VINCULIN LOCALIZATION AND ACTIN STRESS FIBERS DIFFER IN THYROID-CELLSORGANIZED AS MONOLAYERS OR FOLLICLES

In epithelial cells interactions between the actin cytoskeleton and ce ll-cell junctions regulate paracellular permeability and participate i n morphogenesis. We have studied the relationship between supracellula r morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respe ctively, and showed F-actin staining in apical microvilli and a periju nctional ring. In monolayers, actin stress fibers were also observed i n the apical and basal poles of cells, where they terminated in the vi nculin-rich zonula adherens and in cell-substrate focal adhesions, res pectively. Surprisingly, we were unable to detect vinculin localizatio n in follicular cells, which also did not form stress fibers. Immunobl otting confirmed significantly greater vinculin in triton-insoluble fr actions from monolayer cells compared with follicular cells. Incubatio n of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the lev el of immunodetectable vinculin in the zonula adherens, indicating tha t junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 mu M) caused actin filaments to aggregate associated with retraction of cells from one another and th e disruption of cell junctions. Despite morphologically similar pertur bations of actin organization in follicular cultures treated with cyto chalasin D, junctional staining of ZO-1 and E-cadherin was preserved a nd cells remained adherent to one another. We conclude that in culture d thyroid cells structural and functional associations between actin f ilaments and cellular junctions differ depending upon the supracellula r morphology in which cells are grown. One important underlying mechan ism appears to be regulation of vinculin incorporation into adhesive j unctions by cyclic AMP. (C) 1995 Wiley-Liss, Inc.