Tools to reveal the dynamics of molecular forces in living cells

Abstract When imaged using an optical microscope, a live cell will appear still, but in reality, cells are highly dynamic structures that are pulling and pushing on one another and also on their surrounding extracellular matrix. These pulls and pushes are mediated by minuscule forces – at the scale of tens of piconewtons, that are highly transient and dynamic. Nonetheless, these forces have profound impacts as they can modulate receptor conformation and function. This is akin to how a small posttranslational modification can potently activate an enzyme. For example, the rapidly fluctuating forces at the junction between an immune cell, such as a T cell, and a cancer cell can dictate the fate of the cancer cell and determine whether the T cell performs its cytotoxic functions. Despite the importance of mechanics and their dynamics there are limited methods to study forces. This is particularly the case for studying forces at the single molecule scale. My group is addressing this gap in knowledge by developing tools to map the molecular forces applied by cells. The current application will provide new tools to investigate force dynamics. The first aim will develop a universal tension sensor that can measure force loading rate with high resolution. Here we will create a library of probes with narrowed dynamic range that will validate whether loading rates are linear or dynamic and also will provide computational tools to relate force dynamics with downstream signaling. The second aim will develop tools to test the catch bond model which is a counter-intuitive enhancement in ligand-receptor binding when these complexes experience a moderate mechanical load of ~5-20 pN. This has been observed in single molecule force spectroscopy measurements and we seek to validate this observation in live cell-cell junctions using single molecule tracking of force duration and bond lifetimes. Finally, we will develop a new DNA origami probe to investigate models of anisotropic mechanosensing, where the orientation of a molecular force is thought to drive different signaling outcomes. This molecule force orientation sensor will allow us to measure force orientation in realtime. Taken together the application represents an important step toward better understanding the role of biophysical forces in cell signaling.