Engineering mechanotransduction in mammalian cells using the Notch receptor
Sloas, D. Christopher
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Mechanical forces are fundamental regulators of biology. Individual cells can detect environmental forces and transform them into intracellular biochemical actions, which impact gene expression, metabolism, and differentiation. In turn, this phenomenon of “mechanotransduction” at the cellular level affects tissue- and organ-level function and can shape disease progressions. Tools that enable researchers to genetically harness mechanotransduction would therefore be powerful for developing of novel tissue engineering and cell therapy technologies. However, synthetically engineering mechanotransduction in cells has remained difficult. In this thesis, we control how cells respond to molecular forces by engineering modular mechanosensitive receptors. Using a structured-guided approach, we engineered force-sensitive protein domains that, when inserted into synthetic Notch receptors, vary the input-output relationship between mechanical force and cellular action. We demonstrate that the mechanical strength of these domains can be systematically tuned through mutagenesis. We show that our synthetic mechanoreceptors enable the design of signaling networks where tensile forces in the environment are recorded as measurable and specifiable biochemical responses, such as myogenic differentiation in mouse embryonic fibroblasts. We then present additional technologies for modulating the Notch mechanoreceptor’s endogenous mechanical strength, ligand-mediated activation, and protease-regulated activation. Taken together, this dissertation introduces a mechanogenetic framework for synthetically controlling mechanotransduction in mammalian cells, informs the design of future synthetic force-sensitive pathways, and provides valuable tools for the study of Notch signaling in development and disease.