Continual assembly of half-desmosomal structures in the absence of cell contacts and their frustrated endocytosis: a coordinated Sisyphus cycle.

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Abstract
It is widely assumed that the coordinate assembly of desmosomal cadherins and plaque proteins into desmosome-typical plaque-coated membrane domains, capable of anchoring intermediate-sized filaments (IF), requires cell-to-cell contacts and a critical extracellular Ca2+ concentration. To test this hypothesis we studied several cell lines grown for years in media with less than 0.1 mM Ca2+ to steady-state low Ca2+ medium (LCM) conditions, particularly the human keratinocyte line HaCaT devoid of any junctional cell contact (HaCaT-L cells). Using immunolocalization and vesicle fractionation techniques, we found that the transmembrane glycoprotein, desmoglein (Dsg), colocalized with the plaque proteins, desmoplakin and plakoglobin. The sites of coassembly of desmosomal molecules in HaCaT-L cells as well as in HaCaT cells directly brought into LCM were identified as asymmetric plaque-coated plasma membrane domains (half-desmosomes) or as special plaque-associated cytoplasmic vesicles, most of which had formed endocytotically. The surface exposure of Dsg in these half-desmosomes was demonstrated by the binding, in vivo, of antibodies specific for an extracellular Dsg segment which also could cross-bridge them into symmetric quasi-desmosomes. Otherwise, these half-desmosomes were shown in LCM to be taken up endocytotically. Half-desmosomal assemblies were also seen in uncoupled cells in normal Ca2+ medium. We conclude that, in the absence of intercellular contacts, assembly of desmosomal proteins at the cell surface takes place, resulting in transient half-desmosomes which then, in LCM and without a stable partner connection to the adjacent cell, can be endocytotically resumed. This frustrated cycle of synthesis and assembly maintains an ensemble of molecules characteristic of epithelial differentiation and the potential to form desmosomes, even when the final junctional structure cannot be formed. We propose that these half-desmosomal structures are general cell structures of epithelial and other desmosome-forming cells.

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