FITC-70-kDa fragment at 37uC in wells coated with various proteins, cells were lysed with SDS buffer (1% SDS, 50 mM Tris, pH 7.4, 300 mM NaCl, 5 mM EDTA, 1 mM phenylmethyl sulfonyl fluoride, and protease inhibitor cocktail). The lysate proteins were separated by polyacrylamide gel electrophoresis in SDS (SDS-PAGE) and transferred to PVDF membrane (poly- vinylidene fluoride, Millipore, Bedford, MA). RX- or FITC- labeled proteins were immunoblotted with rabbit anti-tetramethly rhodamine (Invitrogen) or anti-FITC as the primary antibody. Phosphorylated FAK Tyr-397 were immunoblotted with Anti- FAK [pY397] (Biosource, CA). Rabbit anti-actin (beta and gamma) rabbit polyclonal antibodies or anti-beta actin rabbit polyclonal antibodies (Abcam, Inc. Cambridge, MA), were also added to allow quantification of a ‘‘housekeeping protein’’ and normaliza- tion of results to amount of lysed cells. Bound primary antibodies were detected with peroxidase-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), chemiluminescence reagent (Western LightningTM, PerkinElmer Life Sciences, Boston, MA), and KodakH X-Omat AR film (Rochester, NY). Film was scanned (ScanJet 6200C, Hewlett Packard, Mountain View, CA), and band density was analyzed by Scion Image (Scion Corporation, Frederick, MD). In some experiments, 1% deoxycholate (DOC) plus protease inhibitors [39] were added to the wells for 5 min, after which the DOC-soluble material was removed by careful aspiration. Fibronectin remaining after the extraction was then solubilized with SDS buffer and quantified by immunoblotting as described above. Normalization of DOC-insoluble material was accom- plished by immunoblotting of actin in cells in replicate wells that were lysed with SDS without prior extraction. Results Fibronectin-null cells assemble exogenous fibronectin when adherent to 160/180 kDa tryptic fibronectin fragments or ‘‘fingerless’’ fibronectin, but not when adherent to 7F3-14F3 or shorter stretches of type III modules Because assembly of fibronectin is intimately related to the organization of the intracellular cytoskeleton [40–42], our original hypothesis for why fibronectin-null cells assemble exogenous fibronectin when adherent to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules 7F3-10F3) was that the heparin-binding HepII domain (modules 12F3-14F3), which enhances formation of actin- containing stress fibers in fibronectin-null cells [22], works alongside modules 7F3-10F3. To test the hypothesis, 12F3-14F3, 7F3-10F3, 7F3-14F3, or 7F3-14F3A was tested as substrates. Fibronectin-null cells adhered well to surfaces coated with all except 12F3-14F3, but after adherence the cells still failed to assemble exogenous RX-fibronectin (Fig. 2 and results not shown). As 7F3-14F3 contains the cell-binding and heparin-binding domains, we concluded that the presence of these integrin- and proteoglycan-binding domains in adsorbed pieces of fibronectin is not sufficient to support fibronectin assembly by adherent fibronectin-null cells. To explore more broadly for the active region(s), proteolytic fragments of plasma fibronectin were tested as adhesive substrates. A coating of the gelatin-binding 160/180 kDa fragments, produced by limited trypsin digestion of native fibronectin, supported assembly very similarly to assembly supported by a coating of intact fibronectin (Fig. 2). As depicted in Fig. 1A, this mixture has 2 fragments in approximately equal amounts that each lack 1F1-5F1 and are monomeric due to cleavages that Self-Help Fibronectin Assembly Figure 2. Fibronectin-null cells assemble exogenous fibronec- tin when adherent to 160/180 kDa fragments or ‘‘fingerless’’ fibronectin, but not when adherent to 7F3-14F3 or smaller type III-module containing proteins. Fibronectin-null cells in DMEM containing 0.2% BSA and 9 mg/ml RX-fibronectin were incubated at 37uC for 4 hr on cover slips coated with fibronectin, 7F3-10F3, 12F3-14F3, 7F3-14F3, 160/180-kDa fragments (160/180 kDa), or ‘‘fingerless’’ Fibro- nectin; at 3 mg/ml. Cells were fixed with paraformaldehyde and observed under the fluorescence microscope. The result is representa- tive of 3 sets of experiments. Bar = 20 mm. doi:10.1371/journal.pone.0004113.g002 remove the C-terminal inter-chain disulfides [24]. The 160-kDa fragment also lacks a 31-kDa C-terminal segment containing modules 15F3 and 10F1-12F1 due to tryptic cleavage within the V region [43], whereas the 180-kDa fragment lacks the V region but includes the 31-kDa C-terminal extension containing 15F3 and 10F1-12F1; both fragments have the gelatin-binding domain and 14 type III modules [44] (Fig. 1A). Finding activity in the 160/180- kDa fragments demonstrated that modules 1F1-5F1, the extreme C-terminal tail, and a dimeric configuration due to the inter- subunit disulfides are not required for supportive activity. So-called ‘‘fingerless’’ fragments, a limited tryptic digest of reduced and alkylated fibronectin produced by a method intended to leave only the trypsin-resistant type III modules intact [25], also were active in supporting fibronectin assembly (Fig. 2). The PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4113