Isolation of Epidermal Desmosomes

When a basal epidermal cell undergoes a commitment to terminally differentiate, it ceases to divide and begins to migrate outward towards the surface of the skin. Dramatic changes in its cytoskeletal architecture take place, accompanied by numerous changes in the expression of keratins, a family of related polypeptides that form 8-nm filaments in these cells. We show here that a shift to the synthesis of unusually large keratins occurs that does not seem to disrupt the ratio of two distinct subfamilies of keratins. Preliminary studies indicate that this differentiation-specific shift may be at the level of transcriptional rather than post-trancriptional regulation. The striking similarities between these large keratins and the type I and type II keratins of basal epidermal cells suggests the important role that both classes of large keratin sequences must play in the assembly of the intermediate filaments within the differentiating keratinocyte.Several laboratories have reported differences between the keratins of stratum corneum and those of the living layers of epidermis (1--4). During the course of terminal differentiation, changes occur in the expression of the keratin polypeptides that comprise the 8-nm tonofilaments (5-10). For the human, keratins of size 46, 48.5, 50, 52, 56, and 58 kd are synthesized by the basal epidermal cells (Fig. 1, lane 1), whereas additional keratins of size 67, 65.5, and 56.5 kd are produced by the terminally differentiating epidermis (lane 3). As the cells pass through the granular layer to the stratum corneum, a slight reduction in the size of the keratins takes place. As judged by in vitro translation of mRNAs isolated from basal (lane 2) and differentiating (lane 4) keratinocytes, the changes in keratin pattern that occur early in the course of terminal differentiation are clearly at the level of mRNA biosynthesis (5).Recently, it was demonstrated that the keratins produced by basal epidermal cells can be divided into two distinct groups based on the ability of their mRNAs to cross-hybridize with two different cloned keratin cDNAs (11). Other epithelia express different subsets of keratins, but most if not all of these seem to be similar to one or the other of the two epidermal keratin subfamilies (12). The small (40-52 kd) and relatively acidic keratins have been named the type I class, and the large (53-58 kd) and more basic keratins have been named the type II class (13,14). At least one member of each of the two keratin types seem to be expressed in all cells at all times, suggesting their combined importance in filament assembly (14). Sequence analyses (13,(15)(16)(17)(18) have shown that while the two types of keratins share only low (<30%) homology, their predicted secondary structures are strikingly similar and are compatible with their playing an essential role in forming the coiled-coil backbone of the protofilament of the 8-nm keratin filament (13,18).The keratins typical of terminally differentiating keratinocytes seem to be unusually large and are not fo...

Section: Discussionmentioning

confidence: 99%

Expression of unusually large keratins during terminal differentiation: balance of type I and type II keratins is not disrupted.

Kim¹,

Marchuk²,

Fuchs³

1984

“…These bands can sometimes be resolved into a number of closely running subcomponents. They presumably correspond to the two periodic acid-Schiff-positive bands reported by Skerrow and Matoltsy (30,31) in their electrophoretic analysis, in which stacking capabilities were lacking. In addition, we shown an additional desmosomal constituent not reported by these authors.…”

Section: Discussionmentioning

confidence: 99%

“…Our modifications of the original technique for the isolation of desmosomes from bovine muzzle epidermis (30) include the use of a nonionic detergent and sonication to free desmosomes from intracellular organelles and from adjacent plasma membrane . Subsequently, through differential centrifugation, we obtain preparations of whole desmosomes morphologically and 246 RAPID COMMUNICATIONS biochemically similar to the desmosome fractions of Skerrow and Matoltsy (31,32), except that our whole desmosomes are comparatively enriched in the very high molecular weight nonglycosylated proteins (205,000 and 230,000 mol wt) .…”

Section: Discussionmentioning

confidence: 99%

“…Skerrow and Matoltsy (30,31) isolated a desmosome preparation from cow nose epidermis and, upon sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of this material, found some 24 protein bands. More recently, Drochmans et al (3), using the same tissue, described an alternative procedure for obtaining fractions containing intact desmosomes and tonofilaments.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Isolation of the intercellular glycoproteins of desmosomes.

Gorbsky

Steinberg

1981

204

111

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

“…Dcsmosomes were isolated from bovine muzzle epidermis by a modification (Gorbsky and Steinberg, 1981) of the method of Skerrow and Matoltsy (1974). Briefly, 5 g of sliced bovine muzzle epidermis from freshly slaughtered animals were homogenized (polytron Instruments type 20; Kinematica GmBH, Luzern, Switzerland) in 125 ml of CASC buffer (0.1 M citric acid-sodium citrate, pH 2.6), containing 0.05 % NP-40 nonionic detergent, protease inhibitors leupeptin (5/~g/mi) pepstatin A (5 #g/ml) (Sigma Chemical Co., St. Louis, MO) 1 mM PMSF, and 1 mM DTT and then stirred for 3 h at 40C.…”

Section: Preparation Of Desmosomesmentioning

confidence: 99%

A protein antigenically related to nuclear lamin B mediates the association of intermediate filaments with desmosomes.

Cartaud

Ludosky

Courvalin

et al. 1990

Abstract. Desmosomes are specialized domains of epithelial cell plasma membranes engaged in the anchoring of intermediate filaments (IF). So far, the desmosomal component(s) responsible for this binding has not been unambiguously identified. In the present work, we have examined bovine muzzle epidermis desmosomes for the presence of protein(s) structurally and functionally related to lamin B, the major receptor for IF in the nuclear envelope (Georgatos, S. D., and G. Blobel. 1987. J. Cell Biol. 105:105-115). By using polyclonal antibodies to lamin B in immunoblotring experiments, we find that a desmosomal protein of 140-kD shares epitope(s) with lamin B. Immunoelectron microscopic and urea extraction experiments show that this protein is a peripheral protein localized at the cytoplasmic side of the desmosomes (desmosomal plaques). Furthermore, this protein binds vimentin in an in vitro assay. Since this binding is inhibited by lamin B antibodies, the epitopes common to the 140-kD protein and to lamin B may be responsible for anchoring of intermediate filaments to desmosomes. These data suggest that lamin B-related proteins (see also Cartaud, A., J. C. Courvalin, M. A. Ludosky, and J. Cartaud. 1989. J. Cell Biol. 109:1745-1752