ABSTRACT Cellular behaviors are modulated by a variety of stimuli in the environment, which can be generally categorized as chemical, physical, and biological factors. Previous work suggests that physical forces, both at the mesoscale of cell-cell interactions and the microscale of ligand-receptor binding events, play a critical role in regulating cell physiology. For example, stem-cell differentiation is regulated by neighboring cells and the stiffness of the extracellular matrix, while T cells are better activated by target cells with a mechanically strong cortex. The T- cell receptor also bears forces exerted from the pMHC, which affects receptor activation in a non-monotonic manner. While a plethora of evidence suggests the critical role of mechanical force in regulating cell fate and activation states, force itself has rarely been considered or exploited as a target for cell engineering and therapeutics development. Major hurdles include the lack of effective sensors that can digitally sense mechanical forces, and the lack of genetically encoded intracellular devices that can convert mechanical forces into a signaling cascade that modulates cell states. In this proposal, we will combine state-of-the-art technologies in protein design, RNA synthetic biology, and cell engineering to develop “mechaswitches”: universal, modular and programmable signal transduction systems that are able to trigger specific cellular actions in response to mechanical signals. Each mechaswitch is composed of a protein-based force sensor inserted within a force- bearing protein of interest and a transducer mRNA that implements a desired cellular action. When the protein of interest is subjected to a defined range of forces, the mechaswitch force sensor responds by changing its conformation. This conformational change is in turn detected by a sensing element within the transducer mRNA that either switches on or off the expression of specific proteins that modulate cell behavior. We will develop sensors covering a wide range of molecular forces and RNA switches that control protein expression with high fidelity. In proof-of-principle experiments, we will apply mechaswitches to program cell differentiation in defined extracellular environments and to modulate T cell proliferation and activation in response to specific antigens. Because of the modularity of mechaswitches, which enables their sensing and output components to be rapidly swapped and recombined, we envision a host of other uses for mechaswitches that could transform the study and application of mechanical forces in cell biology. The successful realization of this project is expected to not only advance basic research in mechanobiology, but also lay the foundation for the first therapeutic strategies informed by the mechanical signals and target mechano-properties of diseased cells and proteins.

NARRATIVE The proposed research aims to engineer cells with force-responsive molecular devices that can modulate cell differentiation and T cell-based immune response. The results could lead to new strategies that improve cancer treatment and regeneration. Thus, this work is relevant to the NIH’s mission to reduce illness and disability.