Occluding junctions and cytoskeletal components in a cultured transporting epithelium

Abstract

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This article explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicine and cytochalasin B, on the degree of tightness of the occluding junctions . To study the degree of tightness, monolayers were prepared by plating MDCK cells on nylon disks coated with collagen . Disks were mounted as flat sheets between two Lucite chambers, and the'sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers . Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation . Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane . Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell . Colchicine causes a profound disruption of microtubules but only a 27% decrease in the electrical resistance of the resting monolayers . Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance . The cortical ring of filaments remains in place but appears fragmented with time . We find that removal of extracellular Ca", which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca". Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible . The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTAopened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity.

“…kept to a large extent the nonextracted cell shape at the phase contrast and EM levels. In agreement with previous observations (18,44), an actin-rich submembrane cytoskeleton was observed associated with both apical and basolateral surfaces of MDCK cells by rhodamine phalloidin fluorescence on semi-thin frozen sections; larger amounts of actin were detected on the apical surface due to the presence of microvilli ( Fig. 1 A).…”

IL8 release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers

“…An important modulator of endothelial and epithelial permeability is the actin cytoskeleton through its interaction with TJ complex components (Meza et al, 1980;Madara, 1991;Lum & Malik, 1996;Blum et al, 1997). In dengue virus-infected HMEC-1, actin fibres forming the peripheral ring at the apical side were rapidly disorganized and possibly fragmented as actin aggregates were observed in the cytoplasm of infected cells.…”

“…It has been shown that disarray of the actin ring tethered to the plasma membrane in the apical side of epithelial and endothelial cells disrupts the organization of the TJ complex and the permeability of confluent monolayers (Meza et al, 1980;Lum & Malik, 1996). Infected HMEC-1 showed disorganization of actin structures and the actin ring.…”

“…Several proteins participate in the organization and function of TJ. Importantly, their localization and interactions are modulated by association with the actin cytoskeleton, in particular the actin ring tethered to the plasma membrane on the apical side (Meza et al, 1980;Madara, 1991;Rao et al, 2002). An increase in cellular permeability could result from alterations in the sealing capacity of TJ, caused by modifications to the TJ complex interactions (Cereijido et al, 1989).…”

Cell-ECM traction force modulates endogenous tension at cell–cell contacts

“…To disrupt the cell-cell contact, we exploited the Ca 2þ dependence of cadherin binding (23). We first determined the cell morphology, traction force, and cell-cell force for cell pairs with mature cell contacts in calcium-containing media.…”