THE STRIATED MUSCULATURE OF BLOOD VESSELS : II. Cell Interconnections and Cell Surface

The interconnections and the surfaces of the striated muscle cells which occur in thoracic and in lung veins of the mouse were studied with the electron microscope. The osmium-fixed tissues were embedded in methacrylate or in araldite and sectioned with a Porter-Blum microtome. Many preparations were stained before embedding with phosphotungstic acid or after sectioning with uranyl acetate. Typical intercalated discs are observed in this muscle. They are similar to the discs found in heart muscle. These intercalated discs represent boundaries between separate muscle cells. Along the discs, cells are joined in planes normal to their myofilaments. The same cells are also joined in planes parallel to the myofilaments by means of lateral interconnections. These lateral cell boundaries are in continuity with the intercalated discs. Three morphologically distinct parts occur within the lateral cell interconnections: One is characterized by small vesicles along the plasma membrane, the second part has the structure of desmosomes, and a third part represents an external compound membrane (formed by the two plasma membranes of the adjoining cells) and is termed "quintuple-layered cell interconnection." Small vesicles and plasma membrane enfoldings along the free surface of muscle cells are interpreted as products of a pinocytosis (phagocytosis) process. Some of them are seen to contain small membrane-bounded bodies or granules. The free cell surface shows a characteristic outer dense layer ("basement membrane") which accompanies the plasma membrane. The topographic relation of this dense layer with the plasma membrane seems to vary in different preparations. The significance of this variation is not well understood. On two occasions a typical arrangement o vesicles and tubules was observed at Z band levels, just beneath the plasma membrane. These structures are believed to represent endoplasmic reticulum. Their possible significance for the conduction of excitation is discussed.     

Observations on the Fine Structure of the Turtle Atrium

The general fine structure of the atrial musculature of the turtle heart is described, including; the nature of the sarcolemma; the cross-banded structure of the myofibrils; the character of the sarcoplasm, and the form and disposition of its organelles. An abundant granular component of the sarcoplasm in this species is tentatively identified as a particulate form of glycogen. The myocardium is composed of individual cells joined end to end at primitive intercalated discs, and side to side at sites of cohesion that resemble the desmosomes of epithelia. Transitional forms are found between desmosomes and intercalated discs. Both consist of a thickened area of the cell membrane with an accumulation of dense material in the subjacent cytoplasm. This dense amorphous component is often continuous with the Z substance of the myofibrils and may be of the same composition. The observations reported reemphasize the basic similarity between desmosomes and terminal bars of epithelia and intercalated discs of cardiac muscle. Numerous unmyelinated nerves are found beneath the endocardium. Some of these occupy recesses in the surface of Schwann cells; others are naked axons. No specialized nerve endings are found. Axons passing near the sarcolemma contain synaptic vesicles, and it is believed that this degree of proximity is sufficient to constitute a functioning myoneural junction.     

Structure and Variation in the Sarcotubular System of the Extraocular Muscle Fibres in the Shark Scyliorhinus canicula L.

The microscopic anatomy of the extraocular eye muscles of the shark Scyliorhinus canicula is described. In contrast to swimming muscles, the fibres are not differentiated into distinct fibre type groups. The sarcoplasmic reticulum has narrow terminal cisternae in the Z band region, longitudinal tubules and a fenestrated cisterna in the H band region. Triads or dyads are mostly located at the Z discs. A number of variations from this general pattern is described, with main emphasis on triad location, orientation and structure. Since triads in vertebrates usually are located either at the Z disc or at the A-I junction, and with a fixed structure, these variations in shark muscles are of phylogenetic interest, as they may provide basis for an explanation of development to the two different triad locations.     

Ultrastructure of atrial and ventricular myocardium in the pike Esox lucius L. and mackerel Scomber scombrus L. (Pisces)

Summary Atrial and ventricular muscle in the pike and mackerel hearts consists of narrow, branching cells. The atrial cells in the two species are similar whereas the ventricular cells differ. The sarcolemma is attached to the Z and M lines of the sarcomere. Intercalated discs are common, and the transverse parts display desmosomes and intermediate junctions. Nexuses are uncommon and only occur in the longitudinal parts of the intercalated discs. The sarcoplasmic reticulum forms a regular hexagonal network on the myofibrillar surface. Subsarcolemmal cisternae form peripheral couplings at the I-A level. Junctional processes are usually inconspicuous, but an electron dense substance is present between the sarcolemma and the Junctional sarcoplasmic reticulum. Specific heart granules are common in atrial cells of both species and in ventricular cells of the pike, but are very scarce in mackerel ventricular muscle.This work was supported by grants from the Norwegian Research Council for Sciences and the Humanities     

