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

Biochemical and functional characterization of intercellular adhesion and gap junctions in fibroblasts. Am J Physiol Cell Physiol 279: C147-C157, 2000.--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 [Ca.sup.2+] and was [is greater than]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 ([is greater than][10.sup.5]) 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