Figure 8. Vinculin co-localizes with assembled fibronectin on supportive substrates. Fibronectin or the indicated recombinant fibronectin constructs, 20 nM, were coated on surfaces of cover slips in 24-well tissue culture plates. After 1 hr incubation of fibronectin-null cells at 37uC on adherent substrates, 9 mg/ml FITC-fibronectin was Self-Help Fibronectin Assembly added to culture medium, and cells were incubated for an additional 3 hr. Cells were assessed by fluorescence microscopy as described for Fig. 2. Panels under ‘‘Merge (FITC-FN & vinculin)’’ were merged images of vinculin (red) and FITC-FN (green). Panels under ‘‘Vinculin’’ or ‘‘Vinculin+FITC-FN’’ represent 4-fold enlargement of the white rectan- gular box. Bar = 20 mm. The result is representative of 2 sets of experiments. doi:10.1371/journal.pone.0004113.g008 Denaturation or mutagenesis of 1F3 exposes cryptic sites for association with other parts of fibronectin [50,54,55] and formation of a ternary complex with heat-denatured 10F3 and the N-terminal 70-kDa fragment [50,56]. 1F3 has also been shown to bind to 7F3 and 15F3 [54]. Treatment of purified fibronectin in solution with a recombinant protein that contains 1F3 lacking the N-terminal part of the module results in multimerization of fibronectin [51,57]. The truncated 1F3 binds to modules 1F3-3F3 and 11F3, probably after a spontaneous opening of these modules [58]. Such spontaneous opening followed by domain swapping may be a key event in assembly of fibronectin fibrils [58]. Force– induced unfolding of 1F3 and other modules has been considered as a possible step in fibronectin fibril formation [59], and N- terminally truncated 1F3 has been suggested to mimic an intermediate in a simulated forced unfolding of the intact 1F3 module [60]. Nevertheless, recombinant rat fibronectin lacking either 1F3 or 1F3 through 7F3 is assembled into fibrils [49,53]. Although these fibrils have some structural differences, this observation indicated that fibronectin-fibronectin interactions mediated by 1F3 are not essential for formation of fibronectin fibrils. Identification of 1F3 as one of the regions in substrate-adsorbed fibronectin required for support of assembly of exogenous fibronectin by adherent cells represents a new activity of 1F3 in fibronectin assembly. Relating our finding to studies of mutated, truncated, or denatured 1F3 is difficult because 1F3-containing proteins in our experiment contained other type III modules, including 3F3, 10F3, and 11F3, and were present in the adhesive substratum rather than in solution. In addition, the activity of coatings of pieces of fibronectin containing 1F3 is mimicked by coatings of laminin [11,12] or type I collagen [61,62]. On fibronectin-null cells, fibril extension occurs on elongated focal adhesions near the cell periphery and the sites where 70K binds [11]. Cells formed similar looking (vinculin-stained) focal adhesions when plated on any of the fibronectin constructs tested. However, cells were only capable of matrix assembly and 70K binding when the construct contained 1F3 and the C terminus. One possibility is that there is an unknown receptor that recognizes 1F3 in this context and initiates a signaling pathway that activates matrix assembly. Mechanisms for the action of 1F3 have been proposed that involve direct interactions with cells [63– 65]. We speculate, therefore, that 1F3 and unknown regions of laminin and type I collagen engage receptors on adherent cells, generating signals that make the cells competent to assemble fibronectin. Laminin or type I collagen may not necessarily interact with the receptor(s) that recognize(s) 1F3 of fibronectin. Identified receptors in the C-terminal part of fibronectin include a4b1 [66,67], syndecan proteoglycans [68,69], and RGD- independent a5b1 interaction induced by the urokinase plasmin- ogen activator receptor (uPAR) [70,71]. We could not find evidence that integrin a4b1 is an adhesion receptor in the fibronectin-null cells used in the present experiments, i.e., the cells did not adhere to a coating of vascular cell adhesion molecule (VCAM) (results not shown). Syndecans and uPAR are known to influence fibronectin assembly by fibroblasts and bind to 12F3-14F3 PLoS ONE | www.plosone.org 10 January 2009 | Volume 4 | Issue 1 | e4113