CLICs become polarized during and are necessary for efficient cellular migration. (A) Confluent monolayers of WT MEFs or NIH3T3s were scratched and cells were allowed to migrate for 8–12 h. CTxB-555 and Tf-647 were pulsed into migrating cells for 2 min in the presence of anti-CD44 (WT MEF), anti-GFP (GFP-GPI expressing WT MEF), or anti-Thy-1 (NIH3T3) antibodies or cells were labeled for Cav-1 (WT MEF). Dotted lines indicate leading edges. Arrows show colocalization between anti-CD44 or anti–Thy-1 and CTxB-555 but not Tf-647 at the leading edge. Bar, 20 µm. (B) Quantitation of average pixel intensity from the leading to trailing edges for CTxB-555, Cav-1, and Tf-647. Inset shows the concentration of tubular, Cav-1, and Tf-negative CTxB-labeled CLICs at the leading edge. A rectangular area, outlined, was used to calculate the average pixel intensity (along the y axis) across the leading to trailing edge (along the x axis) for each endocytic marker. Plot profiles identify a concentration of CTxB in the leading edge and Cav-1 in the trailing edge whereas Tf shows uniform intensity across the cell. Bar, 10 µm. (C) Electron micrographs of a migrating WT MEF. Large arrow indicates direction of migration. Magnifications from the leading edge and the trailing edge show representative images of CTxB-HRP–labeled CLICs (1, 2) and surface-connected caveolae (3, 4). Bar, 500 nm. (D) Quantitation of the number of endocytic structures at the leading and trailing edge of cells treated as in C. Budded caveolae and CCVs are positive for CTxB-HRP label, whereas caveolae and CCPs are not. Error bars show SEM. (E) WT MEFs were incubated with or without CTxB-HRP for 2 min. The DAB reaction was performed on live cells for 5 min. Cells were washed and allowed to grow for a further 4 h. CTxB-555, anti-CD44 antibodies, and Tf-647 were added directly to cells for 2 min of uptake before acid stripping and fixation. Bar, 10 µm. (F) 12–15 cells treated as in E were quantitated for average fluorescence intensity of CTxB-555, Tf-647, or goat anti–mouse-488 for mouse–anti-CD44. (G) Confluent monolayers of WT MEFs were scratched and cells were allowed to migrate into the space for 1 h. Cells were incubated with serum alone or serum with 10 µg ml⁻¹ CTxB-HRP for 2 min and the DAB reaction was performed as in E. After 4 h of migration, cells were fixed and distance migrated was determined by measuring the distance of the gap between both sides of the wound at time zero and after 4 h of incubation. Error bars show SEM from three independent experiments.