Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

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

“…Those bands of 48,000-68,000 mol wt are largely prekeratin proteins derived from the tonofilaments (3,5,34) . Preparations of purified cores are highly enriched in a very broad band -150,000 mol wt, two broad bands of 115,000 and 100,000 mol wt, and a 22,000 mol wt component (Fig.…”

Section: Resultsmentioning

confidence: 99%

Isolation of the intercellular glycoproteins of desmosomes.

Gorbsky

Steinberg

1981

205

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.

“…This might suggest that the cytokeratins of epithelial cells of internal organs are more related to each other than to epidermal prekeratins . Yet, the relationship of the various cytokeratins is even more complicated since significant differences of cytokeratin polypeptide composition can also be found among various internal organs (these authors, manuscript in preparation), and different prekeratin polypeptides are expressed even in different layers of skin and in epidermal keratinocytes grown in culture (1, 11,17,[65][66][67][68][69] . We conclude that the tonofilamentous structures, which are indistinguishable in different epithelial cells, can be formed by completely different sets of proteins of the cytokeratin family .…”

Section: Franke Et Almentioning

confidence: 99%

“…Isolated tonofilaments were used directly or after reconstitution (see above) for negative staining with uranyl acetate or phosphotungstic acid (11,14) .…”

Section: Electron Microscopymentioning

confidence: 99%

Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border.

Franke¹,

Winter

Gründ

et al. 1981

Self Cite

117

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-border fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1 .5 M KCI) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SIDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of M r 55,000, 48,000, and 40,000 was noted. On two-dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (M r 48,000 and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the M r 55,000 protein which focused at pH values higher than 6.4 . The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins . Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell . The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue-specific patterns of their protein subunits .