TRANSLATIONAL DIFFUSION MEASUREMENTS OF A FLUORESCENT PHOSPHOLIPID BETWEEN MDCK-I CELLS SUPPORT THE LIPID MODEL OF THE TIGHT JUNCTIONS

The diffusion of a lipid fluorescent analogue across tight junctions in MDCK-I cells was studied by FRAP measurements. Cells were labelled with C-6-NBD-PC at the apical cell membrane. Transepithelial resistance of MDCK-I cells was found to be higher than 3000 Omega x cm(2), which indicates the presence of a very tight epithelium. After bleaching of large areas, fluorescence recovery was detected by confocal laser scanning microscopy, and diffusion coefficients were calculated. The lipid probes were able to diffuse to neighbouring cells as long as tight junctions between cells were intact. The average diffusion coefficient of the lipid was determined to be 5 x 10(-9) +/- 3.5 x 10(-9) cm(2)/s (n = 12) at room temperature. In controls, where the tight junctions were opened by complexing Ca2+ ions with EDTA, no recovery was observed. Redistribution of C-6-NBD-PC into the bleached apical membrane did not occur in experiments at temperatures below 10 degrees C. We conclude that the tight junction structure can be described by the lipid model, assuming the formation of an intermembranous hexagonal cylinder or lipids for the cell contact. In this model the exoplasmatic leaflets of the plasma membranes are continuous, thus allowing free diffusion of suitable lipids from one cell to another.