Display of Cell Surface Sites for Fibronectin Assembly Is 1 Modulated by Cell Adherence to F3 and C-Terminal Modules of Fibronectin Jielin Xu1,2,3., Eunnyung Bae1,2,3., Qinghong Zhang1,2,3¤, Douglas S. Annis1,2,3, Harold P. Erickson 4 , Deane F. Mosher1,2,3* 1 Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 2 Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 3 Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 4 Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America Abstract Background: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules 7F3-10F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules 7F3-10F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. Methodology/Principal Findings: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to 7F3-10F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules 2F3-14F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module 1F3 or the 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 best mimicked the activity of a coating of intact fibronectin. Constructs 1F3-C V0, 1F3-C V64, and 1F3-C D(V15F310F1) were all able to support fibronectin assembly, suggesting that 1F3 through 11F1 and/or 12F1 were important for activity. Coatings in which the active parts of 1F3-C were present in different proteins were much less active than intact 1F3-C. Conclusions: These results suggest that 1F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells. Citation: Xu J, Bae E, Zhang Q, Annis DS, Erickson HP, et al. (2009) Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin. PLoS ONE 4(1): e4113. doi:10.1371/journal.pone.0004113 Editor: Rory Edward Morty, University of Giessen Lung Center, Germany Received July 18, 2008; Accepted December 4, 2008; Published January 1, 2009 Copyright: 2009 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIH 21644. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dfmosher@wisc.edu ¤ Current address: Department of Dermatology, University of Colorado, Denver, Colorado, United States of America . These authors contributed equally to this work. Introduction Fibronectin is a dimer of nearly identical subunits composed largely of types I (F1), II (F2), and III (F3) fibronectin modules (Fig. 1A). Fibronectin is present as an abundant soluble protein in blood plasma and an insoluble extracellular matrix (ECM) protein. Assembly of fibronectin into ECM is a cell-dependent process that is initiated at specialized sites on cell surfaces [1]. Display of these sites requires active integrins [2–10]. The ‘‘handle’’ in soluble fibronectin that cells use to initiate assembly, however, is located in the N-terminal F1 and F2 modules, not the RGD-containing F3 module recognized by a5b1 and several other integrins [11]. Assembly of exogenously added soluble plasma fibronectin by adherent fibronectin-null cells is dependent upon the substrate to which the cells are attached; cells adherent to surface-adsorbed fibronectin or laminin are competent to assemble exogenous fibronectin whereas cells adherent to surface-adsorbed vitronectin or the cell-binding domain of fibronectin (7F3-10F3), which contains the RGD sequence recognized by integrins, assemble exogenous fibronectin poorly [9,12–14]. A similar difference is true for binding of the N-terminal 70-kDa fragment of fibronectin that binds to the surface sites that initiate fibronectin assembly [11]; fibronectin-null cells adherent to surface-adsorbed fibronec- tin or laminin-1 bind 70-kDa fragment whereas cells adherent to surface-adsorbed vitronectin do not [11]. Co-coating experiments demonstrated that the presence of vitronectin on a surface suppresses the ability of surface-adsorbed fibronectin or laminin to support fibronectin assembly by adherent fibronectin-null cells whereas 7F3-10F3 lacks such suppressive activity [12]. These results indicate that the interaction of adherent cells with regions of adsorbed fibronectin other than the RGD-containing integrin modules recognized by a5b1 is needed to support initiation of fibronectin assembly. Such regions could include the V (CS) region or 4F3-5F3 recognized by a4b1 integrin [15,16], the 12F3-14F3 PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4113