Figure 4. Fibronectin (FN)-null cells spread and formed focal contacts on FN-central cell-binding domain (FNIII_8–11) similar to cells on intact FN or contiguous arrays of FN-CCBD and FN-growth factor-binding domains (GFBDs) (FNIII_1–11 or FNIII_8–v15) for at least 24 hours.

FN-null fibroblasts were cultured at 8,000 cells per 35-mm dish that had been precoated with 0.125 µm intact FN or glutathione S-transferase (GST)-tagged FNIII_8–11, FNIII_1–11, or FNIII_8–v15 in DMEM for 4, 24, 48, or 72 hours at 37 °C. After the appropriate incubation times, cells were fixed, permeabilized, and stained for vinculin and actin as described in Materials and Methods. (a) FN-null cells had formed vinculin-positive focal contacts (green) and actin stress fibers (red) on all precoated surfaces by 4 hours. (b) Vinculin-positive focal contacts and actin stress fibers persisted in all cells at 24 hours. (c) By 48 hours, FN-null cells plated on GST-FNIII_8–11 were less well spread and had only a few vinculin-positive focal contacts, whereas cells on intact FN, FNIII_1–11, or FNIII_8–v15 remained well spread with prominent actin stress fibers and many focal contacts. (d) By 72 hours, FN-null cells on dishes coated with GST-FNIII_8–11 had become sparse and those that remained were mostly rounded with minimal evidence of actin bundles or focal contacts, whereas cells on intact FN, FNIII_1–11, or FNIII_8–v15 continued to be spread with prominent actin stress fibers and focal contacts. Bar = 20 µm.