Self-Help Fibronectin Assembly Figure 1. Diagram of proteolytic fragments, recombinant proteins, location of antigenic epitopes, and relevant features of fibronectin. (A) Diagram of modular make-up of plasma fibronectin, proteolytic fragments, and locations of antigenic epitopes and sites of proteolytic cleavage. N-termini (N) are on the outsides, and C-termini (C) are in the middle. The bulk of each subunit consists of 12 type I (thin rectangles), two type II (triangles), and 15 type III modules (thick rectangles). The subunits are joined near the C-termini by a pair of disulfide bridges. Sites of proteolytic cleavage are shown for the subunit on the right (Cath D, cathepsin D). The subunit to the left contains the alternatively spliced V region that is susceptible to trypsin. Locations of antigenic epitopes and gelatin-binding activity are shown for the subunit on the left. Monomeric fragments generated uniquely from the two subunits by trypsin, without or with (‘‘fingerless’’ fragments) prior reduction and alkylation, are shown below the subunits. Fragments generated from both subunits are shown only on the right. (B) Diagram of recombinant pieces of fibronectin. The layout is as described in panel A. Key F3 modules are indicated for the subunit on the left. These proteins were made without or with AF3 inserted between 11F3 and 12F3 (arrowhead), as described in the text. The pieces shown were generated using cDNA encoding subunits with the V89 version of the V region and no BF3. Modular compositions of the various recombinant proteins produced in High Five cells or E. coli are shown below. Recombinant proteins containing the C-terminus form dimers (such as 1F3-C) and recombinant proteins without the C-terminus were expressed as monomers (such as 1F3-14F3). (C) Diagram of the different alternatively spliced forms of the V region that we expressed in 1F3-C dimers. doi:10.1371/journal.pone.0004113.g001 modules (HepII domain) that bind syndecans [17], the gelatin- binding domain that binds to cellular (type 2) transglutaminase [18,19], the GNGR sequences in type I modules that have the potential to isomerize to GisoDGR recognized by avb3 [10,20], or the IGD sequences in type I modules important for the motogenic activity of fibroblasts [21]. Assembly of fibronectin by cellular fibronectin-expressing cells is less sensitive to the nature of the adherent substrate [12]. One explanation for the insensitivity is that newly secreted endogenous fibronectin mimics activities of substrate-coated fibronectin [11]. To search for the hypothesized supportive region(s) in fibronectin, sets of proteolytically generated or recombinant pieces of fibronectin were tested. Surprisingly, pieces as large as one comprising modules 2F3-14F3, which contain the heparin-binding and cell adhesion domains, were only minimally active in supporting fibronectin assembly. In contrast, pieces in which the 1st type III module (1F3) and C-terminal modules (including V region, 15F3, and 10F1-12F1) flanked 2F3-14F3 (see Fig. 1B) were nearly as active as full-length fibronectin. Materials and Methods Cells Fibronectin 2/2 (fibronectin-null) fibroblastic cells from fibronectin 2/2 mouse embryonic stem cells were derived and handled as described previously [12,22]. Preparation of adhesive substrates and cells for experiments The adhesive proteins were usually diluted to 20 nM in phosphate buffered saline, pH 7.4 (PBS) prior to coating overnight at 4uC of glass cover slips or wells of 24-well tissue culture plates. PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4113