Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication.

Cell-to-cell communication is achieved by passage of small molecules through gap junction membrane channels. The expression of the transforming gene from Rous sarcoma virus, v-src, induces a rapid and dramatic reduction in cell-to-cell communication in cultured cells. To determine whether connexin43, a major gap junction protein expressed in fibroblasts, is a target for the v-src protein tyrosine kinase activity, we examined the phosphorylation state of connexin43 in cells expressing variants of src.

Junctional intercellular communication is cooperatively inhibited by oncogenes in transformation.

We examined the actions of the cellular src (c-src) and adenovirus E1A genes on junctional cell-to-cell communication. Neither gene causes complete transformation of NIH3T3 cells on its own, but the two do so in conjunction with one another. This cooperation goes hand in hand with summation of the actions of the two genes on junctional communication: junctional permeability is reduced when the cells are transfected with either gene; it is reduced significantly more when they are transfected with both.

The cellular src gene product regulates junctional cell-to-cell communication.

Overexpression of the cellular src gene in NIH 3T3 cells causes reduction of cell-to-cell transmission of molecules in the 400- to 700-dalton range. This down-regulation of gap junctional communication correlates with the activity of the gene product, the protein tyrosine kinase pp60c-src. The down-regulation was enhanced by point mutation of Tyr527 (a site that is phosphorylated in pp60c-src and that inhibits kinase activity) or by substitution of the viral-src for the cellular-src carboxyl-terminal coding region.

Polyomavirus middle T antigen downregulates junctional cell-to-cell communication.

We examined the effect of polyomavirus middle T antigen on cell-to-cell communication in rat F cells transfected with an inducible middle T recombinant DNA or infected with a conditional mutant virus. Junctional permeability fell (reversibly) when middle T transcription was induced or when middle T was switched to the transformation+ form. The effect correlates with the rise of protein tyrosine kinase activity.

Tropomyosin in peripheral ruffles of cultured rat kidney cells.

Tropomyosin distribution has been studied in two normal lines and one transformed line of rat kidney cells during the early phases of substrate attachment and growth. One non-motile normal line, which spreads rapidly after attachment, immediately begins to assemble prominent stress fibers that contain tropomyosin. It displays small peripheral ruffles that are not noticeably stained with anti-tropomyosin.

Cyclic AMP effects on cell-to-cell junctional membrane permeability during adipocyte differentiation of 3T3-L1 fibroblasts.

Mouse 3T3-L1 fibroblast cells, also know as preadipocytes, differentiate in vitro into adipocytes when treated with promoting agents and acquire numerous properties characteristic of mature fat cells. We studied junctional cell-to-cell communication by measuring the incidence of electrical coupling and transfer of carboxy-fluorescein among these cells. When 3T3-L1 cells were induced to differentiate into adipocytes, they lost virtually all cell-cell communication.

Translation and functional expression of cell-cell channel mRNA in Xenopus oocytes.

mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected into Xenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled.

Intercellular communication and the control of growth: XII. Alteration of junctional permeability by simian virus 40. Roles of the large and small T antigens.

We studied the action of temperature-sensitive mutant simian virus 40--a transformation-inducing DNA virus--on the junctional permeability to mono-, di- and triglutamate in rat embryo-, pancreas islet (epithelial)-, and 10T1/2 cell cultures. Junctional permeability was reduced (reversibly) in the transformed state. To dissect the genetics of this alteration, we used two kinds of mutant virus DNA.

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by the src gene in a revertant cell with normal cytoskeleton.

To learn whether the reduction of cell-to-cell communication in transformation is a possible primary effect of pp60src phosphorylation or secondary to a cytoskeletal alteration, we examined the junctional permeability in transformed cells with normal cytoskeleton. The permeability to fluorescent-labelled mono- and diglutamate was compared in clones of Faras' vole cells--clones transformed by Rous sarcoma virus and reverted from that transformation. One revertant clone (partial revertant), had the high level of pp60src kinase activity and tumorigenicity of the fully transformed parent clone, but had lost the cytoskeletal alterations of that clone.

