Figure 7. V region, 15F3, and 10F1 were not required for support of fibronectin assembly. 1F3-C V89 or the indicated other recombinant fibronectin constructs, 20 nM, were coated on surfaces of cover slips in 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. The result is representative of 3 sets of experiments. (B) Assembly of FITC-fibronectin was assayed by immunoblotting as described for Fig. 5B, 3 sets of duplicate experiments (n = 6). doi:10.1371/journal.pone.0004113.g007 Self-Help Fibronectin Assembly cell with regions of adsorbed fibronectin outside of modules 7F3-10F3 are required for display of the cell surface binding sites that initiate fibronectin assembly. Coatings of various proteolytic fibronectin fragments or recombinant fibronectin segments, each containing 7F3-10F3 and flanking modules, were tested for effects on fibronectin assembly by adherent cells or binding of the N- terminal 70-kDa fragment to assembly sites on these cells. A coating of recombinant 2F3-14F3, which contains the 12F3-14F3 heparin-binding domain as well as the cell adhesion domain, was not effective in supporting fibronectin assembly. Addition of module 1F3 or C-terminal modules to modules 2F3-14F3 resulted in some activity, and addition of both 1F3 and the C-terminal modules resulted in a construct, 1F3-C, that approached the activity of intact fibronectin. Co-coating experiments revealed that the required modules work best when present in the same piece of fibronectin. The fact that 1F3-C V0, 1F3-C V64, and 1F3-C D(V15F310F1) all supported fibronectin assembly indicates that the V region, 15F3, and 10F1 are not needed for supportive activity. These results suggest that 1F3 acts together with fibronectin C- terminal modules to cause adherent cells to assemble fibronectin. There was no requirement for two features that are necessary for soluble fibronectin to be assembled: the N-terminal 70-kDa portion, which directs soluble fibronectin to cell surface assembly sites [11], and a dimeric configuration, which presumably is required to perpetuate ‘‘head-to-tail’’ interactions between fibro- nectin subunits during fibril growth [48]. The grouping of fibronectin modules required for a substrate to support assembly of soluble fibronectin by adherent cells, therefore, is novel and different from the groupings required for optimal cell adhesion or for soluble fibronectin to bind to and become insolubilized at assembly sites. Are the results due to differences in the conformation in which the purified fragments or recombinant proteins adsorb to surfaces? Pieces of fibronectin that failed to support fibronectin assembly strongly supported cell adhesion per se, demonstrating that the RGD sequence in 10F3 of the various adsorbed proteins is available to bind integrins. Similar composition/activity relation- ships were found for the set of proteolytic fragments and the set of recombinant proteins, even though the recombinant proteins all had the same N- and C-terminal ‘‘tails’’ introduced by the cloning strategy [38] as compared to the different and variable surrounding sequences in the fragments generated by limited tryptic digestion of native and of reduced and alkylated fibronectin. Similar effects were seen on glass cover slips and tissue culture dishes. Finally, in results not presented, we coated 1F3-C V89 or 2F3-14F3 in different coating concentration varying from 5 nM to 80 nM and detected the amount of protein adsorbed to the wells by ELISA using an antibody (MabIII10) recognizing the 10F3 module. 2F3-14F3 adsorbs similarly as 1F3-C V89 (results not shown), suggesting the difference in fibronectin assembly is not due to difference in protein adsorption when coating. Thus, the results were consistent despite changing variables that would be expected to influence conformations that proteins adopt upon adsorption to surfaces. We therefore favor the hypothesis that 1F3-C V89, the 160/180-kDa fragments, or the Peak 3 ‘‘fingerless’’ fragments imparts information to adhering cells that is missing in 2F3-14F3 or smaller pieces of fibronectin. Recognition of 1F3 and the C-terminal region by adhering cells results in a phenotype in which binding sites for the N-terminal region of soluble fibronectin are expressed at specific regions on the surface of the adherent cell. 1F3, along with 2F3, is known to influence fibronectin assembly in cells expressing fibronectin and is thought to work, at least in part, by mediating fibronectin-fibronectin interactions [26,49–53]. PLoS ONE | www.plosone.org 9 January 2009 | Volume 4 | Issue 1 | e4113