Gap junction (GJ) dynamics revealed by photoconverting expressed Cx43-Dendra2. (A) GJs consisting of many densely packed channels visible as green lines and puncta between HeLa cells expressing Cx43-Dendra2 (panel 1). GJs orient perpendicular providing a view onto their edge (circled in center, panel 1; also shown in Figures 1B, C), or horizontally providing a view onto their surface (circled on right; remaining panels, Figure 1A). GJs are dynamic structures. Their channels are replaced within several hours, as demonstrated by photoconverting Dendra2-tagged Cx43. A region containing two horizontally oriented GJs was photoconverted (entire field shown), and green and red channels were recorded over time. Within 1-hour post conversion a widening, homogenous green line of channels appeared along the GJs (panels 2-5). (B) Following photoconverted GJs for longer periods resulted in a steady loss of red fluorescence from the photoconverted area, and a simultaneous recovery of green fluorescence (circled in panel 1), suggesting that older channels are continuously removed from central GJ areas, while newly synthesized channels are simultaneously added to their periphery. Fluorescence intensity profiles for red and green channels measured along lines traversing the photoconverted GJs are shown. (C) Photoconversion allows estimation of GJ channel turnover. A portion of a perpendicular oriented Cx43-Dendra2 GJ was photoconverted (circled). Over time, red fluorescent puncta appeared adjacent to the converted GJ area (arrow-heads, panels 3, 4). Puncta were not detected immediately post-conversion (panel 2), suggesting that they were released from the photoconverted GJ area. Puncta correspond to degradative endocytic vesicles that are generated by the release of small GJ channel packets from GJs . (D) Schematic representation of GJ turnover as shown experimentally in (A-C).