Identification of desmosomal surface components (desmocollins) and inhibition of desmosome formation by specific fab′

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Abstract
Specific antibodies against the components of desmosomes, the adhesive junctions of epithelial cells, have been used to determine which components are located on the cell surface. Three criteria have been used: fluorescent antibody staining, immuno-gold labelling and electron microscopy, and quantitative measurements of antibody binding using [125I]protein A. When these techniques were applied to living Madin-Darby bovine kidney (MDBK) cells, antibodies against only two des-mosomal components, glycoproteins of approximately 115×103.r and 100×103Mr, bound to the cell surface. Antibodies against all other components, the 230 and 205×103Mr proteins (desmo-plakins), the 150×l3Mr glycoprotein and the 82 and 86×103Mr proteins reacted in fluorescent antibody staining only after cells had been fixed and made permeable. MDBK cells were cultured in the presence of univalent fragments (Fab′) of anti-desmosomal antibodies for periods from 24 h to 72h. After these times cells were fixed, made permeable, and stained with anti-desmoplakin antibody to assay for desmosome formation. Fab′ derived from anti-100×103Mr protein specifically inhibited desmosome formation, whereas Fab′s from anti-desmoplakin, anti-150× 103Mr and anti-82 and 86×103Mr proteins were without effect. We conclude that the 100×103Mr and the immunologically related 115 ×103Mr components are located on the cell surface and are directly involved in cell-cell adhesion. We have named them desmocollins to denote that they are involved in the adhesive function of desmosomes. The modulation of desmocollin distribution during monolayer formation and establishment of epithelial polarity has also been studied. Fluorescent and immuno-gold labelling using Fab′ or IgG at 4 °C revealed that desmocollins were initially evenly dispersed over the cell surface. Staining with IgG at 37 °C caused the desmocollins to ‘patch’ but not to ‘cap’. With the establishment of con-fluency, desmocollins were gradually removed from the upper surfaces of the cells (or masked and rendered inaccessible to antibody) being confined to the lateral and probably basal regions of the cells. Treatment of confluent monolayers with 3mM-EGTA rendered the desmocollins stainable, probably by causing their release from lateral constraint. Desmocollin staining at the cell surface was not appreciably reduced during 5 h of EGTA treatment, suggesting that desmocollins, unlike desmosomal plaques, may not be internalized after junction breakdown.