Clathrin localization and dynamics during MT-induced focal adhesion disassembly. (a) TIRF image of a nocodazole-treated NIH3T3 fibroblast immunostained with antibodies against pY397 FAK and clathrin heavy chain. The merged image shows pY397 FAK (red) and clathrin (green). The region outlined by the box is shown at higher magnification on the right. The histogram shows the number of focal adhesions with clathrin puncta. (b) The left panel shows a merged TIRF image of an NIH3T3 cell line stably expressing GFP-clathrin (green) and transiently transfected with RFP-FAK (red) 18 min after MT regrowth to stimulate focal adhesion disassembly. The outlined region is shown at higher magnification in the panels on the right during time points after MT regrowth (minutes:seconds). Note that clathrin puncta colocalize with and accumulate on the disassembling focal adhesion. (c) Quantification of clathrin and FAK fluorescence during disassembly of the focal adhesion shown in b. (d) TIRF images from a video of GFP-clathrin accumulation at focal adhesions initially devoid of clathrin during MT regrowth in RFP-FAK–expressing cells as in b. Time is in minutes:seconds after MT regrowth. (e) Quantification of clathrin and FAK fluorescence during disassembly of the large focal adhesion in the top row of d. (f) Histogram indicating the fold clathrin accumulation during disassembly of focal adhesions (FA) initially containing low or high levels of clathrin before MT regrowth. Data are from 25–30 focal adhesions. Error bars are SEM. (g) TIRF images from a video of GFP-clathrin and focal adhesions (RFP-FAK) in a cell treated with nocodazole for 4 h and imaged while still in nocodazole. Time is in minutes:seconds. (h) Quantification of clathrin and FAK fluorescence of the large focal adhesion in the top row of g. noco, nocodazole. Bars: (a, b [insets], d, and g) 2 µm; (b) 15 µm.