Adhesive Force Regulation and Mechanotransduction

Mechanical forces regulate diverse cellular functions, and integrin receptor-based adhesive complexes have emerged as central players in force transmission from the extracellular matrix (ECM) to the cytoskeleton and conversion of forces into biochemical signals. These adhesive signals direct cell fate and regulate tissue formation, homeostasis, repair, and even pathogenesis. Cell adhesion to ECM components is primarily mediated by the integrin receptor family. Following ligand binding, integrins cluster into nanoscale focal adhesion (FA) structures that function as foci for the transmission of anchorage and propulsive forces. These FA complexes consist of integrins and actins vertically separated by a ~40 nm core that includes cytoskeletal proteins and signaling molecules. FAs function as structural links, providing strong adhesive forces, and signal transduction elements between the cell and its ECM.

Mechanical interactions between a cell and its ECM comprise coupled spatiotemporal force components. This dynamic balance is governed by the size and distribution of cell-ECM adhesive structures, cytoskeletal architecture, and actomyosin contractile forces. We have established novel, integrated bioengineering platforms to analyze cell adhesion mechanobiology. These platforms include hydrodynamic adhesion assays, deformable force arrays, photo-triggerable adhesive ligands, and nano/micropatterned substrates as well as unique cell lines expressing FA protein mutants (integrin β1, vinculin, talin, FAK) to analyze the regulation of adhesive forces and mechanotransduction. This research has provided unique findings into mechanotransduction that cannot be obtained using conventional assays such as cell morphology, spreading, and migration.

As an example of the ability to our integrated platforms to provide novel insights into mechanobiology, we have recently demonstrated using vinculin-null cells expressing vinculin-mutants that this FA protein regulates traction force and adhesive force through several mechanisms (Fig. 1). The vinculin head domain (VH) enhances adhesion strength, but not traction forces, by increasing ECM-bound integrin-talin complexes; this activity of the VH domain requires talin-binding activity, but does not require interaction with actin or myosin contractility. A full length, autoinhibition-deficient vinculin mutant (T12) increases adhesion strength compared to VH alone, implying a role for the actin-binding activity of the vinculin tail domain.  Autoinhibition of vinculin regulates integrin-FN complex/FA assembly and, unlike VH, is subject to contractility-dependent adhesion strength modulation. In contrast to adhesive force, transmission of traction force is entirely dependent on a full length vinculin molecule and is also regulated by autoinhibition. Strikingly, the residence time of wild-type vinculin in FAs, but not T12 or VH, correlates with applied force (Fig. 2), supporting a mechanosensitive model for vinculin activation and stabilization in which forces applied across vinculin also maintain the molecule in its active conformation. These results provide new mechanistic insights into how vinculin’s structure, biochemistry, and binding partners interact with actomyosin contractility to regulate transmission of traction and adhesion forces.

 

Figure 1: Vinculin regulates traction forces. (A) Cells spread on mPADs (posts labeled red) showing localization of vinculin (eGFP) to FAs (top) and spreading (yellow outline) and force vectors (cyan arrows) (bottom, scale bar 4 µm). (B) Box-whisker plot for cell area. Kruskal-Wallis p<0.0001, * p<0.05 vs. null, # p<0.05 vs. WT, † p<0.05 vs. VH; § p<0.05 blebb vs. control. (C) Box-whisker plot for total traction force per cell. Kruskal-Wallis p<0.0001, * p<0.022 vs. null, # p<0.01 vs. WT, † p<0.01 vs. VH; § p<0.05 blebb vs. control. (D) Relationship between traction force and cell area showing linear regression fits.

 

Figure 2: Vinculin residence times in FAs correlate with applied force and require head-tail interactions. FRAP was performed on FAs on mPAD posts with known applied forces. (A) FRAP recovery curves for WT localized to FAs transmitting different forces. (B) Correlation between recovery time (t1/2) and applied force for WT. Linear regression: t1/2 = 1.56*force + 20.0, p < 0.0001.