Mapping cell-matrix stresses during stretch reveals inelastic reorganization of the cytoskeleton

Abstract

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actin-myosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.

FIGURE 1

Stretching device. Collagen gels with embedded fluorescent microbeads were jellified on flexible-bottomed wells. The wells were positioned on a sample holder based on a hollow cylindrical loading-post concentric with the objective of the epi-fluorescence microscope. The application of a negative pressure underneath the annular outer region of the sample resulted in a homogeneous and equibiaxial strain of the central area.

FIGURE 2

Experimental protocol. (A) Scheme of the traction microscopy protocol. A well containing a cell-cultured collagen gel was placed on the stretching device and bright field and fluorescence images of an isolated cell were acquired (I). The gel was stretched and bright field and fluorescence image acquisition was done again (II). The stretch was released and two final bright field and fluorescence images of the cell were acquired (III). After trypsin cell removal, a fluorescence reference image (RI) was recorded to assess the cell-free unstrained gel (IV). The gel was stretched again, and a fluorescence reference image was recorded to observe the cell-free strained gel (V). (B) Time course of the experimental protocol before cell detachment. (C) In traction recovery experiments, the protocol was extended to continue fluorescence image acquisition for 8 min (1 image/min) after stretch release.

FIGURE 3

Effect of stretch on cell-matrix stresses. Traction fields exerted by an adhered cell in baseline (left), during stretch (11%) (middle) and after stretch release (right). Inset shows a bright field image of the cell in baseline. Color scale indicates the magnitude of traction forces and arrows depict the direction and relative magnitude of traction force. Tractions were calculated with 2.6 μm spatial resolution. For clarity, arrows are displayed with 5.2 μm spacing. Scale bar is 20 μm.

FIGURE 4

Effect of stretch on cell traction force. Average cell traction (T) measured before (baseline, gray bars), during (black bars) and after stretch release (white bars). Measurements were done at 0% strain (control), 5.5% strain, and 11% strain. Standard deviation of traction values within each cell was on average 3.6 Pa (42% CoV). Data are mean ± SE (n = 6). *p < 0.05 and **p < 0.01 versus baseline, respectively.

FIGURE 5

Dependence of local traction forces after stretch release on baseline traction. All data of traction fields of cells subjected to the same stretch level were pooled together and sorted by the magnitude of traction forces exerted under baseline conditions. The median of each group is plotted versus the median of the pooled traction forces in the same spatial points in stretch release configuration. Black circles correspond to cells subjected to no stretch (control), red circles to 5.5% stretch, green triangles to 11% stretch, and yellow triangles to cells pretreated with ML7 and subsequently subjected to 5.5% stretching. Traction forces below 1.5 Pa are not plotted because data were noisy. Data are mean ± SE (n = 6). Lines are linear fits to the data. Inset: Values of the slopes obtained from linear fit of data in control (black bar), 5% stretch (red bar), 11% stretch (green bar), and ML7+5% stretch (yellow bar). Error bars are the standard error of the slopes provided by the fit ML7 + 5.5%. Dashed line corresponds to 1.

FIGURE 6

Effect of actomyosin inhibition on the cell stress response to stretch. Mean traction (T) of non-pretreated cells (solid bars) and cells pretreated with ML7 (dashed bars) and subsequently subjected to 5.5% strain. Gray bars correspond to baseline configuration, black bars to stretch configuration, and white bars to stretch release. Data are mean ± SE (n = 6). *p < 0.05 and **p < 0.01, respectively, versus baseline non-pretreated cells (solid gray bar). ⁺⁺⁺p < 0.001 versus baseline pretreated cells (dashed gray bar). (Inset) Ratio of T values under stretch or release conditions versus T under baseline conditions. Ratios were computed for each cell and then averaged. Solid bars indicate nonpretreated cells and dashed bars indicate cells pretreated with ML7. Dashed line corresponds to baseline.

FIGURE 7

Cell-matrix stress recovery after stretch release. (A) Time course of traction force (T) recovery after stretch release of cells subjected to 5.5% strain. Data are mean ± SE (n = 6). Time zero corresponds to stretch release. Dotted line is baseline T. (B) All data of traction fields were pooled together and sorted by the magnitude of traction forces exerted under baseline conditions. Time course recovery of each group is plotted as the median of the pooled traction forces after stretch release divided by the median of the same group under baseline conditions. Black circles correspond to points of the traction map initially exerting low tractions (1.5–4.5 Pa), white triangles to mid traction (4.6–9), and black squares to high traction (>9). Traction forces below 1.5 Pa are not plotted because data were noisy. Dashed line indicates the identity.