FIGURE 7:
TGFβ-induced EMT alters adhesion organization and stability. (a) Live-cell imaging of paxillin-mEmerald and tensin-mCherry to visualize focal adhesion turnover in MCF10AT cells with and without TGFβ treatment. Top row: control, bottom row: TGFβ-treated. Scale bar, 10 µm (main image), 1 µm (inset). (b) Cumulative distributions of adhesion lifetimes. Inset, quantification of mean adhesion lifetime; mean ± SEM (***p < 0.001; >150 adhesions from five cells total). (c) Focal adhesion microstructure of MCF10AT seeded on MTSs overexpressing paxillin-eGFP and imaged with TIRF and 3B superresolution microscopy. Scale bar, 3 µm (main image), 1 µm (zoom). (d) Quantification of mean adhesion length of MCF10AT cells. TGFβ-treated cells have significantly longer adhesions; mean ± SEM (***p < 0.001; >100 adhesions from five cells). (e) Tensin-positive fibrillar adhesion formation in MCF10AT overexpressing paxillin-mEmerald and tensin-mCherry. Scale bar, 3 µm. (f) Quantification of the spatial colocalization between tensin and paxillin in adhesions. TGFβ-treated cells have significantly more paxillin-tensin colocalization (*p < 0.05; >10 cells). Pearson’s correlation was calculated based on pixels within the focal adhesion areas. (g) Paxillin-mEmerald (N-terminal) axial position in focal adhesions in MCF10As measured with SAIM. Paxillin position is plotted as the relative axial distance from the silicon wafer coated with fibronectin. Scale bar, 5 µm. (h) SAIM axial height measurements of adhesion proteins tagged with fluorescent proteins in MCF10As with and without TGFβ treatment. Paxillin is the only protein to significantly change axial position >10 nm, suggesting that the position of paxillin moves relative to that of the adhesion as a whole. (i) Quantification of paxillin-GFP axial position in MCF10As with and without TGFβ treatment. Paxillin in treated cells is located at a significantly higher axial nanoscale position (*p < 0.05; >10 cells). (j) Representative images of the axial position of paxillin-mEmerald (N-terminal) in focal adhesions in PyMT and Met1 cells. Scale bar, 5 µm.