Investigating the Role of Force and Stiffness in Controlling Cell Behavior

Forces and the mechanical properties surrounding eukaryotic cells have been increasingly shown to play crucial roles in modulating biological functions, from healthy tissue development to disease progression. This work focuses on three aspects of cellular responses to their mechanical microenvironment: stiffness sensing, force regulation, and the effect of cancer progression on tissue architecture and elasticity. To make quantitative measurements of cell properties, I developed a novel technology based on atomic force microscopy (AFM) that is capable of dynamically controlling microenvironmental stiffness, as well as measuring forces and displacements. I first observed that cells nearly instantly change their contraction in response to step changes in stiffness (Chapter 2). I then determined that this process requires a mechanical equilibrium that balances contraction of the viscoelastic cytoskeleton with deformation of the extracellular matrix. This seconds-timescale equilibration provides a lower bound to the rate of whole-cell scale stiffness sensing (Chapter 3). Extracellular matrix stiffness has been identified as a key driver of tumorigenesis, but elucidating the link between tissue architecture and elasticity has remained experimentally difficult. Using AFM, I observed a near 2-fold stiffening of pre-malignant breast epithelial spherical structures, called acini, which are responsible for milk production, as well as are the initial site of many breast cancer cases. Further, the specific apparent microenvironmental stiffness for an epithelial cell within this acinar structure was calculated using 3D computational modeling, revealing a marked difference in compliance independent of extracellular matrix changes (Chapter 4). A widely cited concept in cellular mechanosensing to explain the response of cells to changes in their mechanical microenvironment is "tensional homeostasis", whereby adherent cells will actively respond to compression or extension by maintaining some previously established tension. However, no experiments have yet directly shown this behavior at the single cell level. Using a combination of protein patterning and AFM, I provide the first evidence of this homeostatic behavior. Notably, I found that cells do not maintain an absolute setpoint, but rather work to counteract force changes at a slow rate, beyond which force will freely change, a process I term tensional buffering. In addition, after rapid loading, cells will work to maintain their new level of tension, not returning to a previous setpoint (Chapter 5).Ultimately, these advances further our understanding of the role of mechanics in both healthy and diseased cellular processes, laying the groundwork for tissue engineering approaches for growing healthy tissues or therapeutic targets for a new array of hard to treat diseases