Self-Help Fibronectin Assembly Figure 6. Binding of the 70-kDa N-terminal fibronectin fragment has the same requirement of adherent substrates. Fibronectin (FN) or recombinant fibronectin constructs, 20 nM, were coated on surfaces of coverslips or 24-well tissue culture plates. After 1 hr incubation at 37uC of fibronectin-null cells on the above proteins, 2.7 mg/ml FITC-70-kDa fragment was added to culture medium, and cells were incubated for an additional 3 hr. (A) Cells were assessed by fluorescence microscopy as described for Fig. 2. Bar = 20 mm. The result is representative of 3 sets of experiments. (B) Binding of FITC-70-kDa fragment was assayed by immunoblotting after SDS-PAGE of total extracts. Anti-FITC and anti-beta actin polyclonal antibody were used for detecting bound FITC labled 70-kDa fragment and loading control, respectively. The bar graphs represent relative amounts of bound FITC- 70 kDa fragment in proportion to fibronectin as adherent substrate after normalization with actin in the same extract and were compiled from 3 experiments as in Fig. 5B (n = 6). doi:10.1371/journal.pone.0004113.g006 cells adherent to various substrates. Cells adherent to fibronectin, 1F3-C V89, 2F3-C V89, 1F3-14F3, 2F3-14F3, 7F3-10F3, or 1F1-3F3+2F3-C V89 all showed localization of vinculin in focal adhesions near the edges of cells (Fig. 8). The increased amount of FITC-Fibronectin assembled by cells adherent to fibronectin or 1F3-C V89 as compared to cells adherent to 2F3-C V89, 1F3-14F3, or 1F1-3F3+2F3-C V89 co- localized with coalesced vinculin (Fig. 8). However, similar patterns of vinculin-containing focal contacts were present in fibronectin-null cells plated on all constructs, including cells on 2F3-14F3 or 7F3-10F3 that completely lacked assembled FITC- fibronectin (Fig. 8). Explorations of mechanisms The localization of vinculin into focal contacts of both fibronectin-assembly competent and incompetent cells raised questions of whether incompetency can be overcome. LPA is a stimulator of fibronectin assembly, which activates Rho and induces actin stress fiber formation [46]. Fibronectin-null cells adherent to 2F3-14F3 showed an increase in fibronectin assembly when stimulated with 500 nM LPA; however, the increased assembly was much less than on 1F3-C V89 (results not shown). The same was true when cells were stimulated with 10 mg/ml 9EG7 which activates b1 integrins [47] or both LPA and 9EG7 together; fibronectin assembly by cells on 2F3-14F3 was increased, but the increase was much less than similarly treated cells on 1F3- C V89 (results not shown). 9EG7 was also used to stain active b1 integrins. Fibronectin-null cells adherent to 1F3-C V89 had more active b1 integrins localizing to the periphery of the cells than cells on 2F3-C V89, with assembled fibronectin fibrils co-localizing with active b1 integrins (results not shown). However, for cells on 2F3-14F3, which could not assemble fibronectin, strong 9EG7 staining occurred at focal adhesions (results not shown). Finally, we found that phosphorylation level of FAK Tyr-397 was the same for fibronectin-null cells adherent to 1F3-C V89 or 2F3-14F3, regardless of assembly time ranging from 15 min to 3 hr (results not shown). Taken together, these results indicate that formation of focal adhesions by interaction with an adhesive protein maybe necessary but it is not sufficient for fibronectin assembly. Discussion In short-term assays, fibronectin-null cells assemble exogenous fibronectin when adherent to a substrate coated with intact fibronectin but not when adherent to fibronectin’s cell-binding domain (modules 7F3-10F3) recognized by a5b1 and other integrins [12]. We hypothesized that interactions of the adherent PLoS ONE | www.plosone.org 8 January 2009 | Volume 4 | Issue 1 | e4113