Gap junctions between Novikoff hepatoma cells following dissociation and recovery in the absence of cell contact

Using thin-section and freeze-fracture techniques, the fates of formation plaques with and without gap junctions [designated FP(+), FP(-)] have been studied following the dissociation of Novikoff hepatoma cells with EDTA. Quantitative analysis shows dissociated cells to possess less numerous FP(+)'s and FP(-)'s than undissociated suspension cultures. Both EM techniques showed gap junctions remaining on cell surfaces, after one junctional membrane had been ripped from its cell and had apparently resealed forming a bleb. These structures were only infrequently observed to invaginate from the cell surface, suggesting that endocytosis is rare. Following the 30-min dissociation, some cells were placed in growth medium and incubated on a gyratory shaker (200 rpm) for 5-90 min. Under these conditions, the cells "recovered" from the dissociation process, but failed to reaggregate. FP(-)'s, dramatically reduced in dissociated preparations, completely disappeared by 30 min of recovery. FP(+)'s were reduced by dissociation, continued to fall over the first 15 min of recovery, and declined slowly to a low level by 90 min of recovery. Substantial numbers of the larger gap junctions were surrounded by numerous smaller junctional aggregates, an unusual configuration for undissociated cells. From these data, we suggest that: (a) a fraction of the FP(+)'s persists on cell surfaces for some time after dissociation; (b) FP(-)'s are more labile and are apparently not produced during breakdown; (c) FP(-)'s do not develop in the absence of prolonged cell contact; and (d) irregular junctions with adjacent, smaller junctions may represent junctional breakdown.