Intercellular communication and the control of growth: X. Alteration of junctional permeability by the src gene. A study with temperature-sensitive mutant Rous sarcoma virus.

To study changes of junctional membrane permeability associated with transformation, the junctions and the nonjunctional membranes of quail embryo-, chick embryo- and mouse-3T3 cell cultures, infected with temperature-sensitive mutant Rous sarcoma virus, were probed with fluorescent-labelled glutamate. Junctional permeability fell in the transformed state. In the quail cells, the fall was detectable within 25 min of shifting the temperature down to the level (permissive) at which tyrosine-phosphorylation by the viral src gene product is expressed.

Induction of cell--cell channel formation by mRNA.

Intercellular junctional communication is very common in normal organized tissue. It provides a pathway for transmission of electrical signals, especially in heart muscle, and may be important in differentiation and growth control. The hydrophilic channels which enable cell--cell communication have been well characterized by biophysical methods, and there is now good evidence that they are contained in the nexus (gap junctions).

Cell junction and cycle AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type.

The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other - a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop.

De novo construction of cell-to-cell channels.

Nexus (gap junctions), which are considered to contain cell-to-cell channels, are newly formed in uterine smooth muscle during parturition or in response to estrogen treatment of virginal animals. A mRNA preparation was isolated from estrogen-dominated rat myometria and was encapsulated into liposomes. Subsequently the liposomes were fused with cultured cells of a mouse cell line CL-1D.

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells.

The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of "gap junction"; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related.

Intercellular communication and tissue growth: VII. A cancer cell strain with retarded formation of permeable membrane junction and reduced exchange of a 330-dalton molecule.

A cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5 per cent of the contacts (as against 100 per cent normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels.

The role of divalent cations in activation of the sea urchin egg. I. Effect of fertilization on divalent cation content.

The Ca and Mg content of unfertilized sea urchin eggs (3 and 21 mumole/ml eggs) remains remarkably constant over periods of hours, even when the eggs are suspended in Ca- or Mg-free sea water. After fertilization the Ca content of eggs in regular sea water increases sharply by about 20%, followed by a decrease to the unfertilized level by 40 minutes. However, if the fertilized eggs are washed three minutes in Ca-free sea water a sharp decrease in the Ca content occurs amounting to 30% of the total in the first 40 minutes, with little change thereafter.

Autotransplantation of retinal pigment epithelium in intravitreal diffusion chamber.

Retinal pigment epithelial (RPE) cells were autotransplanted intravitreally in diffusion chambers in rabbit eyes. The RPE cells underwent transformation; first into macrophages, later into spindle-shaped cells with collagen production having epithelial cell characteristics. DNA replication, shown by autoradiography, and loss of pigment granules indicated proliferation of these cells.

The membrane junctions in communicating and noncommunicating cells, their hybrids, and segregants.

Human Lesch-Nyhan cells, which are coupling and have gap junctions, were fused with mouse cl-lD cells, which are noncoupling and lack gap junctions. The resulting hybrid cells were coupling and had gap junctions while they contained the nearly complete complement of parent chromosomes. As the hybrid cells lost human chromosomes, clones appeared among the segregants, which had reverted to the noncoupling and junction-deficient trait of the mouse parent cell.

Intercellular communication and tissue growth : V. A cancer cell strain that fails to make permeable membrane junctions with normal cells.

A method is described for testing communication between a normal and a cancerous cell in culture without inserting microprobes into either cell; microprobes are put into other normal cells coupled to the normal cell in question. It is shown with this method that a cell strain (class-A), of epithelial morphology, isolated from Morris' liver tumor (H-5123) fails to make communicative junctions with several types of normal cells; small inorganic ions and fluorescein do not pass from the normal cells to the class-A cells (they do pass from the normal cells to normal cells, even between normal cells of different type). The class-A cells also appear incapable of junctional communication among themselves.