Probe Sensitivity to Cortical versus Intracellular Cytoskeletal Network Stiffness

Abstract

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.

Figure 1

Confocal images of confluent monolayer of vehicle-only control (top row) as compared with 1-μM-dexamethasone-treated (bottom row) SC cells; F-actin (red) (A and D), phosphorylated myosin (green) (B and E), nucleus (blue). Cortex and stress fibers are seen in both groups, but the cortex is more prominent in treated SC cells (white arrows in (D)). Also, although p-myosin is present in both groups, it is more concentrated at the cortex of treated cells (white arrows in (E)). (C) and (F) are overlaid images.

Figure 2

Measurements of cell biophysical properties of SC cells after (A and B) treatment with varying concentrations of dexamethasone as compared with vehicle alone or (C and D) overexpression of α-actinin or RhoA as compared with GFP or control. (E)–(H) show comparison of wild-type (WT) MEFs with vimentin KO. Superconfluent monolayers were used for the dexamethasone studies on SC cells (A and B), confluent for the α-actinin or RhoA expression studies on SC cells (C and D), and both confluent (E and F) and sparsely seeded (G and H) cells for the MEF studies, as described in the Materials and Methods. Geometric mean ± standard error about geometric means is shown. Curves shown in (A) and (B) are best regression fits to the data; the equations are given in Supporting Materials and Methods. Data for individual cell strains are found in Figs. S2 and S3. To see this figure in color, go online.

Figure 3

Confocal images of SC cells. (A) WT-non-transduced, (B) GFP-transduced, (C) α-actinin-transduced, and (D) RhoA-transduced cells are shown; F-actin (red), nucleus (blue). Control and GFP groups had similar F-actin distribution and cortex structure (A and B). The α-actinin-transduced cells showed an altered morphology and had a relatively thicker cortex (white arrow in (C)). The RhoA-transduced cells showed significant accumulation of stress fibers at peripheral regions and a more prominent cortex (white arrow in (D)).

Figure 4

Confocal images showing F-actin (red), nucleus (blue) (A and B), and vimentin intermediate filaments (green) (C and D) of WT MEFs (A and C) and vimentin-KO MEFs (B and D). (D) shows no staining in a vimentin-KO MEF. (E) shows a merged image of (A) and (B). (F) is a structured illumination micrograph (SIM) of a WT MEF at the basal cortex level, showing the close association between actin stress fibers and vimentin intermediate filaments. A magnified image of the inset in (F) showing vimentin intermediate filaments surrounding and interconnecting actin stress fibers is given (G).

Figure 5

Confocal cross-sectional representative images of typical WT (A) and vimentin-KO (B) MEFs show that the vimentin-KO MEFs are thinner than the WT MEFs.

Figure 6

Strain-energy distribution (log scale) for indentation into a cell of a sharp AFM tip (A and B), a 0.8 μm diameter rounded AFM tip (C and D), a 10 μm diameter rounded AFM tip (E and F), and a 4.5 μm OMTC bead (G and H). The indentation is smaller in cases (A) and (B) (80 nm) because of numerical limitations, as discussed in the Materials and Methods, than for the other AFM tips (400 nm). The OMTC bead is embedded 25% of its diameter into the cell and twisted by a torque of 60 Pa applied in a counterclockwise fashion. (A), (C), (E), and (G) are for cases with E_cortex = E_{intracellular}; (B), (D), (F), and (H) are for E_cortex = 50 × E_{intracellular}. The cortex in each panel is the narrow region between the two horizontal black lines and has a thickness of 400 nm before indentation. The strain-energy distribution in each panel is normalized to the maximal strain energy in that panel, and a log scale is used. Cell thickness is 5 μm.

Figure 7

(A) E_apparent/E_{intracellular} as a function of E_cortex/E_{intracellular} for an AFM round tip of diameter 0.8–10 μm with an indentation of 400 nm. Inset shows result for AFM sharp tip with indentation of 80 nm. Cell thickness is 5 μm. (B) E_apparent/E_{intracellular} as a function of E_cortex/E_{intracellular} for an OMTC probe of diameter 4.5 μm for embedding depths of 10, 25, or 50% of bead diameter is shown. Cell thickness is 5 μm. Inset shows results for an OMTC probe embedded 50% into a cell with E_cortex/E_{intracellular} = 50 for cell thicknesses ranging from 2.75 to 30 μm. To see this figure in color, go online.