Self-Help Fibronectin Assembly described for Fig. 2. Bar = 20 mm. (C) After 4 hr incubation of fibronectin-null cells with 9 mg/ml Rx-fibronectin at 37uC on cover slips coated with Peak 1, 2, or 3 protein, 3 mg/ml, cells were lysed with buffer containing 1% SDS, anti-tetramethly rhodamine IgG fraction and anti-actin (beta and gamma) polyclonal antibody were used for detecting deposited labeled fibronectin and loading control, respectively. All panels are representative of at least 3 sets of experiments. doi:10.1371/journal.pone.0004113.g003 fragment preparation contained a mixture of 180- to 200-kDa fragments when analyzed by SDS-PAGE (results not shown). These could be separated by FPLC (Fig. 3A). The 200-kDa fragment was found in Peak 3, and the smaller fragments were in Peaks 1 and 2 (results not shown). Fragments from all three peaks supported cell adhesion, but only a substrate coated with Peak 3 fragments supported assembly of fibronectin by adherent fibro- nectin-null cells (Fig. 3B and 3C). The compositions of the separated fragments were therefore determined by immunoblot- ting with monoclonal antibodies (results summarized in Fig. 1A). Figure 4. 1F3 and the C-terminal segment are important and need to be on the same molecule for substrate activity. Assembly of RX-fibronectin by fibronectin-null cells adherent to 6F1-C V89, 1F3-C V89, 2F3-C V89, 1F3-14F3, 1F1-3F3+2F3-C V89, or 1F3-14F3+31 kDa, 3 mg/ml, was assessed by fluorescence microscopy as described for Fig. 2. The result is representative of 3 sets of experiments. Bar = 20 mm. doi:10.1371/journal.pone.0004113.g004 Peak 3 fragments contained epitopes for L8 and 9D2, which recognize 9F1-1F3 and 1F3, respectively [26], whereas Peak 1 and 2 fragments lacked the epitopes. IST-7, which recognizes 15F3 [26], detected proteins in Peaks 2 and 3. C4B10, which recognizes 12F3-14F3 [26], detected the protein in all three peaks. Peak 3 fragments were also recognized by monoclonal antibody 5C3 but not by 4D1. Antibody 5C3 recognizes an epitope in 7F1-9F1, whereas 4D1 recognizes an epitope in 1F1-3F1 (unpublished data). The 5C3 and L8 epitopes were destroyed by reduction of Peak 3 fragments with b-mercaptoethanol (results not shown). Approxi- mately 50% of the protein in Peak 3 was found to bind to gelatin- agarose. These results, therefore, were interpreted as showing that a portion of Peak 3 fragments contains 1F3 and sequences from the gelatin-binding domain whereas Peak 1 and 2 fragments do not. The bands were diffuse even after purification (result not shown), which is compatible with size heterogeneity within each fraction. Further, binding of Peak 3 to gelatin and its recognition by L8 and 5C3, which occur only under non-reduced conditions [26,45] (and results not shown), indicated that at least part of the gelatin- binding domain had not been completely reduced, alkylated, and digested during preparation of the fragments. The incomplete modifications fortuitously generated informative fragments that suggested that 1F3 and perhaps 7F1-9F1 of the gelatin-binding domain are necessary for the supportive activity of surface- adsorbed fibronectin fragments on fibronectin assembly. 1F3 and C-terminal modules are necessary for optimal supportive activity To define the contribution of the gelatin-binding domain and modules 7F1-9F1, 1F3, 15F3, and 10F1-12F1; recombinant 6F1-C V89, 6F1-CA V89, 1F3-C V89, 1F3-CA V89, 1F3-14F3, 2F3-14F3, and 2F3-C V89 were purified as secreted proteins after baculovirally directed expression (Fig. 1B) and tested as adherent substrates for fibronectin-null cells. The cells assembled exogenous fibronectin equally well when adherent to 6F1-C V89, 6F1-CA V89, 1F3-C V89, or 1F3-CA V89 (Fig. 4 and results not shown). These results, therefore, demonstrated that the gelatin-binding domain or 7F1-9F1 is not required for supportive activity. Fibronectin-null cells adherent to 1F3-C V89, 2F3-C V89, or 1F3-14F3 all bound FITC- or Rx-labeled fibronectin, but the fluorescent microscopic images of the bound protein were different for cells on 1F3-C V89 compared to cells on 1F3-14F3 or 2F3-C V89 (Fig 4 and Fig 5A). Fibrils associated with cells on 1F3-C V89 were numerous and aligned in an organized manner perpendic- ular to the edge of the cell. In contrast, cells on 2F3-C V89 or 1F3-14F3 bound fibronectin in fibrils that were less numerous, less elongated, and poorly organized. Proteolytic fibronectin fragments and/or recombinant proteins were mixed together and used as substrates for culture of fibronectin-null cells to test if combinations of proteins containing 1F3 or the C-terminal modules are as effective as when both determinants of activity are present in the same protein. Co- coating of 1F1-3F3 with 2F3-C V89 or of 1F3-14F3 with 31-kDa fragment supported fibronectin assembly only minimally more than substrate coated with 2F3-C V89 or 1F3-14F3 alone (Fig. 4). A mix of recombinant 1F1-3F3, recombinant 2F3-14F3, and the 31- kDa tryptic fragment that contains 15F3 and 10F1-12F1 also poorly supported assembly (results not shown). PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4113