John D. Pitts

Permeability of junctions between animal cells: Intercellular transfer of nucleotides but not of macromolecules☆

Abstract Many cell types growing in tissue culture form intercellular junctions which are permeable to nucleotides but not to RNA, DNA or protein.

Isolation and characterisation of arthropod gap junctions

Gap junctions have been isolated from the hepatopancreas of the crustacean arthropod, Nephrops norvegicus (Norway lobster). SDS‐PAGE of these preparations shows two major protein bands, mol. wt. 18 000 (18 K) and mol. wt. 28 000 (28 K). The 18‐K and 28‐K proteins are interconvertible, cannot be distinguished by two dimensional tryptic and chymotryptic peptide mapping, and therefore appear to be different (most likely monomeric and dimeric) forms of the same protein. The protein can also aggregate to higher multimeric forms mol. wt. 38 000 (presumed trimer), and mol. wt. 52 000 (presumed tetramer). The buoyant density of the isolated gap junctions in continuous potassium iodide gradients is 1.260 g/cm3. The junctions are progressively solubilized in increasing SDS concentrations, mostly between 0.1% and 0.2% SDS, and this is accompanied by the release of the 18‐K and 28‐K forms of the junctional protein. The Nephrops hepatopancreas 18‐K junctional protein has antigenic determinants in common with the vertebrate 16‐K junctional protein as shown by cross‐reactivity with two different affinity purified antibody preparations. However, no detectable similarity can be seen between the major 125I‐labelled tryptic and chymotrytpic peptides of the Nephrops hepatopancreas 18‐K protein and the mouse liver 16‐K protein.

The role of junctional communication in animal tissues

SummaryPermeable intercellular junctions are a common feature of most animal tissues. These junctions allow the free exchange of small ions and molecules between all the cells in coupled populations. Such limited syncytial interaction contributes to the integration of individual cells into organized tissues.

Metabolic interactions between animal cells through permeable intercellular junctions

Abstract In mixed cultures of HGPRT− (lacking hypoxanthine: guanine phosphoribosyltransferase activity) and wild-type mammalian cells coupled by permeable intercellular junctions, the purine nucleotide pools equilibrate between the two cell types prior to incorporation into nucleic acid. As a consequence, in the presence of exogenous hypoxanthine, the mutant cells stimulate the activity of HGPRT pathway in the wild-type cells and the de novo pathway of purine nucleotide biosynthesis is inhibited in both cell types. That is, the metabolic activity of a mixed culture of coupled cells is a unique characteristic of the mixture, not simply the sum of the activities of the component cells, whereas the activity of a mixed culture of cells which are not coupled by permeable intercellular junctions is simply the sum of the activities of the two cell types.

Analysis of vertebrate gap junction protein.

A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X‐100. The major band on SDS‐polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. (ii) The junctions are progressively solubilised by increasing concentrations of SDS (in the range 0.1‐0.5%) and the dissolution of the junctional structure, observed by electron microscopy, parallels the release of the 16 K protein. (iii) Glutaraldehyde fixation of intact junctions cross‐links the 16 K protein. (iv) The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in V79 cell cultures exposed to 4beta‐phorbol 12‐myristate 13‐acetate.

Replication of polyoma virus DNA: I. A resting cell system for biochemical studies on polyoma virus

Abstract A technique is described for adapting mouse embryo cells to the resting state by using medium supplemented with only 0.5% serum. In cells maintained in low serum medium for 4 days the rates of DNA and RNA syntheses, and the activities of DNA polymerase and DNase, are very low. A large proportion (90%) of such cells can be infected with polyoma virus at an input multiplicity of 20 PFU/cell. After infection there are large increases in the rates of DNA and RNA syntheses and DNA polymerase activity, but DNase activity remains very low.

Binding of bovine papillomavirus type 4 E8 to ductin (16K proteolipid), down-regulation of gap junction intercellular communication and full cell transformation are independent events

The E8 open reading frame of bovine papillomavirus type 4 encodes a small hydrophobic polypeptide that contributes to primary cell transformation by conferring to cells the ability to form foci and to grow in low serum and in suspension. Wild-type E8 binds in vitro to ductin, a component of gap junctions, and this binding is accompanied by a loss of gap junction intercellular communication in transformed bovine fibroblasts. However, through the analysis of a panel of E8 mutants, we show here that binding of E8 to ductin is not sufficient for down-regulation of gap junction communication and that there is no absolute correlation between down-regulation of gap junction communication and the transformed phenotype.

Patterns of junctional communication in skin: studies on cultured keratinocytes

Junctional communication has long been suggested to play a role in coordinating the development of multicellular tissues. A better understanding of the patterns of communication between cells in such tissues is important for the identification of areas where this process may have a role. We have investigated the patterns of communication in cultures of human epidermal keratinocytes by iontophoretic injection of Lucifer Yellow CH, using involucrin expression as a marker of cells undergoing terminal differentiation. Cells that lack involucrin (i.e., the basal, proliferating cells) transfer dye preferentially to other involucrin-negative cells, whereas involucrin-positive cells either are not coupled or transfer dye with similar frequency to involucrin-positive and involucrin-negative neighbors. This decrease in communication associated with terminal differentiation was observed in both the presence and the absence of assembled desmosomes. Our observations lead us to speculate that loss of junctional communication may influence the commitment of basal keratinocytes to terminal differentiation.

Junctional Permeability and Its Consequences

For the last 100 years attention has been focused on the cell as the unit of life, and indeed this important concept has formed the basis for much of our understanding of structure and function in biological systems. Although this concept is meaningful and accurate for prokaryotes and other unicellular organisms, the cell in a highly developed multicellular organism must lose much of its independence in the milieu of the composite tissues. We must now accept that multicellular organisms are populations of interacting and interdependent cells with properties which cannot be assigned specifically to individual units but instead must be collectively assigned to all the different cells which together are the developing embryo or the adult tissue.

Permeability of junctions between animal cells. Intercellular exchange of various metabolites and a vitamin-derived cofactor.

Abstract A variety of metabolites and a vitamin-derived cofactor are shared between cells coupled by permeable intercellular junctions (gap junctions). The molecular species which pass through the junctions have been identified as 2-deoxy-glucose or 2-deoxy-glucose-6-phosphate, phosphoryl choline or CDP-choline, proline or its precursors and lower glutamated forms of tetrahydrofolate. The active tetraglutamate form of tetrahydrofolate (molecular weight 960) is not shared between coupled cells. These observations support the concept that junctional permeability is selective only with respect to molecular size and that populations of coupled cells will become syncytial with respect to their low molecular weight cytoplasmic components.