Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent p rotein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP w as transfected into communication-competent normal rat kidney cells, Cx43-n egative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed , transported, and assembled into gap junctions that exhibited the classica l pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed fu nctional gap junction channels when transfected into otherwise communicatio n-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells r evealed that many gap junction plaques remained relatively immobile, wherea s others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 mu m and 0.5-1.5 mu m. In some cases, the larger intracellular Cx43 -GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represen t other routes of trafficking to or from the plasma membrane. The localizat ion of Cx43-GFP in two transport compartments suggests that the dynamic for mation and turnover of connexins may involve at least two distinct pathways .