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

In this work, FRAP measurements were performed on MDCK cells under three different conditions. (a) Adding the fluorescent probes to the apical domain of confluent monolayers. This set of measurements was carded out on cells grown either on glass coverslips or collagen/Nitex filters (see below). No differences were found between both conditions. (b) The fluorescent probes were added to the free surface of sparse cells incubated in D-MEM or confluent monolayers prevented from establishing intercellular contacts by incubation in S-MEM on glass coverslips. (c) To allow access of probes to the basolateral surface, confluent MDCK monolayers were formed on 15-1~m mesh nylon filters (type Nitex; Tetko, Elmsford, NY), glued to acrylic plastic rings, and covered with native type I collagen gels (24) . Previous studies (12, 63) have shown that filter grown MDCK cells display 200-300 O.cm 2 transmonolayer electrical resistance and are impermeable to antibodies. The cells were plated on the internal chamber at immediate confluency (2-3 × 10 ~ cells/cm 2) in D-MEM 10% horse serum, which was changed 2 h later to protein-free, dye-free D-MEM and immediately before the experiment to Moscona's saline solution (140 mM NaCI, 2.7 mM KCI, 4 x 105 M, sodium phosphate, 11.9 mM NaHCO3, 9.4 mM glucose) supplemented with 50 mM Hepes (pH 7.5), 0.1 mM CaC12, 1 mM MgC12, and 50 I~g/ml pre-immune, electrophoretically pure, mouse IgG (washing buffer). After 20 min on ice, fluorescein-conjugated Fab fragments were added to the same medium up to a final concentration of 30 lag/ml. Labeling by B2-m: subconfluem or confluem cells were incubated with 10 -7 M FITC-B2-m in medium containing 0.1% BSA for 8-12 h and then washed free of excess B2-m. To enhance the fluorescent signal from fluorescent Fab fragment mAb, a fluorescein-conjugated Fab fragment from a goat anti-mouse IgG (Organon Tecnika-Cappel), was occasionally added. In this case, pre-immune mouse IgG was not present in the incubation medium. When the probes were added from the apical side, the total incubation time was 1 h, followed by four washes in washing buffer. When the fluorescent probes were added from the basolateral side, the incubation time was 3-7 h followed by 3 x 1-h washes in washing buffer. Previous studies indicate that the monolayers remain tight under these conditions. These long incubation periods were required to allow diffusion of the antibody through the unstirred layers trapped in the filter (45, 63) . All these procedures were performed on ice to prevent endocytosis of the labeled probes. The nylon filters were detached from the plastic ring and the cells were mounted in washing buffer under a glass coverslip facing the microscope objective. FRAP measurements were performed usually at 190C, immediately after washing away free antibody.