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, 110 (3), 881-6

Multidimensional Traction Force Microscopy Reveals Out-Of-Plane Rotational Moments About Focal Adhesions

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Multidimensional Traction Force Microscopy Reveals Out-Of-Plane Rotational Moments About Focal Adhesions

Wesley R Legant et al. Proc Natl Acad Sci U S A.

Abstract

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Experimental setup and computational methods. (A) Volume rendering of an EGFP-actin–expressing mouse embryonic fibroblast (MEF) fully spread and polarized on a planar PEG hydrogel with fluorescent beads imbedded (magenta). (B and C) Shear and normal components of bead displacement trajectories color-coded by magnitude. (D) Inset outlined in C magnified showing the normal component of bead displacement trajectories. The bottom figure is the cross-sectional view of the inset outlined above showing the multidimensional bead displacement trajectories. (E) Schematic of the finite element model to reconstruct the Green’s function. (All scale bars, 20 μm.)
Fig. 2.
Fig. 2.
Multidimensional traction stress and cytoskeletal localization. (A and B) Shear and vertical traction stress vectors generated by a MEF expressing EGFP-actin. The vectors are color-coded by magnitude. Both components of traction stresses are localized to the cell periphery. (C and D) Inset outlined in A magnified showing traction stress vectors color-coded by shear and normal components. Maximum shear tractions are detected at the termini of actin stress fibers, whereas the upward and downward normal tractions are applied in front of and behind the fiber ends, respectively. (E and F) Shear and normal traction stress vectors generated by a MEF expressing paxillin-EGFP are localized to focal adhesions in broad peripheral regions and a narrow retracting tail. (G and H) Inset outlined in E magnified showing shear and normal traction stress vectors relative to focal adhesions. Maximum shear stresses are detected directly over elongated focal adhesions, whereas the upward-to-downward gradient of normal stresses forms a rotational moment around the adhesions. (All scale bars, 20 μm.)
Fig. 3.
Fig. 3.
Dynamic measurements of 2.5D traction stress. (A) Time-lapse images depicting traction stress vectors color-coded by the normal component generated by a migrating MEF expressing mEGFP-farnesyl. As the cell moves (toward right), rotational moments are applied in the protruding front as well as the sides. (B) Time-lapse cross-sectional views of the inset outlined in A showing dynamic rotational moments that move with the thin protruding cellular body during cell migration. (C) Time-lapse images of mEGFP-farnesyl–expressing MEF undergoing spreading. No significant vertical traction stresses are detected until the cell extends thin protrusions and flattens against the substratum. Minimal tractions are detected under the nucleus. (D) Time-lapse cross-sectional views of the inset outlined in C. Comparable to migrating cells, rotating moments progress outward with the leading edge and remain localized to the cell periphery. (All scale bars, 20 μm.)
Fig. 4.
Fig. 4.
Finite element models of focal adhesion rotations. (A) Cartoon depicting the key elements in the FEM model. A contracting actin fiber generates shear traction on the upper surface of a focal adhesion (FA), which is modeled as a rigid plate connected to the PEG hydrogel. The shear load is applied uniformly on the top side of the FA, which is sufficient to induce both the horizontal and vertical substrate displacements comparable to experimental data. (B) Finite element renderings showing deformed configurations of the PEG hydrogel and focal adhesion. Contour maps along the symmetry plane show both horizontal and vertical displacements within the hydrogel. (C) Scatter plots of the measured hydrogel displacements from individual FAs (n = 121, 10 cells) with the average (green lines). Only beads residing within the first 0–2 μm of the hydrogel are shown. The x axis was normalized by the major axis of the fitted ellipse to each adhesion, so the adhesion itself spans from −1 to 1 (SI Appendix). The average FEM simulated hydrogel displacements within 0–2 μm from the hydrogel surface are shown for comparison (magenta lines, with the x axis normalized by the modeled FA length). (D) Scatter plots of the normalized experimental traction stresses from individual FAs with the average (green lines). The x axis is normalized by the fitted major axis length and the y axis of both plots is normalized by the mean shear traction surrounding each adhesion. (E) Top-down graphical maps of the averaged experimentally measured traction stresses from D with the same spatial dimensions. The inner ellipse depicts the normalized area of the FA, and the average shear and normal tractions within and outside of the FA are shown. The shape of the inner ellipse is scaled to match the mean major and minor axis lengths of the ellipses fit to the adhesions (8.3 ± 3.9 μm and 2.17 ± 0.9 μm, respectively). Note that the maximum shear traction stress is centered on the FA, whereas the maximum upward and downward traction stresses are at the distal and proximal ends of the FA, respectively.

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