Fluorescent Dye Transfer Research Articles

Gap junction messenger RNA expression by vascular wall cells.

Gap junctions between vessel wall cells provide a pathway for the intercellular exchange of ions and small molecules. Pure cultures of microvascular and macrovascular endothelial and smooth muscle cells, vascular pericytes, and several nonvascular cell lines were tested for junctional communication by fluorescent dye transfer. All of the vascular wall cells were capable of dye transfer. Since gap junctions are formed by a family of related proteins (connexins) whose unique domains may confer physiological regulatory properties, we tested total RNA from these cultures by Northern blot analysis for expression of the currently available, characterized, and cloned mammalian gap junction proteins: connexin26, connexin32, and connexin43. All of the vascular wall cells expressed connexin43 messenger RNA. Connexin43 was expressed in vascular cells from bovine, porcine, rat, and human sources. Several nonvascular cell lines of mesenchymal origin also expressed connexin43 messenger RNA. When high stringency Northern blots were used, messenger RNAs for connexin32 or connexin26 were not detected in any of the vascular wall cells but were expressed in several cell lines of epithelial origin. Freshly isolated and purified aortic endothelial and smooth muscle RNA preparations similarly contained only connexin43 messenger RNA, excluding the possibility of culture-induced alterations in gene expression. The expression of connexin43 by all vascular wall cells may provide a mechanism for the functional integration of the vessel wall by gap junctions.

Changes in junctional communication associated with cell cycle arrest and differentiation of trochoblasts in embryos of Patella vulgata

In early embryos of molluscs, different clones of successively determined trochoblasts differentiate into prototroch cells and together contribute to the formation of a ciliated ring of cells known as the prototroch. Trochoblasts differentiate after cell cycle arrest, which occurs two cell cycles after the commitment of their stem cell. To study the changes of junctional communication in embryos of Patella vulgata in relation to commitment, cell cycle arrest, and differentiation of the trochoblasts, we have monitored electrical coupling as well as transfer of fluorescent dyes. The appearance of dye coupling in embryos of Patella occurs after the fifth cleavage (at the 32-cell stage), when the cell cycles of all embryonic cells become asynchronous and longer. At the 32- and 64-cell stages all cells are well coupled. However, after the 72-cell stage dye transfer to or from any cell of the four interradial clones of four primary trochoblasts becomes abruptly reduced, whereas electrical coupling between these cells and the rest of the embryo can still be detected. From scanning electron microscopical analysis of the cell pattern we conclude that this change in gap junctional communication coincides with cell cycle arrest and with the development of cilia in all four clones of primary trochoblasts. Similarly, after the 88-cell stage the four radial clones of accessory trochoblasts stop dividing, reduce cell coupling, and become ciliated. By the formation of the prototroch, the embryo becomes subdivided into an anterior (pretrochal) and a posterior (posttrochal) domain which will develop different structures of the adult. At the 88-cell stage, the cells within each of these two domains remain well coupled and form two different communication compartments that are separated from each other by the interposed ring of uncoupled trochoblasts. The relations among control of cell cycle, changes in junctional communication, and differentiation are discussed.

Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis

We have stripped small (3 x 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.

Induction of intercellular communications in epithelial cell cultures

A popular criterion of cell-cell communication in tissue cultures is dye coupling: the ability of the injected fluorescent dye of low molecular weight to be transferred from one cell to another. We report about a new factor which induces cell-to-cell dye coupling in previously uncoupled epithelial sheets. Paradoxically it is the standard fluorescent microscopy itself (that is, blue light of 320- to 480-nm wavelength) which induces rapid morphological alterations of cell culture followed by the transfer of fluorescent dye from one cell to another. Thus monitoring cell-cell dye coupling by fluorescent microscopy may itself induce the dye coupling in previously uncoupled epithelial cells.

