Fluorescent membrane probes and the mechanism of maintenance of cellular asymmetry in epithelia

Abstract

Individual epithelial cells are asymmetric with respect to morphology, distribution of enzymes, lipid composition, ionic permeability, and sensitivity to drugs and hormones on the apical (mucosal) versus basolateral (serosal) membranes. It has been suggested that the tight junction, which forms the morphological boundary between apical and basolateral surfaces, helps to maintain this cellular asymmetry by forming a barrier to lateral diffusion of membrane constituents across these membranes. We have directly tested this hypothesis by selectively labeling either the apical or basolateral membrane with fluorescent probes and ascertaining 1) whether the probe can diffuse laterally in the membrane, and 2) whether it can pass through the tight junction region to the side of the cell initially not labeled. Our results show that membrane-bound lectins and some lipid probes are incapable of passing through the tight junction region. The lectins are immobile on the cell surface, but the lipid probes diffuse freely in the membrane. We propose that the ability of a lipid probe to pass through the tight junction is correlated with its ability to flip-flop to the inner monolayer of the cell membrane bilayer, and that the tight junction thus forms a barrier to lipids in the outer monolayer only. Differences in diffusion rates of different lipid probes also appear to reflect different physical characteristics of the inner versus outer membrane leaflet.