Gap junctions: A new level of detail revealed by shadowing at -170° to -182°C in a JEOL JFD-9000 equipped with cryopump and liquid nitrogen-cooled shrouds

Abstract

In conventional freeze-fracture replicas, vertebrate gap junctions (GJ) are visualized as patches of 9 nm P-face particles with center-to-center spacings of 9-11 nm. Occasionally IMPs have a central “dimple”. On E-faces, GJ are represented by 4-6 nm diameter conical pits separated by 5-7 nm wide membrane ridges. In addition to the disparity of sizes of IMPs and pits, there is no discernable substructure in the pits corresponding to the dimples in the IMPs. In this report, we present replicas revealing a new level of detail for gap junctions, including evidence for channels in both P-face IMPs and E-face pits.Mice were fixed by perfusion with 2.5% glutaraldehyde in phosphate buffer. Samples were gradually infiltrated to 30% glycerol, frozen by plunging into liquid propane, fractured at -105° to -182°C (measured by thermocouple in sample stage), and shadowed at an angle of 45° in a JEOL JFD-9000 freeze-etch machine equipped with electron beam guns. The JFD-9000 was modified by the addition of interlocking liquid nitrogen-cooled shrouds for specimen and knife and a CTI-8F cryopump.