Fig. 4.

Fn fiber breakage for fibers that are adherent to an elastic silicone substrate. (A) A spatially resolved FRET map of manually deposited Fn fibers subjected to a 2× increase in substrate length shows areas of fiber breakage and large areas of mechanically induced unfolded Fn Type III modules, as confirmed via (B) a histogram of every pixel’s acceptor intensity / donor intensity (I_A/I_D, see Materials and methods) and calibrated to a guanidinium hydrochloride (GdnHCL) denaturing curve. The absolute strain scale represents the average I_A/I_D values of Fn fibers that are fully relaxed or extended to the point where they begin to break (5–6×), as described in the text and in Fig. 3. (C) Zooming in on an example breakage location in a fiber as noted by the red box in (A). (D) A fluorescence image of the break, especially a (E) contrast-enhanced imaged, reveals that a small number of individual strands are being pulled apart and left behind in the breakage gap that are not detected in our FRET measurements due to the necessary analysis thresholding (see Materials and methods). (F) AFM image confirming the presence of a small amount of fibrillar Fn left on the substrate and a tapered end to the broken fiber. Inset zoomed-in image of a fractured fiber end shows the receding strands. (G) Some breaks also show only a small amount of Fn left behind on the substrate and show more blunted fibers ends. Scale bar in (A)=30 µm, Scale bar in (D)=15 µm and is the same scale as the images in (C) and (E).