Figure 2.

Fluorescence images and spectra of Fn-D/A in fibroblast matrix fibrils. (A–C) Fn-D/A was added to the culture medium of NIH 3T3 fibroblasts and incorporated into their fibrillar matrix. Excess unlabeled Fn was added to prevent intermolecular energy transfer. Fibrils visibly separated from other fibrils and from cells were analyzed. Arrows indicate beginning and end points of a series of spectra collected along a fibril. Spectra were integrated from 0.5-μm fibril segments spaced roughly 1 μm apart. (Scale bars, 10 μm.) (D) FRET varied between fibrils and along individual fibrils. Spectra labeled A, B, and C correspond to fibrils in images. Other fibrils that were analyzed are designated by lowercase letters, d–l. The shaded region indicates the range of FRET observed for Fn-D/A in fibrils of cells treated with cytochalasin D, a reagent that disrupts cytoskeletal tension (n = 78, 8 fibrils). The dashed line at the bottom indicates the level of FRET from Fn-D/A in 8 M Gdn⋅HCl in PBS.