Physical and Molecular Pathways Involved in Cellular Sensing of Mechanical Signals

Mechanical properties of the extracellular environment provide important cues that regulate cell behavior. Particularly, the cellular response to substrate rigidity has become an important parameter to consider in disease treatment as well as tissue engineering. The goal of this thesis is to understand how adherent cells sense and respond to external rigidity cues. It has been challenging to study the mechanism that drives the preferential migration of cells towards stiffer substrates at a rigidity border due to difficulties in capturing cells as they transiently encounter a rigidity interface. Using a model system developed for testing cellular responses at a simulated rigidity border, I find that NIH 3T3 cells preferentially localize to the rigid portion of the model system. Cells use filopodia extensions to probe substrate rigidity in front of the leading edge and use substrate strain to determine whether the filopodia protrusions retract or expand to occupy the area. Myosin II mediated contractility is necessary to generate forces for both probing the substrate and retraction in response to substrate strain. Focal adhesion kinase null (FAK -/-) cells, known to be defective in durotaxis, are able to readily cross the rigidity border, while reexpression of focal adhesion kinase (FAK) rescues rigidity sensing. The model experimental system allows efficient analyses of conditions affecting rigidity sensing of cells. The results suggest that enhanced Rho activity, likely through Rho downstream effector mDia1, may underlie many rigidity sensing defects including those caused by FAK deficiency and microtubule disassembly. Additionally, I show that probing mechanisms at the front of a cell are used not only for probing rigidity but for sensing the state of migration. Design of a new checkerboard micropattern with alternating adhesive and non-adhesive areas revealed that the appearance of new traction forces and focal adhesions at the leading edge promotes the downregulation of pre-existing traction forces and focal adhesions that lag behind. These results suggest that in migrating cells continuous protrusion and mechanical probing directly in front of existing adhesions modulates traction force build up and serves as a key mechanism for regulating mechanical output in response to physical cues.