CONTINUAL ASSEMBLY OF HALF-DESMOSOMAL STRUCTURES IN THE ABSENCE OF CELL CONTACTS AND THEIR FRUSTRATED ENDOCYTOSIS - A COORDINATED SISYPHUS CYCLE

It is widely assumed that the coordinate assembly of desmosomal cadher ins and plaque proteins into desmosome-typical plaque-coated membrane domains, capable of anchoring intermediate-sized filaments (IF), requi res cell-to-cell contacts and a critical extracellular Ca2+ concentrat ion. To test this hypothesis we studied several cell Lines grown for y ears in media with less than 0.1 mM Ca2+ to steady-state low Ca2+ medi um (LCM) conditions, particularly the human keratinocyte Line HaCaT de void of any junctional cell contact (HaCaT-L cells). Using immunolocal ization and vesicle fractionation techniques, we found that the transm embrane glycoprotein, desmoglein (Dsg), colocalized with the plaque pr oteins, desmoplakin and plakoglobin. The sites of coassembly of desmos omal molecules in HaCaT-L cells as well as in HaCaT cells directly bro ught into LCM were identified as asymmetric plaque-coated plasma membr ane domains (half-desmosomes) or as special plaque-associated cytoplas mic vesicles, most of which had formed endocytotically. The surface ex posure of Dsg in these half-desmosomes was demonstrated by the binding , in vivo, of antibodies specific for an extracellular Dsg segment whi ch also could cross-bridge them into symmetric quasi-desmosomes. Other wise, these half-desmosomes were shown in LCM to be taken up endocytot ically. Half-desmosomal assemblies were also seen in uncoupled cells i n normal Ca2+ medium. We conclude that, in the absence of intercellula r contacts, assembly of desmosomal proteins at the cell surface takes place, resulting in transient half-desmosomes which then, in LCM and w ithout a stable partner connection to the adjacent cell, can be endocy totically resumed. This frustrated cycle of synthesis and assembly mai ntains an ensemble of molecules characteristic of epithelial different iation and the potential to form desmosomes, even when the final junct ional structure cannot be formed. We propose that these half-desmosoma l structures are general cell structures of epithelial and other desmo some-forming cells.