title: Two-color fluorescent analysis of connexin 36 turnover: relationship to functional plasticity.
abstract: 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 HaloTag 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.
authors.affiliation: Ruiz Department of Ophthalmology & Visual Science, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA John.OBrien@uth.tmc.edu Yanran.Wang@uth.tmc.edu.
authors.forename: Helen Yanran
authors.initials: HY
authors.lastname: Wang
authors.affiliation: Ruiz Department of Ophthalmology & Visual Science, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Lin
authors.affiliation: Ruiz Department of Ophthalmology & Visual Science, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Cheryl K
authors.initials: CK
authors.lastname: Mitchell
authors.affiliation: Carl Zeiss Microscopy LLC, Thornwood, NY 10594, USA.
authors.forename: Sripad
authors.initials: S
authors.lastname: Ram
authors.affiliation: Ruiz Department of Ophthalmology & Visual Science, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA John.OBrien@uth.tmc.edu Yanran.Wang@uth.tmc.edu.
authors.forename: John
authors.initials: J
authors.lastname: O'Brien
mesh_terms: D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002417:Cattle; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D017630:Connexins / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D000097004:Gap Junction delta-2 Protein
chemical_list: D020126:Brefeldin A; D017630:Connexins; D000097004:Gap Junction delta-2 Protein
keywords: Confocal microscopy; Connexin; Gap junction; Membrane trafficking; Pulse-chase; Tracer coupling