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Am J Physiol Cell Physiol 279: C147-C157, 2000;
0363-6143/00 $5.00
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Vol. 279, Issue 1, C147-C157, July 2000

Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts

Kevin Ko, Pamela Arora, Wilson Lee, and Christopher McCulloch

Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5S 3E2

Despite their significance in wound healing, little is known about the molecular determinants of cell-to-cell adhesion and gap junctional communication in fibroblasts. We characterized intercellular adherens junctions and gap junctions in human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion required Ca2+ and was >30-fold stronger than cell-to-fibronectin adhesion at 15 min. Electron micrographs showed rapid formation of adherens junction-like structures at ~15 min that matured by ~2-3 h; distinct gap junctional complexes were evident by ~3 h. Immunoblotting showed that HGF expressed beta -catenin and that cadherins and connexin43 were recruited to the Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal microscopy localized the same molecules to intercellular contacts of acceptor and donor cells. There was extensive calcein dye transfer in a cohort of Texas red dextran-labeled cells, but this was almost completely abolished by the gap junction inhibitor beta -glycyrrhetinic acid and the connexin43 mimetic peptide GAP 27. This donor-acceptor cell model allows large numbers (>105) of cells to form synchronous cell-to-cell contacts, thereby enabling the simultaneous functional and molecular studies of adherens junctions and gap junctions.

adherens junctions; fluorescence dye; cell adhesion


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