Mechanical loading plays a fundamental role in tissue remodeling by regulating cell behaviors in a manner dependent on the modality, frequency, and amplitude of loading. However, the mechanisms by which cells are able to differentiate dynamic signals in vivo remain unknown. By altering cytoskeletal-receptor-matrix interactions within focal adhesions in response to mechanical stimuli, cells may sense and respond to dynamic loading in a manner dependent on magnitude and frequency and specific substrate-integrin pair involved. To test this hypothesis, the objectives of this proposal are to: (1) develop a single fiber force-measuring device with bound extracellular matrix proteins to apply dynamic loads to receptors on a cell surface in vitro, (2) quantify the assembly of focal adhesions at the surface of fibers using GFP-paxillin and measure the development cytoskeletal reinforcements in response to loading protocols over a range of physiologic frequencies, and (3) determine the effects of different ligands on focal adhesion formation and the development of reinforcements at the surface of fibers. An understanding of cellular response, in terms of number and distribution of focal adhesions and force generation resulting from cytoskeletal reinforcement, to dynamic loads applied through various extracellular matrix fibers will elucidate the mechanisms by which cells are able to differentiate mechanical loading of various frequencies and amplitudes in vivo.
