FIGURE 3:

The relationship between α6β4, paxillin, and vimentin in the protrusive processes extended by repair cells at the wound edge. Vimentin (E, green) colocalized and extended to and inserted into prominent paxillin-rich adhesion plaques (A, D, red) at the tips of these protrusive processes, which contained little actin (B, green) filaments. (C) Overlay of paxillin and actin. (F) Overlay of paxillin and vimentin. Arrows identify paxillin-rich focal adhesions preferentially linked to vimentin. Inset, vimentin and paxillin colocalization at higher magnification (D–F). (G) Coimmunoprecipitation analysis of wounded explants at D1–D3 in culture examined in wounded explants after separation into OAZ and CMZ fractions. Samples were immunoprecipitated for paxillin and the immunoprecipitates immunoblotted for vimentin and paxillin. Blots included whole-cell lysates (WCLs) blotted for total vimentin and paxillin expression in each fraction. Results were quantified over three independent experiments and are represented as the ratio of vimentin to paxillin in each fraction (H). Results demonstrate that vimentin interacts with paxillin predominately in the CMZ, with the greatest interaction occurring at D2, a time of significant motility for wound closure (H). Cells were labeled for α6 integrin (I, red) or β4 integrin (L, red) and colabeled with vimentin (J, green) or F-actin (M, green), respectively, with overlays in K and N, at 1 d after injury. Both α6 integrin and β4 integrin were concentrated in the protrusive processes of the repair cells, into which vimentin intermediate filaments but few F-actin filaments were extended (arrows). All images are confocal projected images except insets of paxillin, vimentin, and overlay (D–F, insets) and α6 integrin, vimentin, and overlay (I–K). (A–F, and I–N) were taken at the leading edge of the CMZ. Bar, 20 μm, for inset, 5 μm. Secondary antibody controls were performed, which demonstrated specificity of antibody staining (Supplemental Figure S1).