Person: Gee, Elaine Pei-San
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Elaine Pei-San
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Gee, Elaine Pei-San
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Publication Biophysical and Molecular Determinants in Cell Tension-Mediated Fibronectin Unfolding that Drive Fibrillogenesis(2013-02-20) Gee, Elaine Pei-San; Ingber, Donald Elliott; Stultz, Collin Melveton; Blacklow, Stephen; Shih, William; Smith, Michael; Hogle, JamesAssembly of the extracellular matrix (ECM) protein fibronectin (FN) is a mechanical process that involves cell binding to FN through cell surface integrin receptors and application of tensional forces generated in the cell's contractile actin cytoskeleton. Deformation-induced exposure of cryptic sites, defined as buried molecular recognition sites, in FN has been proposed as a mechanism by which cell tension drives FN fibrillogenesis. The primary integrin attachment site on FN is the RGD loop in the 10FNIII domain. In this thesis, I set out to define the molecular biophysical mechanism by which cell tension application at the RGD site promotes unfolding and thereby induces FN-FN self-assembly leading to matrix fibril formation. Chapter 1 of this dissertation provides an overview of the current knowledge behind the biophysical and molecular basis of FN assembly in the ECM and its key role in development and disease. In Chapter 2, steered molecular dynamic simulations show that the 10FNIII domain under force applied through its N-terminus and RGD loop (N-to-RGD) unfolds to a preferred kinetic intermediate with solvent-exposed N-terminal hydrophobic residues in a manner different from past analyses in the literature where force through the N- and C- termini leads to multiple unfolding pathways. Use of single-molecule atomic force spectroscopy in Chapter 3 experimentally reveals that a mechanically stable intermediate of 10FNIII exposed by N-to-RGD pulling shows a length extension that agrees with the predicted kinetic intermediate. Results of biochemical and cellular studies using synthetic peptides with sequences from the 10FNIII intermediate show in Chapter 4 that the twenty-three amino acid sequence that spans the unraveled N-terminus of the predicted intermediate mediates FN multimerization and contains a minimal seven amino acid sequence we call the multimerization motif that is sufficient to induce FN-FN multimer assembly. Finally, Chapter 5 summarizes the new insights supported by this work regarding the role that mechanical forces applied at the cell binding site in 10FNIII plays in the physiological unfolding of FN with respect to FN fibrillogenesis and ECM assembly.Publication Fibronectin Unfolding Revisited: Modeling Cell Traction-Mediated Unfolding of the Tenth Type-III Repeat(Public Library of Science, 2008) Gee, Elaine Pei-San; Ingber, Donald; Stultz, CollinFibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10th type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly.