Localization of phosphorylated connexin 43 by serial section immunogold electron microscopy

Abstract

Gap junction turnover occurs by the internalization of both plasma membranes of a gap junction plaque to form a double membrane-enclosed vesicle, or connexosome. Phosphorylation has a key role in regulation, but further progress requires clearly distinguishing gap junctions and connexosomes and precisely localizing proteins to them. We examined by electron microscopy serial sections of mouse preovulatory ovarian follicles collected with an automated tape collecting ultramicrotome (ATUM). We found connexosomes may form from adjacent cell bodies, from thin cell processes, or from the same cell. By immunolabeling serial sections, we found S368 of connexin 43 is phosphorylated on gap junctions and connexosomes, whereas S262 is phosphorylated only on some connexosomes. These data suggest that S262 phosphorylation contributes to connexosome formation or processing, and 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 for investigating the three-dimensional organization of cells in their native environment.