Intercellular mechanotransduction through adherens junctions in fibroblasts

Cells in mechanically active environments form extensive intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown how cells transmit mechanically-induced signals through intercellular junctions. The objectives of this thesis were to: (1) develop an in vitro model system that facilitates study of intercellular adhesion and communication in cells from soft connective tissues; (2) use this model to study how physical forces directed through intercellular adherens junctions mediate force-induced calcium signals. To study the formation of intercellular adherens junctions and gap junctions, I developed a model in which suspended human gingival fibroblasts were plated on established homotypic monolayer cultures. These cells rapidly formed adherens junctions (∼15 min.) mediated by cadherins. Distinct gap junctional complexes were evident within 3 hr. of cell-cell contact. Intercellular cadherin-binding induced calcium influx through membrane channels and release of Ca2+ from internal calcium stores. These [Ca2+]_i changes were temporally correlated with increased recruitment of intercellular junctional proteins into the cytoskeleton and movement of GFP-actin to sites of cell-cell contact. Buffering intracellular Ca2+ with BAPTA/AM significantly inhibited intercellular adhesion as well as recruitment of cadherins and β-catenin to the actin cytoskeleton. Depolymerization of actin filaments by cytochalasin D dramatically reduced intercellular adhesion and recruitment of cadherins and catenin to the actin cytoskeleton. These results indicated that calcium signals regulate intercellular adhesion through remodeling of cortical actin and recruitment of cadherins and β-catenin into intercellular junctions. To study intercellular mechanotransduction, controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. Force application induced robust Ca2+ transients (65 ± 9.4 nM above baseline) that depended on influx of extracellular Ca2+ through mechanosensitive channels as both Ca2+ chelation and gadolinium chloride abolished the response and MnCl₂ quenched fura-2 fluorescence after force application. Similar Ca2+ transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. At intercellular contacts, force application induced accumulation of microinjected rhodamine-actin which was inhibited by buffering intracellular calcium fluxes. My results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. Consequently I conclude that in fibroblasts, N-cadherins are intercellular mechanotransducers.