Gap junction internalization and processing in vivo: A 3D immuno-electron microscopy study

Abstract

Gap junctions have well-established roles in cell-cell communication byway of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. Highpressure frozen, serial-sectioned tissue was immunogold labeled for connexin 43 (Cx43, also known as GJA1). Within a volume corresponding to ≈35 cells, every labeled structure was categorized and had its surface area measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of cathepsin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.