Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin's mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vimentin-null (vim(-/-)) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim(-/-)mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin's enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim(-/-)mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim(-/-)mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of vim(-/-)mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young's moduli of normal and vim(-/-)mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.
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