Isolated-polarized epithelial cells as an experimental system for cell physiology studies.

Transporting epithelial cells are endowed with polarity, that is, different regions of the cell differ structurally and functionally. The most important aspect of epithelial cell polarity is that the transport proteins expressed in the apical and basolateral membrane are different. This property is essential for epithelia to perform transepithelial net transport (vectorial transport) when both sides are bathed in solutions of the same composition. Epithelial cell polarity involves also other biochemical differences (e.g., different phospholipid composition of the two plasma membrane domains), as well as structural differences between apical and basolateral regions, particularly in the cytoskeleton and distribution of organelles (Fig 1). Epithelial cells contact their neighbors via tight junctions, desmosomes, and gap junctions. Desmosomes attach the cells mechanically, by binding of cell adhesion molecules (CAMs) of the adjoining cells. Cadependent CAMs are called cadherins and are integral membrane glycoproteins with a single transmembrane domain. The one expressed in epithelia is cadherin E or uvomorulin. The intracellular domain of E-cadherin interacts with the cytoskeleton in a complex fashion (reviewed by Buxton, 1993). Gap junctions are channels that communicate the cytosol of adjacent cells. Each cell contributes a connexon, which is a hexamer of transmembrane proteins called connexins (a family consisting of at least 14 members. In the connexon the six connexin molecules are parallel to each other, perpendicular to the plasma membrane, leaving a space in the middle. A gap junction (cell-to-cell channel) is formed by two connexons belonging to adjacent cells, joined in series. Gap junctions allow the permeation of molecules up to about 1 kDa. Their functional regulation is isoform-dependent and may involve phosphorylation, and effects of intracellular pH, intracellular pCa and/or membrane voltage (Bennett et al., 1991). A functional epithelium requires also contacts with the basal lamina, a region at the interface with the underlying connective tissue, via hemidesmosomes and focal contacts. Both forms of contact involve integrins, transmembrane proteins that bind to motifs in molecules of the extracellular matrix. In the intracellular side, hemidesmosomes attach to intermediate filaments via a desmoplakin-like protein and focal contacts attach to actin filaments via a complex including talin, a-actinin and vinculin. In epithelial cells, integrins are involved in the initiation of cell polarization following plating of dissociated cells (Rodriguez-Boulan & Nelson, 1989; Ruoslahti et al., 1994). The apical and basolateral membrane domains are separated by the junctional complexes, which include the zonula occludens (“tight junction”) and the zonula adherens. The zonula occludens is formed by transmembrane proteins arranged in linear polymers, bound to similar proteins in the adjacent cell. This is visualized in freeze-fracture microscopy as strands arranged in parallel, with branches and anastomoses. It is thought that in the junctions the outer leaflets of the plasma membranes are fused. However, although in many epithelia the Correspondence to: L. Reuss