Focal adhesions as mechanotransducers: Differential localization of focal adhesion components in response to mechanical strain.

摘要

The fields of regenerative medicine and cellular engineering hold tremendous promise in the treatment of a large number of medical conditions. The key to these efforts lies in understanding how cells sense and respond to stimuli in their environment. One critically important stimulus that guides the development and function of a variety of tissues is mechanical force. The goal of this thesis is to improve the current understanding of how cells receive and process mechanical signals. Previous work suggests that cell adhesion structures called focal contacts may play a central role in the transduction of mechanical signals. The hypothesis guiding the studies in this thesis is that mechanical force in the form of strain, applied via the extracellular matrix, will cause changes in the composition and signaling activity of focal contacts. To address this hypothesis, and the mechanisms involved in the cell response, an experimental system was developed to apply strain to smooth muscle cells cultured on flexible silicone rubber substrates coated with adhesion proteins. Following strain application, the distribution of the focal contact components vinculin, α-actinin, and paxillin between the detergent-insoluble cytoskeletal pool and the soluble cytoplasmic pool were evaluated by immunofluorescence microscopy and western blotting. Cyclic strain, but not single step changes in strain, were found to increase insoluble vinculin and paxillin, as well as the tyrosine phosphorylation state of paxillin. In order to determine whether a soluble signaling step was involved in this process, incorporation of fluorescent vinculin into the focal contacts of permeabilized cells was observed during strain. Again, cyclic strain, but not step changes in strain, caused enrichment of vinculin in cell adhesions. These results suggest that a threshold number of mechanical perturbations must be exceeded to alter net focal contact composition, and that one part of the mechanism is mediated by a direct effect of force on adhesions. A better understanding of force-induced changes in cell adhesions may provide insight into the role of mechanical signals in health and disease, and may allow the rational application of these signals for therapeutic use in guiding cell function in cell and tissue engineering.