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. 2020 Nov 18;119(12):2558–2572. doi: 10.1016/j.bpj.2020.10.043

Figure 5.

Figure 5

Fibroblast traction forces lead to substrate deformations but not fibronectin fibril assembly. (A) Epifluorescence and phase contrast images of live pHDF seeded for 3 h on elastomers coated with fluorescently labeled FN (FFN). The intensity of FFN increased under the cell body on the softer (ζ = 0.7) elastomers. (B) Normalized FFN fluorescence intensity under the cell body normalized against background FFN intensity. Mean and SD from two independent experiments are presented (n = 10). (C) Confocal microscopy images of live pHDF seeded on FFN-coated elastomers. On the softer elastomers (ζ = 0.7), surface out-of-plane deformations were observed. Dashed lines and arrows are visual aids. (D) Confocal microscopy images of live pHDF seeded for 2 h on elastomers with immobilized fluorescent beads and coated with FN. (E) Particle image velocimetry (PIV) analysis from bead displacements on elastomers seeded with pHDF for 1 h was used to produce substrate deformation fields. The magnitude of the color-coded vectors is given in pixels, and the white line denotes the cell outline. (F) Bead displacements (deformations) as a function of distance from the cell center was calculated as detailed in the Methods for each cell (gray lines) and averaged (circles with SD). The data (mean ± SD, n = 9 for ζ = 0.7, and n = 14 for ζ = 1.0) were fitted using an exponential decay function (red line) starting from a distance of 20 μm, which corresponds to the typical cell radius. One of two independent experiments is presented. (G) Quantification of two parameters reflecting total substrate deformation and the deformation boundary from the cell edge based on the fitted curves from (F). A threshold value of 1 μm for the deformation vectors was selected to calculate the distance from the cell edge at which deformations drop below the threshold and the area under the fitted curve (AUC) correlating with total substrate deformation. Both the boundary distance and AUC were lower for the stiffer elastomers. Each data point corresponds to an independent experiment and the line the mean. (H) Confocal microscopy image of beads immobilized on a soft elastomer (ζ = 0.7), which is being deformed by a living cell. Orthogonal views are presented to better visualize the crater-like structure formed. Scale bars, 50 μm for (A) and 20 μm for (C, D, E, and H). To see this figure in color, go online.