Figure 7. Adhesion composition correlates with size in 3D.

U2OS cells were co-transfected with DsRed-paxillin and with GFP-dSH2, GFP-vinculin, or GFP-zyxin. (a–b) Cells were cultured in 2 mg/ml bovine collagen gels. The ratio of dSH2 to paxillin intensities for each adhesion was determined and presented as a heatmap image (a) and plotted as a function of adhesion length (b, gray dots). Black dots show the median values for adhesions < 1 μm, 1–2 μm, and > 2 μm (error bars are 25^th and 75^th percentiles). There is no trend of dSH2 levels with adhesion size of cells in collagen matrices. (c) Because intensity ratio values can only be compared within a single cell, data from multiple cells were summarized by dividing the median intensity ratio values for adhesions < 1 μm by those of adhesions > 1 μm. Error bars are the 95% confidence intervals. Cells cultured in collagen matrices, on glass, or on PCL scaffolds did not show a significant difference in dSH2 levels between small and large adhesions (the 95% confidence intervals include 1 (identical intensity ratios); from left to right, n = 6, 3 and 9 cells). See Fig. S5c–d for glass and PCL scaffold images. (d–h) A similar analysis is presented for cells expressing vinculin/paxillin (d–e) or zyxin/paxillin (f–g) and cultured on PCL scaffolds. Both vinculin and zyxin show increased levels (relative to paxillin) with increasing adhesion size. Insets show small adhesions in locations with limited adhesive area. (h) Quantification across multiple cells by dividing intensity ratios of adhesions < 1 μm by those of adhesions > 2 μm. Both vinculin and zyxin levels are significantly higher in larger adhesions (n = 9 cells for each group). Bars, 10 μm (for insets, 2 μm).