MICROTUBULE INTEGRITY IS NECESSARY FOR THE EPITHELIAL BARRIER FUNCTION OF CULTURED THYROID-CELL MONOLAYERS

Vectorial transport in the thyroid epithelium requires an efficient ba rrier against passive paracellular flux, a role which is principally p erformed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton . Although the contribution of the actin cytoskeleton to tight junctio n physiology has been intensively studied, less is known about possibl e interactions with microtubules. In the present study we used electro physiological and immunohistochemical approaches to investigate the co ntribution of microtubules to the paracellular barrier in cultured thy roid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm . cm(2)). Colchicine (1 mu M) caused a prog ressive fall in electrical resistance to <10% of baseline after 6 h an d depolarization of the transepithelial electrical potential differenc e consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhi bit the major apical conductances of thyroid cells but was reversed up on removal of the drug. Immunofluorescent staining for tubulin combine d with confocal laser scanning microscopy demonstrated that thyroid ce lls possessed a dense microtubule network extending throughout the cyt oplasm which was destroyed by colchicine. Colchicine also produced cha nges in the localization of the tight junction-associated protein, ZO- 1: its normally continuous junctional distribution was disrupted by st riking discontinuities and the appearance of many fine strands which e xtended into the cytoplasm. A similar disruption in E-cadherin stainin g was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in thi s electrically tight, transporting epithelium. (C) 1995 Academic Press , Inc.