Changes in tertiary and quaternary structure of proteins within the actin cytoskeletal network are a likely way cells read mechanical signals from their environment. However, showing that these conformational changes occur as a result of mechanical stress and that such changes are important to the function of the cell is a major challenge. This thesis seeks to address these questions using a cohort of molecular biophysical and cell biological methods applied in increasingly complex contexts. First, the importance of force-driven unfolding to function and how changes in unfolding pathway correlate with diseased states was determined with single molecule Atomic Force Microscopy on nano-constructs of wild-type and mutant forms of dystrophin. Biophysical studies showed that the ability to fold into mechanically stable, spectrin-type helical bundle domains and the preservation of cooperative unfolding were common characteristics of functional truncated dystrophins. Second, a newly developed in-cell cysteine labeling technique demonstrated stress-enhanced repeat unfolding within spectrin in wild-type red blood cells under shear stress versus static conditions, thus demonstrating that forced unfolding is not just an in vitro phenomena. The importance of the cytoskeletal network to spectrin function was also demonstrated in mutant, 4.1R-null red blood cells, where the intrinsic properties of spectrin remain intact but the network integrity is compromised by absence of 4.1R. Loss of network integrity was evident in a decrease in spectrin unfolding under stress. Repeat unfolding was accompanied by changes in associations of spectrin with its binding partners in a time- and stress- dependent manner, indicating that the erythrocyte cytoskeleton exhibits a graded response to stress. Lastly, with cardiomyocytes derived from embryonic stem cells, the importance of stress to quaternary structure of actinin within the sarcomeric cytoskeleton and its effects on cell-wide function was tested in cells adhered to elastic substrates. Substrate stiffness sets the load on these spontaneously contracting cells, and differences in load lead to cytoskeletal reorganization with significant effects on cardiogenic development. Taken together, these findings present evidence of various cytoskeletal proteins – especially in the spectrin superfamily – as mediators of mechanical signaling within cell.