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Fig. 6. Close molecular interactions between Cx43, ZO-1 and Src during HCH-induced endocytic internalization of Cx43-GFP gap junction plaques. (A) Percentage of two adjacent cells in which one Cx43-GFP transfected (T) cell is in contact with a wild-type cell expressing endogenous Cx43 (Wt), or in which the two cells express Cx43-GFP (T/T). The transfected cells were identified by the presence of GFP within cells. (B,C) Localization of immunoreactive ZO-1 between two adjacent cells that (B) express Cx43-GFP, or between (C) one cell that expresses Cx43-GFP (T) and a wild-type cell (Wt) that expresses only endogenous Cx43. Dashed lines correspond to the zone of contact between the two adjacent cells. (D-K) High-resolution deconvolution fluorescence microscopy analysis of the interaction of Cx43-GFP with ZO-1 and Src during the internalization of gap junction plaques in response to 50 µM HCH. (D) Side view of a gap junction plaque (green) decorated on both sides by ZO-1 (red). Colocalization is shown in yellow. (E) Presence of ZO-1 only on one side of the plaque after HCH treatment, indicating the beginning of the plaque endocytosis. (F) Localization of ZO-1 on the internal side of the gap junction plaque during internalization. (G) View of an early Cx43-GFP annular gap junction associated with ZO-1 in its center. (H) Cx43-GFP gap junction plaque (green) and distribution of Src (red) in control cells. (I) Side view of a gap junction plaque associated at one side with Src after HCH exposure. Colocalization is shown in yellow. (J) At the beginning of plaque internalization the presence of Src (yellow) was mainly detected on the external side of the gap junction plaque. (K) View of an early Cx43-GFP annular gap junction associated in its periphery with Src (yellow). Images are representative of five different experiments.
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