Increased electrotonic coupling in aged rat hippocampus: A possible mechanism for cellular excitability changes

The effects of aging on the intercellular transfer of the low molecular weight fluorescent dye 5,6-carboxyfluorescein was studied in subfields fascia dentata, CA1, and CA3 of rat hippocampal slices maintained in vitro. All three areas exhibited a statistically significant increase in dye coupling with age. The increased dye coupling was accompanied by an apparent increase in postsynaptic excitability as assessed by the ratio of the population spike to EPSP components of the extracellulary recorded field potential. The possibility that artifactual dye coupling due to cell fusion contributed significantly to these results was ruled out by the demonstrations that a high molecular weight, dextran-coupled fluorescein compound did not produce multiple fills and that dye coupling with carboxyfluorescein could be prevented by prior intracellular loading with Ca++, a procedure that decouples gap junctions in other tissue. The increase in extent of electrical coupling suggested by these data may largely account for the apparent increase in cellular excitability of this tissue with age and may reflect the mechanism by which the senescent hippocampus compensates for the loss of afferent input during the course of normal aging. The additional possibility is raised that increased electrical coupling may reflect a mechanism for permanent associative storage of information in this system.

Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell.

Lens epithelial cells communicate with two different cell types. They communicate with other epithelial cells via gap junctions on their lateral membranes, and with fiber cells via junctions on their apices. We tested independently these two routes of cell-cell communication to determine if treatment with a 90% CO2-equilibrated medium caused a decrease in junctional permeability; the transfer of fluorescent dye was used as the assay. We found that the high-CO2 treatment blocked intraepithelial dye transfer but not fiber-to-epithelium dye transfer. The lens epithelial cell thus forms at least two physiologically distinct classes of gap junctions.

Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP.

The effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell-cell communication between mouse Balb/c 3T3 cells and between Chinese hamster V79 cells was inhibited almost completely by tumor-promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor-mediated, since specific binding of 3H-phorbol-12,13-dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.

Control of gap junction formation in early mouse embryos

Intercellular communication via gap junctions begins in the eight-cell stage in early mouse embryos. We have studied the timing of this event in relation to compaction, and have begun to explore some of the possible control mechanisms underlying it. Gap junction formation was inferred by measuring ionic coupling as well as by observing the intercellular transfer of fluorescent dye. Embryos were obtained early on Day 3 of pregnancy by flushing the oviducts of HA/ICR mice that had been mated with CB6 F 1 J males. Gap junctions were detected only in those embryos which had achieved the fully compacted state. Inhibition of protein synthesis by cycloheximide treatment beginning as early as the late four-cell stage failed to block compaction or the acquisition of gap junctions, demonstrating that the necessary proteinaceous components are present in advance of these events. In order to test the possibility that gap junctions could be induced to form prematurely, fully compacted, communication-competent eight-cell embryos were aggregated with two- or four-cell embryos. Even after 10 hr of aggregation, no interembryonic gap junctions could be detected. Fully compacted eight-cell embryos when aggregated with each other, however, became ionically coupled within 3–5 hr. The number of interembryonic junctional channels was judged to be effectively small, since the aggregated embryos exhibited obvious ionic coupling but very weak dye coupling. In contrast to gap junction formation within embryos, junction formation between embryos was blocked by cycloheximide. These results demonstrate that gap junction formation in early mouse embryos is under precise temporal control, involving the assembly or mobilisation of preexisting components. This stockpile of components is either unavailable or insufficient to allow the formation of additional gap junctions between aggregated communication-competent embryos without new protein synthesis.

Gap junction formation between normal and reaggregated endoderm cells ofXenopus laevis neurulae.

Using freeze-fracture electron microscopy and fluorescent dye injection we have analysed the contacts between cells of the deeper endoderm taken from neurulae ofXenopus laevis. Endodermal cells in situ have large 1.5 μm diameter gap junctions composed of 8 nm P-face particles and corresponding E-face pits. Beside gap junctions, particle aggregates typical of desmosomal plaques are present but there are no tight junctions. The dissociation of endoderm into single cells involves profound structural alterations in the surface membrane including the complete disappearance of junctional structures among them gap junctions. The reaggregation of endoderm cells leads to the restoration of the surface membrane IMP (Intra Membrane Particle) pattern and, after ca. 30 min, to the establishment of functional pathways allowing for the intercellular transfer of fluorescent dye. Concomitantly gap junctions reappear. The observation that the dissociation and reaggregation of endodermal cells involves IMP alterations which go beyond the cell junctions themselves is discussed as an adaptation of the plasma membrane to changing environmental conditions.