FIGURE 5:

GJ plaques assembled by AP-2 binding–deficient Cx43 mutants are internalization impaired. (A) HeLa cells were transfected with GFP-tagged wild type, S2, S3, and S2+3 mutant constructs and imaged every 2 or 5 min for up to 72 h (phase-contrast and green fluorescence channels). GJ plaque formation, maturation, and endocytosis were captured (depicted by arrows). Selected merged image frames of wild type, F²⁶⁸A (S2), Y²⁸⁶H (S3), and F²⁶⁸A+Y²⁸⁶H-GFP and ΔL^254–290-GFP (S2+3) are shown (see also Movies S1–S5). (B) Quantitative analysis of time periods required for GJ plaques to constitutively turn over (form, mature, and be removed) revealed that GJ plaques assembled of wild type Cx43, on average, turned over in ∼5 h (green bar), while GJ plaques assembled from S2 and S3 mutants turned over in 18–19 h (blue bars), and GJ plaques assembled from S2+3 double mutants were removed after ∼30 h (red bar). (See Table S1 for complete data set.)