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Brenton D. Hoffman : Assessing the Effects of Protein Load on Protein Function in Living Cells

Cells exist in a complex mechanical environment that is both a source of applied forces and a means of mechanical support. An incomplete understanding of the mechanisms cells use to detect mechanical stimuli, a process termed mechanotransduction, is currently preventing advances in tissue engineering and hindering the understanding of several mechanosensitive disease states. Mechanical stimuli are sensed at focal adhesions (FAs), complex dynamic structures comprised of several hundred types of proteins that mediate physical connections between the extracellular matrix and the cytoskeleton. Detection of mechanical cues is thought to be mediated by mechanically-induced changes in protein structure, which, in elegant in vitro single molecule experiments, have been shown to induce new biochemical functions, such as changes in binding affinity as well as the formation of distinct protein-protein interactions. However, the existence and role of these mechanically-induced changes in protein function in living cells are not well understood. To enable the visualization of protein loading, we create Forster Resonance Energy Transfer (FRET)-based tension sensors that emit different colors of light in response to applied forces. The next step in the development of this technology is the use of these sensors to study the effects of mechanical loading on protein functions in living cells. To begin this process, we have refined two commonly used and powerful approaches, Fluorescence Recovery After Photobleaching (FRAP) and fluorescence co-localization to be compatible with FRET-based tension sensors. Initial efforts have focused on the mechanical linker protein vinculin due to its established role in regulating the response of FAs to mechanical loading. These novel techniques reveal that force affects both vinculin turnover as well as its ability to form distinct protein-protein interactions. Further use of these techniques should enable a wide variety of studies in mechanobiology involving different load-bearing proteins, subcellular structures, extracellular contexts, and cellular functions.

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