Cells sense the rigidity of their substrate; however, little is known about the physical variables that determine their response to this rigidity. Here, we report traction stress measurements carried out using fibroblasts on polyacrylamide gels with Young's moduli ranging from 6 to 110 kPa. We prepared the substrates by employing a modified method that involves N-acryloyl-6-aminocaproic acid (ACA). ACA allows for covalent binding between proteins and elastomers and thus introduces a more stable immobilization of collagen onto the substrate when compared to the conventional method of using sulfo-succinimidyl-6-(4-azido-2-nitrophenyl-amino) hexanoate (sulfo-SANPAH). Cells remove extracellular matrix proteins off the surface of gels coated using sulfo-SANPAH, which corresponds to lower values of traction stress and substrate deformation compared to gels coated using ACA. On soft ACA gels (Young's modulus <20 kPa), cell-exerted substrate deformation remains constant, independent of the substrate Young's modulus. In contrast, on stiff substrates (Young's modulus >20 kPa), traction stress plateaus at a limiting value and the substrate deformation decreases with increasing substrate rigidity. Sustained substrate strain on soft substrates and sustained traction stress on stiff substrates suggest these may be factors governing cellular responses to substrate rigidity.

Leukocytes and tumor cells migrate via rapid shape changes in an amoeboid-like manner, distinct from mesenchymal cells such as fibroblasts. However, the mechanisms of how rapid shape changes are caused and how they lead to migration in the amoeboid mode are still unclear. In this study, we confined differentiated human promyelocytic leukemia cells between opposing surfaces of two pieces of polyacrylamide gels and characterized the mechanics of fibronectin-dependent mesenchymal versus fibronectin-independent amoeboid migration. On fibronectin-coated gels, the cells form lamellipodia and migrate mesenchymally. Whereas in the absence of cell-substrate adhesions through fibronectin, the same cells migrate by producing blebs and "chimneying" between the gel sheets. To identify the orientation and to quantify the magnitude of the traction forces, we found by traction force microscopy that expanding blebs push into the gels and generate anchoring stresses whose magnitude increases with decreasing gap size while the resulting migration speed is highest at an intermediate gap size. To understand why there exists such an optimal gap size for migration, we developed a computational model and showed that the chimneying speed depends on both the magnitude of intracellular pressure as well as the distribution of blebs around the cell periphery. The model also predicts that the optimal gap size increases with weakening cell membrane to actin cortex adhesion strength. We verified this prediction experimentally, by weakening the membrane-cortex adhesion strength using the ezrin inhibitor, baicalein. Thus, the chimneying mode of amoeboid migration requires a balance between intracellular pressure and membrane-cortex adhesion strength.

The tumor suppressor p53 induces activation of the mitochondrial protease HtrA2/Omi and prevents Ras-driven invasion by modulating the actin cytoskeleton.