In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

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