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

Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [35S]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.

Heterotrimeric GTP-binding proteins (G proteins) participate in cellular signaling and regulate a variety of physiological processes. Disruption of the gene encoding the G protein subunit alpha13 (Galpha13) in mice impaired the ability of endothelial cells to develop into an organized vascular system, resulting in intrauterine death. In addition, Galpha13 (-/-) embryonic fibroblasts showed greatly impaired migratory responses to thrombin. These results demonstrate that Galpha13 participates in the regulation of cell movement in response to specific ligands, as well as in developmental angiogenesis.

Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90–100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.

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