Weller
R. O.
(
1987
).
Antidesmosomal monoclonal antibody in the diagnosis of intracranial tumours.
J. Pathol
153
,
265
273
Peifer
M.
,
McCrea
P. D.
,
Green
K. J.
,
Wieschaus
E.
,
Gumbiner
B. M.
(
1992
).
The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the segment polarity gene armadillo form a multigene family with similar properties.
J. Cell Biol
118
,
681
691
Rayns
D. G.
,
Simpson
F. O.
,
Ledingham
J. M.
(
1969
).
Ultrastructure of desmosomes in mammalian intercalated disc; appearances after lanthanum treatment.
J. Cell Biol
42
,
322
326
Rezniczek
G. A.
,
de Pereda
J. M.
,
Reipert
S.
,
Wiche
G.
(
1998
).
Linking integrin alpha6beta4-based cell adhesion to the intermediate filament cytoskeleton: direct interaction between the beta4 subunit and plectin at multiple molecular sites.
J. Cell Biol
141
,
209
225
Riggleman
B.
,
Wieschaus
E.
,
Schedl
P.
(
1989
).
Molecular analysis ofthe armadillo locus: uniformly distributed transcripts and a protein with novel internal repeats are associated with a Drosophila segment polarity gene.
Genes Dev
3
,
96
113
Roberts
I. M.
(
1975
).
Tungsten coating—a method of improving glass microtome knives for cutting ultrathin sections.
J. Microsc
103
,
113
119
Ruediger
R.
,
Hentz
M.
,
Fait
J.
,
Mumby
M.
,
Walter
G.
(
1994
).
Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens.
J. Virol
68
,
123
129
Ruhrberg
C.
,
Watt
F. M.
(
1997
).
The plakin family: versatile organizers of cytoskeletal architecture.
Curr. Opin. Genet. Dev
7
,
392
397
Rutman
A. J.
,
Buxton
R. S.
,
Burdett
I. D.
(
1994
).
Visualisation by electron microscopy of the unique part of the cytoplasmic domain of a desmoglein, 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.
,
Franke
W. W.
(
1999
).
Plakophilin 3—a novel cell-type-specific desmosomal plaque protein.
Differentiation
64
,
291
306
Shimizu
H.
,
Masunaga
T.
,
Ishiko
A.
,
Kikuchi
A.
,
Hashimoto
T.
,
Nishikawa
T.
(
1995
).
Pemphigus vulgaris and pemphigus foliaceus sera show an inversely graded binding pattern to extracellular regions of desmosomes in different layers of human epidermis.
J. Invest. Dermatol
105
,
153
159
Skalli
O.
,
Jones
J. C.
,
Gagescu
R.
,
Goldman
R. D.
(
1994
).
IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions.
J. Cell Biol
125
,
159
170
Small
J. V.
,
Furst
D. O.
,
De Mey
J.
(
1986
).
Localization of filamin in smooth muscle.
J. Cell Biol
102
,
210
220
Smith
E. A.
,
Fuchs
E.
(
1998
).
Defining the interactions between intermediate 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.
,
Green
K. J.
(
1992
).
The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks 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.
,
Green
K. J.
(
1993
).
Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.
J. Cell Biol
123
,
691
705
Steinberg
M. S.
,
Shida
H.
,
Giudice
G. J.
,
Shida
M.
,
Patel
N. H.
,
Blaschuk
O. W.
(
1987
).
On the molecular organization, diversity and functions 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(vinyl alcohol) for cryoultramicrotomy.
Histochem. J
21
,
163
171
Troyanovsky
R. B.
,
Chitaev
N. A.
,
Troyanovsky
S. M.
(
1996
).
Cadherin binding sites of plakoglobin: localization, specificity and role in targeting to adhering junctions.
J. Cell Sci
109
,
3069
3078
Troyanovsky
S. M.
,
Eshkind
L. G.
,
Troyanovsky
R. B.
,
Leube
R. E.
,
Franke
W. W.
(
1993
).
Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.
Cell
72
,
561
574
Troyanovsky
S. M.
,
Troyanovsky
R. B.
,
Eshkind
L. G.
,
Krutovskikh
V. A.
,
Leube
R. E.
,
Franke
W. W.
(
1994
).
Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage.
J. Cell Biol
127
,
151
160
Tsukita
S.
,
Tsukita
S.
(
1985
).
Desmocalmin: a calmodulin-binding high molecular 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.
,
Johnson
K. R.
(
1996
).
Plakoglobin domains that define its association with the desmosomal cadherins and the classical cadherins: 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.
,
Cowin
P.
(
1996
).
Desmosomal cadherin binding domains of plakoglobin.
J. Biol. Chem
271
,
10904
10909
Yap
A. S.
,
Niessen
C. M.
,
Gumbiner
B. M.
(
1998
).
The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn.
J. Cell Biol
141
,
779
789
This content is only available via PDF.