ECM composition and forces are major factors in defining the morphology, expression patterns and the structural integrity of tissues. Cells develop and respond to the forces on extracellular matrix (ECM) fibers because those forces are critical to many aspects of tissue and organ function. In engineering functional tissues, it is critical to understand the effects of matrix stretch or relaxation on signaling and other cell functions. The pathways of response to the stretching forces or oscillations in force are poorly understood, but both ion movements and cytoskeletal changes in response to stretch were reported. Recently, we showed that the cellular responses to stretch are observed in Triton X-100 cytoskeletons of cells attached to collagen. In particular, cytoskeletons reversibly bind cytoplasmic focal contact proteins in response to stretch, analogous to in vivo binding. In addition, relaxation of those cytoskeletons causes other cytoplasmic proteins to bind. Normally, the cell assembles an integrated cytoskeleton as it generates a resting tension (traction forces) on extracellular matrix. Substrate or ECM stretching transmits high stresses to the cytoskeleton, which results in cytoplasmic protein binding to matrix contact sites. One major pathway for binding is the stress-dependent alteration of cytoskeletal proteins. Recent results show that a large number of cytoplasmic proteins bind to cytoskeletons in response to stress changes and we propose now to develop a map of the different binding proteins and the signaling pathways to which they are correlated. In order to create this map, we will first develop methods for rapidly identifying the cytoplasmic proteins that bind to cytoskeletons in response to stretch or relaxation on different matrices. Proteomic methods, 2-D gels, mass spectrometric and Western blot analyses, will be utilized to identify the many cytoplasmic proteins involved. We will then study the signaling that occurs in vivo in response to stretch or relaxation as a function of the matrix to which the cell is bound. By comparing and contrasting the stretch- and relaxation-dependent behavior of Triton cytoskeletons from cells on different substrata, we will gain insights into the mechanisms of cell response to force as well as a quantitative measure of the components in the response pathways. These studies will provide a quantitative description of the force-dependent response pathways that form the basis of in vivo and in vitro organ development, wound healing, adaptive changes and certain cancers.
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