Biochemical and mechanical cues tune fibronectin conformation and function
Hubbard, Brant Clark
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The composition and conformational state of biological molecules have a profound influence on cell behavior and large-scale processes including development and disease progression. Fibronectin fibers are a prevalent component of the extracellular matrix that are believed to adopt a wide array conformations with different functions. Two factors that are hypothesized to regulate fibronectin conformation, and hence fibronectin biological function, are allosteric regulators, such as heparan sulfates, and mechanical strain. However, the relative influence of allosteric regulators and mechanical forces on fibronectin conformation has not been determined. This conformational regulation is especially important in the context of the heparin 2 binding domain (modules III12 to III14), which is known to bind and present numerous growth factors, such as vascular endothelial growth factor, to cells. This thesis will highlight three contributions to this field. First, a new, and remarkably simple technique was developed that permits the detection of the non-equilibrium fibronectin conformations. This technique is founded on the identification of monoclonal antibodies that have altered affinities for fibronectin based on heparin treatment or mechanical strain dependence, or that bind fibronectin equally well in all conditions. Second, the impact of both heparin and mechanical strain on the binding of VEGF to the hep2 region of fibronectin was investigated. It was discovered that both strain and heparin co-regulate VEGF binding. Finally, studies of cell attachment and migration on single fibers of fibronectin with controlled strain states provided the first direct evidence that mechanical strain regulates cell attachment, spreading, and migration on a fibronectin matrix. This body of work demonstrating that the conformational changes in fibronectin lead to altered biological activity has broad impact in a number of fields due to the ubiquitous presence and requirement of fibronectin in cell and tissue function.