CNCS Seminar
Tuesday, September 26, 2017, 3:00pm, 119 Physics
Brenton D. Hoffman (Duke University, Biomedical Engineering)
Assessing the Effects of Protein Load on Protein Function in Living Cells
Abstract:- 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. [video]
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