期刊
JOURNAL OF CELL SCIENCE
卷 128, 期 21, 页码 3888-3897出版社
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jcs.162586
关键词
Gap junction; Confocal microscopy; Connexin; Membrane trafficking; Pulse-chase; Tracer coupling
类别
资金
- National Institutes of Health [EY12857, EY10608]
- Research to Prevent Blindness
- Vale-Asche Foundation through the Frederic B. Asche Endowment
- University of Texas Graduate School of Biomedical Sciences at Houston
Gap junctions formed of connexin 36 (Cx36, also known as Gjd2) show tremendous functional plasticity on several time scales. Changes in connexin phosphorylation modify coupling in minutes through an order of magnitude, but recent studies also imply involvement of connexin turnover in regulating cell-cell communication. We utilized Cx36 with an internal Halo Tag to study Cx36 turnover and trafficking in cultured cells. Irreversible, covalent pulse-chase labeling with fluorescent HaloTag ligands allowed clear discrimination of newly formed and pre-existing Cx36. Cx36 in junctional plaques turned over with a half-life of 3.1 h, and the turnover rate was unchanged by manipulations of protein kinase A (PKA) activity. In contrast, changes in PKA activity altered coupling within 20 min. New Cx36 in cargo vesicles was added directly to existing gap junctions and newly made Cx36 was not confined to points of addition, but diffused throughout existing gap junctions. Existing connexins also diffused into photobleached areas with a half-time of less than 2 s. In conclusion, studies of Cx36-HaloTag revealed novel features of connexin trafficking and demonstrated that phosphorylation-based changes in coupling occur on a different time scale than turnover.
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