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Yanran Wang, John O'Brien, Cheryl K Mitchell; Trafficking and turnover of Cx36 in HeLa cells studied by fluorescent pulse-chase labeling. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2373.
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Electrical synapses formed of the gap junction (GJ) protein Cx36 show a great deal of functional plasticity in the retina, much dependent on changes in phosphorylation of the connexin. However, GJ turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied.
We utilized HaloTag technology to perform pulse-chase analysis of Cx36 turnover in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing specific protein labeling. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt phosphorylation sites or C-terminal protein-protein interactions. HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) ligand. Cells were fixed 30 minutes after initiation of chase labeling.
Cx36-Halo formed large plaques at sites of contact between transfected HeLa cells and was also contained in many intracellular vesicles. Pulse labeled Cx36 was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life of Cx36 in junctional plaques was 2.8 hours. Disruption of the Golgi apparatus with brefeldin A prevented the addition of new connexins to junctional plaques. Two classes of intracellular vesicles were observed. Small chase-labeled vesicles were interpreted to be trafficking Cx36 for exocytosis; large ring-shaped vesicles containing pulse label and occasionally chase label were interpreted to be endocytic and degradation vesicles. Newly synthesized vesicles were added to existing GJs throughout the GJ, not just at edges. Old GJ was removed from the center as well as the ends of the existing plaques. Both classes of vesicle were associated with actin filaments labeled with phalloidin leading to the GJ. Thick actin bundles connected all edges of GJ plaques, but actin filaments were rare within any plaque.
Two-color fluorescent pulse-chase labeling allows discrimination of exocytic and endocytic vesicles and revealed unique aspects of connexin trafficking to and from gap junctions. Turnover of Cx36 could contribute to long-term changes in coupling by changing the number of available channels in a gap junction. This can complement short-term regulation of channel opening by Cx36 phosphorylation.
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