Ning Wang

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-l-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction.

We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-l-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

Here we describe a method for quantifying traction in cells that are physically constrained within micron‐sized adhesive islands of defined shape and size on the surface of flexible polyacrylamide gels that contain fluorescent microbeads (0.2‐μm diameter). Smooth muscle cells were plated onto square (50 × 50 μm) or circular (25‐ or 50‐μm diameter) adhesive islands that were created on the surface of the gels by applying a collagen coating through microengineered holes in an elastomeric membrane that was later removed. Adherent cells spread to take on the size and shape of the islands and cell tractions were quantitated by mapping displacement fields of the fluorescent microbeads within the gel. Cells on round islands did not exhibit any preferential direction of force application, but they exerted their strongest traction at sites where they formed protrusions. When cells were confined to squares, traction was highest in the corners both in the absence and presence of the contractile agonist, histamine, and cell protrusions were also observed in these regions. Quantitation of the mean traction exerted by cells cultured on the different islands revealed that cell tension increased as cell spreading was promoted. These results provide a mechanical basis for past studies that demonstrated a similar correlation between spreading and growth within various anchorage‐dependent cells. This new approach for analyzing the spatial distribution of mechanical forces beneath individual cells that are experimentally constrained to defined sizes and shapes may provide additional insight into the biophysical basis of cell regulation. Cell Motil. Cytoskeleton 52:97–106, 2002.

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness ( G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ( [ ] _t ) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18 [ ] _t + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.

It is increasingly evident that the cytoskeleton of living cells plays important roles in mechanical and biological functions of the cells. Here we focus on the contribution of intermediate filaments...

—Mechanical properties of the cells are important in controlling cell shape, cell migration, and other functions. To understand how cytoskeletal (CSK) filaments interact with one another mechanically, mechanical properties of adherent endothelial cells were analyzed after treatment with CSK-disrupting drugs. CSK stiffness (the ratio of applied stress to strain, a measure of cell resistance to shape deformation), viscosity (an index of intracellular structural damping), and permanent deformation (a measure of “plasticity”) were measured with magnetic twisting cytometry, by which rotational stress was applied directly to integrin receptors with ferromagnetic beads coated with RGD-containing peptide. Treatment with cytochalasin D, which disrupts actin microfilaments inhibited stiffness by 50% and decreased permanent deformation from 70% to 50% but had almost no effect on viscosity. In contrast, nocodazole, a microtubule disrupter, had very little effect on inhibition of CSK stiffness, decreased viscosity by 25%, and had no effects on permanent deformation. Acrylamide, an intermediate filament disrupter, had little effect on inhibition of CSK stiffness, little effect on viscosity, and no effect on permanent deformation. Taxol, a drug that facilitates microtubule polymerization, increased stiffness by 10%, increased viscosity by 10%, and decreased permanent deformation from 70% to 50%. Combinations of cytochalasin D and nocodazole, cytochalasin D and acrylamide, or all three drugs resulted in a synergistic effect on inhibition of CSK stiffness and viscosity but not in permanent deformation. Inhibition of oxidative metabolism with potassium cyanide had no effects on stress-induced stiffening response. Inhibition of tyrosine phosphatase with phenylarsine oxide had no effect on stress-induced stiffening response. We conclude that higher order mechanical interactions of CSK filaments are important in determining the mechanical properties of the cell.