In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a comprehensive approach. We studied two cell types—Schlemm's canal endothelial cells and mouse embryonic fibroblasts (MEFs)—using four different probe technologies: 1) atomic force microscopy (AFM) with sharp tip, 2) AFM with round tip, 3) optical magnetic twisting cytometry (OMTC), and 4) traction microscopy (TM). Perturbation of Schlemm's canal cells with dexamethasone treatment, α-actinin overexpression, or RhoA overexpression caused increases in traction reported by TM and stiffness reported by sharp-tip AFM as compared to corresponding controls. By contrast, under these same experimental conditions, stiffness reported by round-tip AFM and by OMTC indicated little change. Knockout (KO) of vimentin in MEFs caused a diminution of traction reported by TM, as well as stiffness reported by sharp-tip and round-tip AFM. However, stiffness reported by OMTC in vimentin-KO MEFs was greater than in wild type. Finite-element analysis demonstrated that this paradoxical OMTC result in vimentin-KO MEFs could be attributed to reduced cell thickness. Our results also suggest that vimentin contributes not only to intracellular network stiffness but also cortex stiffness. Taken together, this evidence suggests that AFM sharp tip and TM emphasize properties of the actin-rich shell of the cell, whereas round-tip AFM and OMTC emphasize those of the noncortical intracellular network.
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