Expanding the synthetic notch signaling toolkit for regulating gene expression in response to diverse extracellular cues
Embargo Date
2027-06-08
OA Version
Citation
Abstract
Multicellular processes, such as development and tissue regeneration, rely on precise signaling coordination between cells and their environment. In natural systems, this coordination is partly achieved through cell surface receptors. These receptors enable cells to detect external stimuli and translate them into intracellular changes, guiding the appropriate cellular responses. Inspired by the mechanisms of natural receptors, researchers have developed engineered receptors to control and reprogram how cells detect and respond to their neighbors and microenvironments. For example, synthetic Notch (SynNotch) receptors function like natural Notch proteins and can be used to install customized detect-and-respond capabilities into mammalian cells. In this Thesis, I describe the design and validation of new SynNotch-based tools that can be used to endow cells with the ability to detect a diverse set of biochemical and biomechanical information, including those based on cell-generated forces, the activity of matrix metalloproteases (MMPs), and in response to the folding state of naturally-derived extracellular matrix (ECM) proteins. Specifically, the first part of the thesis discusses the design and validation of fluorescein-based signaling adaptors, which were exploited to regulate the activity of a fluorescein-binding SynNotch with dose-dependent and spatiotemporal control. Through such adaptors, cells could be made to detect extracellular chemical transformations (including a bio-orthogonal ligation and a photochemical reaction) and enact customized gene expression activities as a response. This strategy was further applied to enable cells to detect and distinguish between folded and unfolded collagen-I proteins, the latter of which is a biomarker for various disease states. The second part of the thesis discusses important biophysical parameters needed for cells to exchange biomechanical information at cell-cell interfaces, as evaluated using synthetic mechanoreceptors in combination with a Delta-like 1 (DLL-1)-based transmembrane ligand protein. Thirdly, I describe a synthetic strategy for programming cells to detect extracellular proteolytic activities (including that of disease-associated MMP enzymes) and activate customized cell signaling outputs as a response. Using a structure-guided protein engineering strategy, I developed novel and modular transcriptional ‘switches’ through which extracellular protein cleavage events can be converted into actionable information—including the induction of synthetic gene expression activities and the modulation of endogenous cell signaling outcomes. The generality of this approach was demonstrated through the design of distinct protease-sensitive switches, including secreted and receptor-based constructs that can be selectively activated by corresponding proteolytic enzymes with specific and tightly regulated control. Overall, by leveraging chemical, structural, and biophysical insights, this Dissertation introduces new chemogenetic and biomechanical components that can be used to direct synthetic signaling activities in engineered cells or to redirect natural pathways, including those that are associated with disease. The future implementation of these systems will provide powerful approaches for programming cell behaviors in response to their microenvironment.
Description
2025
License
Attribution-NonCommercial-NoDerivatives 4.0 International