Clathrin and clathrin adaptors are required for focal adhesion disassembly and polarized cell migration. (a) Phase images from videos of NIH3T3 cells treated with the indicated siRNA and allowed to migrate into a wound. The same wound edge is shown at 0 and 6 h of migration. (b) Quantification of wound migration velocity of cells depleted of clathrin or clathrin adaptors. The histogram represents two to three independent experiments in which 20 wounds were analyzed per condition. *, P ≤ 0.0001 for each condition compared with GAPDH by Student’s t test. (c) Immunofluorescence of NIH3T3 cells depleted of either clathrin or Dab2 and ARH and immunostained for vinculin. Cells were allowed to migrate into a wound for 9 h before processing for immunofluorescence. Arrows indicate examples of elongated tails. (d) Quantification of tail length in migrating cells depleted of clathrin or clathrin adaptors. The histogram represents two independent experiments in which at least 20 cells were analyzed per condition. *, P ≤ 0.005 for each condition compared with mock by Student’s t test. (e) Fluorescence images from videos of migrating NIH3T3 cells stably expressing GFP-FAK and depleted of clathrin, Dab2, and ARH or mock treated (0-, 20-, or 30-min time points are shown). Small arrows point to focal adhesions that disassemble during 30 min of cell migration. Arrowheads point to focal adhesions that fail to disassemble during 30 min of cell migration. Large arrows indicate the direction of migration. (f) Quantitative analysis of focal adhesion (FA) disassembly in cells depleted of clathrin or ARH and Dab2. The histogram shows the percentage of focal adhesions that disassemble per cell during a 30-min interval. For each of the conditions analyzed, focal adhesion disassembly was quantified for at least 10 different cells (30–50 adhesions per cell) from five to eight separate videos. (b, d, and f) Error bars are SEM. Bars, 15 µm.