Mechanotransduction in Fibroblast

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DESCRIPTION (provided by applicant): All cells sense and respond to mechanical forces. Compared with what we know about the sensing and transduction of chemical signals, how cells sense and transduce mechanical force is poorly understood. The objective of this proposal is to determine molecular components of a mechanotransduction unit in fibroblasts. In electrically excitable sensory cells, mechanotransduction units include a plasma membrane ion transport protein linked to extracellular and intracellular tethers. In non-excitable cells, such as fibroblasts, a critical role for plasma membrane ion transport proteins in mechanotransduction has been proposed but not experimentally confirmed. Also, whether a mechanotransduction unit of an ion transport protein linked to extracellular and intracellular tethers is conserved in fibroblasts is unknown. This proposal investigates the hypothesis that the ubiquitously expressed plasma membrane Na-H exchanger NHE1, which is anchored to the actin cytoskeleton, is an essential component of a mechanotransduction unit in fibroblasts. Mechanical force increases NHE1 activity and phosphorylation, and an NHE1-dependent increase in intracellular pH. Additionally, in response to mechanical force NHE1 is necessary for increased activity of the focal adhesion kinase FAK, for recruitment of FAK to focal contacts, and for F-actin assembly. Two specific aims are designed to investigate the role of NHE1 in mechanostransduction. Aim 1 focuses on mechanosensing by NHE1. The kinetics of NHE1 activity in response to mechanical force will be determined, and whether activation is dependent on NHE1 phosphoryation or actin anchoring, de-novo F-actin assembly, or integrin engagement will be tested. Aim 2 focuses on mechanotransduction by NHE1 and whether its ion translocation and actin anchoring are necessary for the mechanosensitive responses of actin filament assembly and focal adhesion remodeling. How NHE1 regulates actin filament dynamics in response to mechanical force will be determined by testing pH-dependent filament severing activity of cofilin, nucleating activity of the Arp2/3 complex, and the turnover and movement of actin filaments, as determined by fluorescence speckle microscopy. How NHE1 regulates dynamic remodeling of focal adhesions will be determined by imaging the spatial and temporal recruitment of focal adhesion proteins and by asking whether pH-dependent actin binding proteins determine focal adhesion stability. Mechanical forces play important roles morphogenesis, cell proliferation, and determining malignant transformation. Mechanical forces also regulate connective tissue remodeling and reparative responses, which underscores the significance of understanding how fibroblasts sense mechanical force and how they transduce mechanical signals into biochemical events that drive cell and tissue responses.