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

Abstract

<jats:title>Abstract</jats:title><jats:p>Gap junctions have well-established roles in cell-cell communication by way 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 studied the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume of electron micrographs, every labeled structure was categorized and counted. Surface area measurements indicate that large connexosomes undergo fission. Subsequent modifications are separation of inner and outer membranes, loss of Cx43 from the outer membrane, and outward budding of the modified membranes. We also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. We discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication.</jats:p>

Transfer of mitochondria and endosomes between cells by gap junction internalization

“…To definitively establish that these vesicles were internalized gap junctions, isolated ovarian follicles were prepared for post‐embedding immunogold labeling. Serial sections collected on tape were labeled with an antibody to the gap junction protein Cx43 and a 10 nm gold secondary antibody, as previously described 11,12 . Labeling of Cx43 unambiguously identified gap junctions between and within mural granulosa cells.…”

“…Ovarian follicles were manually isolated from 25‐day‐old mice. Follicles measuring 360 to 400 μm in diameter were cultured on Millicell membranes (Millipore, PICMORG50) in MEM alpha medium as previously described 11,12 . After 25 hours of culture, follicles were treated with 300 nM ovine luteinizing hormone (oLH‐26, National Hormone and Pituitary Program) for 30 minutes, and then transferred to brass specimen carriers and high pressure frozen with an EMPACT 2 (Leica Biosystems, Buffalo Grove, Illinois).…”

“…For immunostaining, ribbons of tissue sections on kapton tape were cut to lengths of ~3 inches and attached to a sheet of parafilm with double‐sided carbon tape (Electron microscopy Sciences #77816). Sections were labeled with an anti‐Connexin 43 polyclonal antibody (#C6219, Sigma‐Aldrich, St. Louis, Missouri, ) as described 12 …”

“…Immuno‐labeled sections on tape were attached to a 10 cm diameter silicon wafer (University Wafer, South Boston, Massachusetts) with double‐sided carbon adhesive tape (Electron Microscopy Sciences). Wafers were carbon coated (Denton, Moorestown, New Jersey) and first imaged on a Sigma field emission scanning electron microscope (Zeiss, Thornwood, New York) using a backscatter detector as described 11,12 . Higher resolution images of the sections were taken on an FEI Verios 460L field emission scanning microscope with a backscatter detector.…”

“…One hundred and sixty‐nine internalized gap junctions within four volumes of tissue (each encompassing ~15 000 cubic micrometers [60 square micrometers areas imaged through 68 sections of 65 nm thickness]) were analyzed for the fraction of internalized gap junctions that contained mitochondria and MVEs. The surface areas of internalized gap junctions were measured as described earlier 12 …”