A simple dye-coupling assay for evaluating gap junctional communication: The importance of transcription and translation on the establishment of dye-coupling

Zidell, Robert H.; Loch‐Caruso, Rita

doi:10.1016/0309-1651(90)90041-v

Cited by 4 publications

(1 citation statement)

References 28 publications

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“…To facilitate studies of heterologous communication, we found it necessary to develop a quantitative assay using two dyes. Similar assays of double staining using fluorescent beads (Zidell and Loch-Caruso, 1990) or a different fluorescent dye (Mehta et al, 1992) to prestain the recipient cells have been reported, both using microscopic analysis. Our assay uses flow cytometry to determine the fraction of cells containing two dyes as a result of GJIC between pairs of cells each containing a different fluorochrome, one transferable and the other fixed as a marker of the recipient cell population.…”

Section: Discussionsupporting

confidence: 52%

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

Tomasetto

Neveu

Daley

et al. 1993

The Journal of Cell Biology

119

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Abstract. In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein.The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26-and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

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Section: Discussionsupporting

confidence: 52%

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

Tomasetto

Neveu

Daley

et al. 1993

The Journal of Cell Biology

119

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Nickel-induced increases in gap junctional communication in the uterine cell line SK-UT-1

Loch‐Caruso

1993

In Vitro Cell Dev Biol - Animal

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Previous studies have suggested that gap junctions may have a role in various uterine functions, including parturition. Because nickel has been demonstrated to increase uterine contractility in vitro, the effect of nickel (II) chloride on gap junctional communication was assessed in a tumorigenic uterine cell line, SK-UT-1 (ATCC HTB 114). Cells were exposed in vitro to 25 and 50 microM NiCl2 for 24 h or 100 microM NiCl2 for 3, 12, and 24 h, then functional gap junctional communication was measured as the transfer of Lucifer yellow dye from microinjected donor cells to their primary neighbor cells. Dye transfer was significantly increased only in cell cultures exposed to 100 microM NiCl2 for 24 h, compared to untreated controls, lower doses, and shorter exposure periods. This response was inhibited by the simultaneous co-treatment of SK-UT-1 cells with magnesium by adding 100 microM MgSO4 to the dosing medium. Possible mechanisms and implications for these findings are discussed.

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Structure-Function Relationships in Gap Junctions

Wolburg

Rohlmann

1995

International Review of Cytology

100

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A simple dye-coupling assay for evaluating gap junctional communication: The importance of transcription and translation on the establishment of dye-coupling

Cited by 4 publications

References 28 publications

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

Nickel-induced increases in gap junctional communication in the uterine cell line SK-UT-1

Structure-Function Relationships in Gap Junctions

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