CARDIAC MUSCLE OF THE HORSESHOE CRAB, LIMULUS POLYPHEMUS : I. Ultrastructure

The fine structure of the cardiac muscle of the horseshoe crab, Limulus polyphemus, has been studied with respect to the organization of its contractile material, and the structure of its organelles and the cell junctions. Longitudinal sections show long sarcomeres (5.37 µ at L_max), wide A bands (2.7 µ), irregular Z lines, no M line, and no apparent H zone. Transverse sections through the S zone of the A band show that each thick filament is ca. 180 A in diameter, is circular in profile with a center of low density, and is surrounded by an orbit of 9–12 thin filaments, each 60 A in diameter. Thick filaments are confined to the A band: thin filaments originate at the Z band, extend through the I band, and pass into the A band between the thick filaments. The sarcolemmal surface area is increased significantly by intercellular clefts. Extending into the fiber from these clefts and from the sarcolemma, T tubules pass into the fiber at the A-I level. Each fibril is enveloped by a profuse membranous covering of sarcoplasmic reticulum (SR). Sacculations of the SR occur at the A-I boundary where they make diadic contact with longitudinal branches of the T system. These branches also extend toward the Z, enlarge at the Z line, and pass into the next sarcomere. Infrequently noted were intercalated discs possessing terminal insertion and desmosome modifications, but lacking close junctions (fasciae occludentes). These structural details are compared with those of mammalian cardiac and invertebrate muscles.     

Zeugmatin: a new high molecular weight protein associated with Z lines in adult and early embryonic striated muscle

Monoclonal antibodies were generated to a purified preparation of the fascia adherens domains of the intercalated discs of chicken cardiac cell membranes. One of these antibodies, McAb 20, immunofluorescently labeled the Z lines of adult skeletal muscle, the Z lines and intercalated discs of adult cardiac muscle, and the dense bodies and dense plaques of adult gizzard smooth muscle. In addition, McAb 20 was found to label regenerating muscle cells in a cross-striated pattern much like that of Z lines in 24-h muscle cell cultures before the appearance of Z lines was detectable by phase or Nomarski optics and before the concentration of alpha-actinin occurred at the Z lines. Thus, McAb 20 appears to be directed against an antigen involved in early myofibrillar organization. Preliminary biochemical characterization of the antigen recognized by McAb 20 indicates that it is a high molecular weight doublet of over 5 X 10(5) kD that is highly susceptible to proteolysis. By virtue of its presence in Z lines, and its possible role in the end-on attachment of microfilaments to Z lines and membranes, we have named this protein zeugmatin (xi epsilon nu gamma mu alpha identical to yoking).     

A light and electron microscopic study of he heart of a crayfish, Procambarus clarkii (Giraud). II. Fine structure

The adventitia of the crayfish heart is composed of cells that are separated from each other by an intercellular space about 280 Å wide. Desmosomes are present on apposing surfaces of adjacent cells. A basal lamina underlies the adventitia and consists of a dense, amorphous substance that contains numerous fine filaments. The myocardial cells are striated and an external lamina 0.1 μ thick is present on the surface of the plasma membrane. The nuclei and most of the cytoplasm, glycogen and mitochondria are located at the cell periphery. The myofibrils are composed of thick and thin filaments and confined to the core of the cell. A T system and a well-developed SR are present. Elements of these organelles form dyads at levels that correspond to the H bands, and triads at levels that correspond to the Z bands of the peripheral myofibrils. The relationship of the T tubules to the myofibrils is discussed. Locus cells exhibit a unique pattern of intracellular myofibrillar branching. They branch from a region which has a structure similar to the Z band material. The myofibrils radiate outwardly in various directions and form numerous cellular branches which form intercalated discs with adjacent myocardial cells. These discs are more complex than those observed in poikilothermic vertebrates but are simpler than those in mammals. An endocardium is lacking in the crayfish heart but interstitial cells are present in close association with the myocardial cells and neural elements. Terminal nerve processes deeply embedded in the myocardial cells are described.     

