Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response.cell mechanics ͉ cytoskeleton ͉ mechanotransduction ͉ microrheology T he important role of mechanical and physical cues in determining cell behavior is increasingly recognized. Cell shape can modulate cell differentiation (1), whereas substrate stiffness can affect tissue morphogenesis (2) and myoblast differentiation (3). The mechanisms, however, by which mechanical cues lead to molecular and biochemical responses remain largely undetermined. One approach to studying such mechano-sensing processes is to understand the mechanical properties of cells' constitutive molecules individually. Although protein conformation can respond to locally applied, small-scale mechanical signals such as molecular tension (4), how these signals may be combined to sense larger-scale mechanical properties remains unclear (5). Ultimately, an integrated physical description of cytoskeletal mechanics will be required to connect these molecular and cellular levels of description.In soft-matter and polymer physics, the mechanical response and dynamics of supramolecular assemblies are determined by using rheology, the study of the frequency-dependent elastic and viscous behavior of deformable materials. Reliably interpreting rheology measurements on living cells, however, has proven notoriously difficult. Only after decades of experimental effort have different cell measurements begun to report comparable responses, although many differences remain (6). The rheology of cells is typically inferred from either deformation in response to an applied force (termed active microrheology) or the Brownian motion of embedded or attached tracer particles (termed passive microrheology). The results of both approaches depend on theoretical models for the deformation geometry or coupling between the tracer and the cell. Passive methods will also be confounded by any non-Brownian tracer motion such as intracellular trafficking or cell crawling. The comparison of cell mechanics measurements based on different methods is currently ...

Although actin and myosin are important contributors to cell-force generation, shape change, and motility, their contributions to cell stiffness and frequency-dependent rheology have not been conclusively determined. We apply several pharmacological interventions to cultured epithelial cells to elucidate the roles of actin and myosin in the mechanical response of cells and intracellular fluctuations. A suite of different methods is used to separately examine the mechanics of the deep cell interior and cortex, in response to depletion of intracellular ATP, depolymerization of F-actin, and inhibition of myosin II. Comparison of these results shows that F-actin plays a significant role in the mechanics of the cortical region of epithelial cells, but its disruption has no discernable effect on the rheology of the deeper interior. Moreover, we find that myosins do not contribute significantly to the rheology or ATP-dependent, non-Brownian motion in the cell interior. Finally, we investigate the broad distribution of apparent stiffness values reported by some microrheology methods, which are not observed with two-point microrheology. Based on our findings and a simple model, we conclude that heterogeneity of the tracer-cytoskeleton contacts, rather than the network itself, can explain the broad distribution of apparent stiffnesses.

The results of mechanical measurements on single cultured epithelial cells using both magnetic twisting cytometry (MTC) and laser tracking microrheology (LTM) are described. Our unique approach uses laser deflection for high-performance tracking of cell-adhered magnetic beads either in response to an oscillatory magnetic torque (MTC) or due to random Brownian or ATP-dependent forces (LTM). This approach is well suited for accurately determining the rheology of single cells, the study of temporal and cell-to-cell variations in the MTC signal amplitude, and assessing the statistical character of the tracers' random motion in detail. The temporal variation of the MTC rocking amplitude is surprisingly large and manifests as a frequency-independent multiplicative factor having a 1/f spectrum in living cells, which disappears upon ATP depletion. In the epithelial cells we study, random bead position fluctuations are Gaussian to the limits of detection both in the Brownian and ATP-dependent cases, unlike earlier studies on other cell types.