Clathrin is required for MT-induced focal adhesion disassembly. (a) Western blot of clathrin heavy chain (HC) siRNA– or GAPDH siRNA–treated NIH3T3 fibroblasts showing levels of clathrin heavy chain, FAK, pY397 FAK, and GAPDH. (b) Clathrin heavy chain immunofluorescence and transferrin uptake in GAPDH- and clathrin heavy chain–depleted NIH3T3 fibroblasts. (c) Immunofluorescence of vinculin, actin, and transferrin uptake during MT-induced focal adhesion disassembly in GAPDH- and clathrin heavy chain–depleted NIH3T3 fibroblasts. Images are before MT regrowth (10 µM nocodazole [NZ] for 4 h) and 40 min after nocodazole washed out and MT regrowth. (d) Quantification of focal adhesion (FA) disassembly in clathrin heavy chain (clthc)–, GAPDH-, or mock-depleted NIH3T3 fibroblasts. Cells were scored positive if they retained 10 focal adhesions after MT-induced disassembly. Data are from five independent experiments in which several hundred cells were analyzed for each condition in each experiment. (e) Western blot of clathrin heavy chain, pY397 FAK, total FAK, and GAPDH during MT regrowth after nocodazole washout in cells depleted of either clathrin or GAPDH. Cells were treated with nocodazole for 4 h, and then the drug was washed out, and MTs were allowed to regrow for the indicated times before preparing samples for SDS-PAGE. (f) Immunofluorescence of vinculin and MTs after nocodazole washout (40 min) in cells depleted of clathrin. The region outlined by the box in the merged panel is shown at higher magnification at the right. The arrowhead indicates a focal adhesion targeted by an MT. The histogram shows the number of MT–focal adhesion targeting events in clathrin- or mock-depleted cells. Data are from two experiments in which 20 cells were analyzed per condition. (g) Vinculin immunofluorescence and transferrin uptake in cells expressing FKBP–clathrin LC–GFP (arrows) and treated with or without AP20187. Images shown are before (10 µM nocodazole) and 40 min after MT regrowth. The cell expressing FKBP–clathrin LC is outlined in white to show that it does not endocytose transferrin. The histogram shows quantification of focal adhesion disassembly in FKBP-LC versus nontransfected cells. (d, f, and g) Error bars are SEM. Bars, 15 µm.