Dynamics of gap junctions observed in living cells with connexin43-GFP chimeric protein

To study the aggregation of cell-to-cell channels into gap junctions at ind ividual cell-cell contacts, we transfected cells with an expression vector for a chimeric protein composed of the cell-to-cell channel protein connexi n43 and a green fluorescent protein. The chimeric channel protein was visua lized in the fluorescence microscope and was found to form gap junctions at the cell-cell contacts just like wild-type connexin43. Cells expressing th e chimeric: protein had functional cell-to-cell channels. Using timelapse v ideomicroscopy on live cells we observed individual gap junctions over long periods and recorded the time course of aggregation of the chimeric channe l protein into gap junctions at newly formed cell-cell contacts. We found t hat individual small gap junctions were very dynamic, moving about or becom ing assembled and disassembled in the course of minutes, Larger gap junctio ns were more stable than small punctate ones. In control condition, stable new gap junctions were not formed during observation times of 30 min or Lon ger. But at elevated levels of cyclic adenosine monophosphate, the chimeric channel protein began aggregating at new junctions 5-10 minutes after cell -cell contact and continued to concentrate there for at least one hour. Als o already established junctions grew in size, The fluorescent chimeric chan nel protein will be an excellent tool to investigate the regulation of traf ficking of connexin from and to the membrane and the mechanism of connexin channel aggregation into gap junctions.