Hexagonal Array of Subunits in Intercellular Junctions of the Mouse Heart and Liver

Abstract

Hexagonally packed subunits have previously been observed on the surface of plasma membranes in at least two different systems. They were first described by Robertson (1) in electrical synapses of the Mauthner cells after permanganate and osmium-tetroxide fixation. Benedetti and Emmelot (2) have illustrated almost identical patterns in isolated liver plasma membrane fractions which had been negatively stained. We report here a new technique, based on the observations of Doggenweiler and Frenks (3), wMch has allowed us to demonstrate the presence of similar structures in sections of mouse heart and of mouse liver. When tissue bloqks are treated before dehydration and embedding with a preparation of lanthanum salts, the extracellular space becomes filled with an essentially amorphous electronopaque material. After such treatment hexagonally packed structures are clearly delineated in specialized intercellular junctions of the heart and liver (4, 5). MATERIALS AND METHODSTissues are fixed in a formaldehyde-glutaraldehyde mixture (6), are washed in buffer, and then are postfixed for 2 hr in OsO4-eollidine at pH 7.2-7.4. A 2-4% solution of lanthanum nitrate is brought to pH 7.6-7.8 with 0.01 N NaOH, with vigorous stirring. The addition of base must be made slowly to avoid premature precipitation of lanthanum hydroxide. When the titration is done properly, a faint opalescence or traces of flocculent material appear at pH 7.8, the pH at which lanthanum hydroxide becomes insoluble. An aliquot of the lanthanum solution is added to the fixatives so that the final lanthanum concentration is 0.5-1%. The final pH of the lanthanum-osmium tetroxide-collidine solution is about 7.2 and is faintly cloudy. It seems likely that, as lanthanum solutions are brought to a high pH, a colloidal compound is formed which, as described below, permeates the extracellular space as a tracer. While we deem it best to add lanthanum to all the solutions up to the alcohols, this is not necessary and lanthanum can be added during the OsO4 fixation alone. Adding the lanthanum only to the aldehyde fixing fluid or to the buffer rinse solution is less satisfactory, since the heavy metal seems to be washed out from the blocks again during subsequent exposure to aqueous solutions. The exposure to lanthanum solutions should be done at room temperature. RESULTSSections cut from blocks treated with lanthanum as described above contain a material of high electron opacity which fills the cxtracellular space to a variable degree. In most cases the lanthanum has not penetrated the central portion of the block, but the intermediate zone between the center and tl~e surface of the block is well impregnated. At the periphery of the blocks one finds electron-opaque material consistently only in those regions where the extracellular space is very C7

Selective Deposition of Lanthanum in Mammalian Cardiac Cell Membranes

“…Gap junctions appeared therefore as two dense lines 25-30 A thick, separated by a clear space 100-120 A wide . The latter consists of the middle and outer leaflets of the converging junctional membranes, as well as the 20 A intercellular gap, as described by others (17,19) in tissues treated with uranyl acetate or with colloidal lanthanum . In addition to gap junctions, both micropinocytotic vesicles and coated vesicles of cardiac cell membranes (6) showed lanthanum deposits (Figs .…”

“…The localization of lanthanum in the outer leaflets of plasma membranes of frog nerve fibers was first reported by Doggenweiler and Frenk (4) . In addition, lanthanum used as colloidal suspensions (19) or in ionic form (7,15,16) acts as an electron-opaque tracer of the extracellular space . Revel and Karnovsky (19) used colloidal lanthanum to identify a previously undetected 20 A intercellular space at the gap junctions of mouse heart and liver ; we later obtained similar findings in a variety of epithelial and myocardial tissues (13,15,16) .…”

An electron microscopic study of cell death in molar tooth germ epithelia of mouse embryos.

“…They were stained en bloc with 1% uranyl acetate for 1hr, dehydrated in graded concentrations of ethylalcohol, and embedded in Epon 812. Some specimens of 14th day embryos were fixed with the same fixatives containing lanthanum, an extracellular tracer (REVEL and KARNOVSKY, 1967 Granular substances stained intensely with toluidine blue (NOZUE, 1971) were clearly observed in the epithelial tooth germs from the 12th to the 14th day of gestation. Their size varied from under 1 micron to several micron.…”

Assembly of Gap Junctions During Amphibian Neurulation

“…Although the visualization of gap junctions has been enhanced by utilizing heavy metal tracers and stains (11,22,24,30,32,33,34,63), negative staining (4,30,55,81), and freeze fracturing (12,22,30,46,47,55,61,74,81), these studies have contributed little in describing their construction in developing or mature tissues. While some gap junctions are diminutive and macular (22,47,64,65,66), others exhibit a diversity of contours: some are long, particulate chains such as those in the outer plexiform layer of the retina (62), and still others are small, amorphous, particle aggregates similar to those of normal and transformed fibroblasts (28,58,64).…”

“…The use of tracer and freeze-fracturing techniques confirms that the gap junction (46,66) exhibits a 2 to 4-nm gap between cell membranes and is comprised of 8.5-nm, polygonally packed subunits (4,11,22,30,46,47,51,63). Freeze fracture further reveals that the junction assumes a variety of configurations (22,62,66) and cannot always be identified by thin sectioning alone (22,65).…”