Figure 3. Fluorescence and ultrastructural evidence for autophagic AGJ vesicle degradation. (A) HeLa cells were cotransfected with Cx43-mApple and the mammalian autophagy marker protein GFP-LC3 (panel 1), or the activation-deficient LC3-mutant GFP-LC3(G120A) (panel 2). In cells, a fraction of cytoplasmic LC3 (LC3-I) is conjugated to phagophore-membranes (LC3-II) that localizes to autophagosomes; LC3(G120A) cannot be conjugated and remains cytoplasmic. Representative merged fluorescence images acquired 24 h post transfections are shown. Individual and merged fluorescence signals of the boxed areas are shown below at higher magnification. Robust colocalization of cytoplasmic AGJ vesicles present in Cx43-mApple expressing cells (red puncta) with GFP-LC3-II (green puncta) was observed in GFP-LC3 expressing cells, but not in GFP-LC3(G120A) expressing cells. Representative colocalizing AGJ vesicles are marked with arrows; GJs are marked with arrowheads. Bars = 10 μm. (B) Multiple stages characteristic of progressive autophagosome formation and maturation that formed around AGJ vesicles were revealed by ultrastructural analyses of Cx43-GFP expressing HeLa cell preparations. Double-membrane cisternae (phagophores, marked with arrows) progressively encircled AGJ vesicles (panels a–c), coalesced into phagophores (panels c–e) and fused with lysosomes (L, panels d and e), resulting in AGJ degradation inside the phagosome (panel f).