FINE STRUCTURE OF THE SYNAPTIC DISCS SEPARATED FROM THE GOLDFISH MEDULLA OBLONGATA

Synaptic discs are structures localized in the club ending synapses on the Mauthner cell lateral dendrite of the goldfish medulla oblongata. The synaptic discs present a hexagonal array of particles ~8.5 nm center-to-center when observed in en face view. This lattice covers the entire surface Divalent cations are important in the stabilization of this particular hexagonal array of particles When a synaptic disc-rich fraction is treated with chelating agents (EDTA or EGTA), definite changes occur in the hexagonal lattice. First, the synaptic membranes show zones without particles interspersed with zones covered with the hexagonal array of particles Second, the synaptic discs break down and a new structure characterized by two parallel dense bands (7 nm each), separated by a 4 nm gap, is observed. The negative stain fills the gap region showing striations spaced ~10 nm center-to-center crossing the gap, but it does not penetrate the dense bands This "double band" structure is interpreted as an edge on view of a fragment of the synaptic membrane complex. Further treatment of this fraction with a chelating agent plus 0.3% deoxycholate produces an increase in the number of double band structures. However, EDTA plus Triton X-100 (a treatment known to produce solubilization of membrane proteins) never shows such double band structure An ordered material was observed associated with the cytoplasmic leaflets of the double bands This material consists of rows of beads ~4 nm in diameter and spaced at intervals of ~7 nm. Each of these beads is joined to the band by a thin stalk.     

alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane.     

Ultrastructure of the heart of the sturgeon Acipenser stellatus (Chondrostei)

Summary The ultrastructure of atrial and ventricular myocardial cells from Acipenser stellatus is described. The cells of the atrium are more loosely connected than those of the ventricle. Cell contact is by simple intercalated discs and by desmosomes. The cells are flattened, with peripheral myofibrils and a central region of mitochondria and the nucleus. The sarcoplasmic reticulum consists of subsarcolemmal tubules, that frequently extend towards the central mitochondria. Dyads are small and positioned at any sarcomeric level. No T-tubules are present. Specific granules are restricted to the atrial cell, and are sometimes present within the SR tubules.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

Histological and ultrastructural abnormalities in murine desmoglein 2-mutant hearts

Mice carrying a deletion of the adhesive extracellular domain of the desmosomal cadherin desmoglein 2 develop an arrhythmogenic right ventricular cardiomyopathylike phenotype with ventricular dilation, fibrosis and arrhythmia. To unravel the sequence of myocardial alterations and to identify potential pathomechanisms, histological analyses were performed on mutant hearts from the juvenile to the adult state, i.e., between 2 and 13 weeks. At an age of 2 weeks 30% of mutants presented lesions,which were visible as white plaques on the heart surface or in the septum. From 4 weeks onwards, all mutants displayed a cardiac phenotype. Dying cardiomyocytes with calcification were found in lesions of all ages. But lesions of young mutant animals contained high amounts of CD45+ immune cells and little collagen fibers, whereas lesions of the older animals were collagen-rich and harbored only a small but still significantly increased number of CD45+ cells. Electron microscopy further showed that distinct desmosomes cannot be distinguished in intercalated discs of mutant hearts. Widening of the intercellular cleft and even complete dissociation of intercalated discs were often observed close to lesions. Disturbed sarcomer structure, altered Z-discs, multiple autophagic vacuoles and swollen mitochondria were other prominent pathological features. Taken together, the following scenario is suggested: mutant desmoglein 2 cannot fully support the increased mechanical requirements placed on intercalated disc adhesion during postnatal heart development, resulting in compromised adhesion and cell stress. This induces cardiomyocyte death, aseptic inflammation and fibrotic replacement. The acute stage of scar formation is followed by permanent impairment of the cardiac function.     

The existence of an insoluble Z disc scaffold in chicken skeletal muscle

Extraction of glycerinated chicken skeletal muscle with 0.6 M potassium iodide leaves a framework of insoluble components within each muscle fiber. This framework is composed primarily of planes of in-register Z discs that have been thickened by the accumulation of material on both sides of each disc during extraction. Membrane vesicles, presumably remnants of the T system, remain surrounding the Z discs. When the framework is sheared in a blender, it is preferentially cleaved between Z planes, resulting in the formation of large sheets of interconnected, closely packed Z discs in a honeycomb-like array. Cleavage occurs in regions formerly occupied by the A bands, which have been weakened by the removal of myosin. The existence and stability of these planar Z disc arrays demonstrate the presence and strength of connections between adjacent myofibrils.SDS-polyacrylamide gel electrophoresis reveals that this framework consists primarily of actin and desmin, with lesser amounts of a few proteins including α-actinin, myosin and tropomyosin. Z disc sheets and KI-extracted myofibrils provide a distinct face-on view and side view, respectively, of the Z disc. In indirect immunofluorescence, these two views have revealed that desmin is present at the periphery of each Z disc, forming a network of proteinaceous collars within the Z plane. α-Actinin is localized within each disc, giving a face-on fluorescence pattern that is complementary to that of desmin. Actin is present throughout the thickened Z plane, while myosin and tropomyosin exist only in the insoluble residue that coalesces on both faces of each disc.We conclude that desmin, perhaps in conjunction with actin, is responsible for interlinking Z discs of adjacent myofibrils, and may thus serve as a mechanical and structural integrator of muscle fibers. Its hydrophobic nature and coincident distribution with the T system suggest that it may also be responsible for mediating filament-membrane interactions and anchoring the triad to the Z disc. Its collar-like distribution suggests that it may aid in maintaining the structural integrity of the Z disc and the actin filaments inserted into it.     

