title: Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy.
abstract: Gap junction turnover occurs through the internalization of both of the plasma membranes of a gap junction plaque, forming a double membrane-enclosed vesicle, or connexosome. Phosphorylation has a key role in regulation, but further progress requires the ability to clearly distinguish gap junctions and connexosomes, and to precisely identify proteins associated with them. We examined, by using electron microscopy, serial sections of mouse preovulatory ovarian follicles that had been collected with an automated tape collecting ultramicrotome (ATUM). We found that connexosomes can form from adjacent cell bodies, from thin cell processes or from the same cell. By immunolabeling serial sections, we found that residue S368 of connexin 43 (also known as GJA1) is phosphorylated on gap junctions and connexosomes, whereas connexin 43 residue S262 is phosphorylated only on some connexosomes. These data suggest that phosphorylation at S262 contributes to connexosome formation or processing, and they provide more precise evidence that phosphorylation has a key role in gap junction internalization. Serial section electron microscopy of immunogold-labeled tissues offers a new way to investigate the three-dimensional organization of cells in their native environment.
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA Norris@uchc.edu terasaki@uchc.edu.
authors.forename: Rachael P
authors.identifier: 0000-0003-1711-965X
authors.initials: RP
authors.lastname: Norris
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
authors.forename: Valentina
authors.initials: V
authors.lastname: Baena
authors.affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA Norris@uchc.edu terasaki@uchc.edu.
authors.forename: Mark
authors.identifier: 0000-0003-4964-9401
authors.initials: M
authors.lastname: Terasaki
mesh_terms: D000818:Animals; D018031:Connexin 43 / Q000378:metabolism*; D005260:Female; D017629:Gap Junctions / Q000378:metabolism / Q000648:ultrastructure; D006736:Horses; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron / Q000379:methods*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D018031:Connexin 43; D010768:Phosphoserine
keywords: Annular gap junctions; Connexin; Connexosomes; Immunogold; Phosphorylation; Serial section electron microscopy