Clathrin and adaptor protein localization during polarized cell migration. (a) TIRF image of an NIH3T3 fibroblast migrating into an in vitro wound immunostained with antibodies against pY397 FAK and clathrin. The white line shows the wound edge. The merge image shows pY397 FAK (red) and clathrin (green). The boxed region in the merge image is shown at higher magnification on the right. The arrow indicates a focal adhesion located behind the leading edge that has accumulated clathrin puncta. (b) Representative images used for quantitative colocalization of clathrin and focal adhesions in different regions of an NIH3T3 fibroblast migrating into an in vitro wound and immunostained with antibodies against MTs, pY397 FAK, and clathrin. The MT image shows the cell outline and four regions used for quantification (see Materials and methods). The merged image shows pY397 FAK (red) and clathrin heavy chain (green) and the four regions. Arrowheads indicate focal adhesions that colocalize with clathrin puncta. (c) Quantification of focal adhesion (FA) area that colocalizes with clathrin puncta in the four regions defined in b. The histogram represents data from two independent experiments in which 15–20 individual cells were analyzed. (d) Images of NIH3T3 fibroblasts migrating into an in vitro wound and immunostained for MTs, vinculin or pY397 FAK, and ARH or Dab2. The merged images at right show pY397 FAK or vinculin (red) and ARH or Dab2 (green). An individual migrating cell at the wound edge is indicated by the outline. Arrowheads indicate focal adhesions that colocalized with either ARH or Dab2 puncta. (e) Quantification of adaptor protein colocalization with focal adhesion area in the four regions defined in b. The histogram represents data from two independent experiments in which colocalization was measured in 10–20 individual cells. (c and e) Error bars are SEM. Bars, 15 µm.