Electron microscope study of the developing chick embryo heart

Summary Myofibrillar differentiation in heart myocells of the chick embryo was studied with aid of the electronmicroscope. At early stages (29 hrs.) thin myofibrillar bundles are seen already differentiated in the cytoplasm of the myoblast and a few hours later cross striation can be depicted as ill-defined densities regularly spaced along the myofilaments.At later stages it was seen that some myofibrillar bundles seem to arise from a Z-band belonging to a thicker bundle. This disposition was frequently seen but the number of bundles observed arising from a single Z-band is varied. Cases were found in which two or three bundles were interconnected by myofibrillar bridges. The disposition was nominated Z center and it is interpreted as favouring spreading of the contractil wave from bundle to bundle. Conduction of the contractil wave from cell to cell is insured through intercalated discs.Desmosomes and intercalated discs were observed during the course of this study, but no relationship between both components of the cell membranes nor between desmosomes and myofilaments were found.Fellow working under a local fellowship program supported by Grant No. 56030 of The Rockefeller Foundation.     

ISOLATION OF EPIDERMAL DESMOSOMES

A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55–60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.     

FINE STRUCTURE OF THE MYOEPITHELIUM OF THE ECCRINE SWEAT GLANDS OF MAN

The secretory coils of glutaraldehyde-osmium tetroxide-fixed and Epon-Araldite-embedded eccrine sweat glands from the palms of young men were studied with the electron microscope. The myoepithelial cells lie on the epithelial side of the basement membrane and abut other epithelial elements directly. The irregularly serrated base of the cell has dense thickenings along the plasma membrane which alternate with zones bearing pits; the smooth apical surface lacks dense thickenings, is studded with pits, and conjoined to secretory cells by occasional desmosomes. Masses of myofilaments, 50 A in diameter, fill most of the cell and are associated with irregular dense zones. In cross-section the arrangement of the myofilaments seems identical with that of the I band of striated muscle, and the dense zone has typical Z band structure. A few microtubules and cytoplasmic cores bearing profiles of the endoplasmic reticulum, filamentous mitochondria, and glycogen granules penetrate the fibrillar masses and run parallel to the oriented myofilaments. In the perinuclear zone, Golgi membranes, rough- and smooth-surfaced elements of the endoplasmic reticulum, mitochondria, glycogen, microtubules, lipid, pigment, and dense granules are variable components in the cytoplasm. The interrelationships of the myoepithelial cells with the secretory cells suggest that the former may act as regulators, controlling the flow of metabolites to the secretory epithelium.     

Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC,Microtubules and Lysosomal/Proteasomal Degradation

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds.     

Isolation and symmetrical splitting of desmosomal structures in 9 M urea

A new way of isolating desmosomal structures from various epithelia is described which takes advantage of the unusual resistance of the desmosomal plaque and parts of the desmosomal membrane domain to denaturing agents such as 9 M urea and 5 M guanidinium hydrochloride (Gdn-HCl). The fractions obtained have been examined by electron microscopy and by gel electrophoresis. When cytoskeletal fractions from epithelial cells, notably those from multistratified epithelia such as bovine epidermis or tongue mucosa, are treated with urea or Gdn-HCl most of the cytoskeletal protein, including cytokeratin material, is removed. The desmosomal structures, however, are retained with well preserved plaque organization and desmoglea components and can be harvested by centrifugation. This simple and rapid procedure for the enrichment of desmosomal structures and proteins also express internal desmosomal domains as the result of "splitting" of the desmosome along the midline structure. These split desmosomal halves reveal regular arrays of desmogleal particles of 8 to 15 nm diameter projecting from the membrane surface. Gel electrophoresis of the polypeptides present in these residual structures has shown prominent amounts of desmoplakins I and II as well as components 3 and 5 whereas glycoproteins 4a and 4b are significantly reduced in relation to untreated or citric acid-treated fractions. Using immunoelectron microscopy on desmosomes split in urea we have also demonstrated the specific localization of desmoplakin on the cytoplasmic side. The observations suggest that the architectural components of the desmosome are among the cell structures most resistant to protein-denaturing treatments. The value of this procedure for preparations of desmosomal proteins and for the production of antibodies specifically reacting with internal domains of junctions, i.e., tools that may interfere with cell-to-cell coupling, is discussed.