Figure 5. 1F3 and C-terminal modules enhance supportive activity of fibronectin constructs. 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, 9 mg/ml FITC-fibronectin 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. (B) Assembly of FITC-fibronectin was Self-Help Fibronectin Assembly assayed by immunoblotting after SDS-PAGE of material left after 5 min extraction with 1% deoxycholate (DOC insoluble FN). Anti-FITC and anti-beta actin polyclonal antibody were used for detecting deposited labeled fibronectin and loading control, respectively. The amount of DOC-insoluble fibronectin was normalized with actin in the total extract using duplicate samples and the relative amount is shown as bar graph. Each bar represents mean with standard error, 3 sets of experiments with duplicates in each (n = 6). doi:10.1371/journal.pone.0004113.g005 When the amount of fibronectin present in the deoxycholate- insoluble ECM pool was quantified, more fibronectin was assembled by cells on 1F3-C V89 or 2F3-C V89 than by cells on 1F3-14F3 or 2F3-14F3 (P,0.01) (Fig. 5B). These results, along with the supportive activities of Peak 3 ‘‘fingerless’’ fragments and the mix of 160- and 180-kDa tryptic fragments, provide evidence that 1F3 and the C-terminal modules are needed for optimal activity. Requirements for binding of the 70-kDa N-terminal fragment of fibronectin to cell surface sites of fibronectin assembly are the same as for assembly of intact fibronectin Binding of the N-terminal 70-kDa fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin [24] and is an accurate indicator of the ability of various manipulations to enhance or diminish fibronectin assembly [11]. When FITC- labeled 70-kDa fragment was incubated with fibronectin-null cells adherent to fibronectin, 1F3-C V89, 2F3-C V89, 1F3-14F3, 2F3-14F3, or 7F3-10F3; fluorescence microscopy revealed patterns of deposition similar to the patterns of deposition of intact fibronectin (Fig. 6A, compare Fig. 6A with Fig. 5A). No binding was seen with cells adherent to 7F3-10F3 or 2F3-14F3; binding occurred with cells adherent to 1F3-C V89, 1F3-14F3, or 2F3-C V89; and a substrate of 1F3-C V89 best mimicked the patterns of binding exhibited by cells adherent to fibronectin. Western blotting demonstrated a hierarchy of binding, greatest for cells adherent to fibronectin and less for cells adherent to 1F3-C V89, 2F3-C V89, or 1F3-14F3 (Fig. 6B). V region, 15F3, and 10F1 are not required for support of fibronectin assembly To explore the important modules within the C-terminus, recombinant 1F3-C with alternatively spliced forms of the V region (V0, V64, D(V15F310F1)) were generated with or without the alternatively spliced AF3 module (Fig. 1C). Constructs 1F3-C V0/1F3-CA V0 (lacking the V region), 1F3-C V64/1F3-C V64 (contains the V64 version of V region instead of V89 version), and 1F3-C D(V15F310F1)/1F3-CA D(V15F310F1) (lacking V region, 15F3, and 10F1) all supported fibronectin assembly by fibronectin- cells at the same level (Fig. 7), suggesting that V region, 15F3, and 10F1 are not required for support of fibronectin assembly and a variety of sequences may be present immediately C-terminal to 12F3-14F3. Similar results were obtained in 70K binding experiments (results not shown). Vinculin-containing focal adhesions co-localize with assembled fibronectin on supportive substrates but are also present on non-supportive substrates To find out the relationship between assembled fibronectin fibrils and focal adhesions, dual fluorescence microscopy of assembled exogenous FITC-labeled fibronectin fibrils and vinculin stained by monoclonal antibody against vinculin and a Rhoda- mine-labeled secondary antibody was performed on fibronectin- PLoS ONE | www.plosone.org 7 January 2009 | Volume 4 | Issue 1 | e4113