FIGURE 10:

Cell–cell contact and the subcellular fate of Cx43. (A) Cell–cell contact alters the endocytic itinerary of Cx43. Bx43KD and CP43KD cells stably expressing knocked-in WT-Cx43EGFP were cocultured with Bx43KD and CP43KD cells, and the cultures were immunostained for clathrin (red). Note that Cx43EGFP does not discernibly colocalize with clathrin when the Cx43EGFP-expressing cells are in contact with Cx43KD cells. Occasional puncta that seem to colocalize with clathrin are likely to be undocked connexons that were endocytosed from the noncontacting cell surface. (B) Cx43 connexons fail to dock upon arrival at the cell surfaces of BxPC3 and Capan-1 cells. Bx43KD and CP43KD cells stably expressing knocked-in WT-Cx43EGFP or WT-Cx43mAPPLE were seeded on glass coverslips, as described in Materials and Methods. After 24 h, cells were fixed, stained for 4′,6-diamidino-2-phenylindole (DAPI), and examined for colocalization of Cx43EGFP and Cx43mAPPLE. Note the absence of colocalization at the areas of cell–cell contact, as well as in the cytoplasm. (C) Cx43 is endocytosed as annular gap junctions in assembly-efficient LNCaP cells. LNCaP cells stably expressing Cx43EGFP or Cx43mAPPLE were cocultured for 24 h, fixed, stained for DAPI, and examined for the colocalization of Cx43EGFP and Cx43mAPPLE. Note that both gap junctions (red arrows) and intracellular vesicular puncta (white arrows) are composed of both Cx43EGFP and Cx43mAPPLE.