Figure 5.

LPP targeting to adhesions in response to TGFβ requires SHCA. A and B, ErbB2-NT/SHCA^endo (explant 83) and ErbB2-NT/SHCA^low (explant 87) cells were infected with mCherry-LPP, seeded onto fibronectin-coated glass bottom dishes, and cultured in the absence or presence of TGFβ (2 ng ml⁻¹) for 24 h. Cells were then fixed with 4% PFA and stained with antibodies against vinculin and phalloidin (F-actin). Images were taken on a Zeiss CLSM using a Plan-Apochromat ×63/1.4 numerical aperture oil immersion DIC objective lens (1 pixel = 0.132 μm). The arrowheads highlight examples of small adhesions formed after treatment with TGFβ. Scale bar, 10 μm. C and D, images were imported into Imaris to determine the average number of LPP and vinculin-bearing adhesions over the whole cell for each condition. Data were normalized by dividing the number of adhesions in each cell by the total cell area. Cell area was determined by drawing a contour around each cell. Data represent mean ± S.E. (error bars) from three independent experiments. (*, p = 0.001, Mann–Whitney U test). E and F, adhesions in protrusive cell regions were tracked over time using TIRF microscopy. Average assembly (green) or disassembly (red) rates were determined from changes in mean fluorescence intensity after 24 h with or without TGFβ treatment. Data represent individual assembly and disassembly events from three independent experiments. Coloured n values refer to the number of events while black n values refer to the number of cells. The top and bottom lines of the box indicate the third and first quartile, respectively, whereas the heavy central line indicates the mean. The whiskers extend up to 1.5 times the interquartile range. Black dots represent outliers. *, p < 0.001 calculated from the cell averages for assembly and disassembly rates, two-tailed Student's t test.