Engineering light-inducible molecular tools from fluorophore-binding photoactivatable domains

During development, gene expression profiles are tightly regulated in order to ensure the correct assembly and differentiation of cells into organized multicellular and tissue assemblies. Part of this regulation is mediated via juxtacrine and paracrine signaling pathways, which coordinate in order to provide instructive signals that direct differential gene expression patterns over space and time. In particular, the function of juxtacrine signaling pathways—such as the Notch signaling pathway—serves to inform cells regarding their spatial positioning with respect to adjacent cells. In recent years, researchers have designed synthetic strategies in order to mimic developmental signaling programs and determine the molecular mechanisms involved in dysregulated signaling and disease. Such strategies have involved the implementation of synthetic gene circuits, artificial morphogen gradients, as well as engineered tissue microenvironments. These systems have provided powerful approaches for studying intercellular communication, enabling researchers to test hypotheses regarding natural mechanisms of cell-cell communication and to uncover the “design” principles underlying signal reception and propagation during tissue morphogenesis. In addition to their applications in basic science, these systems also possesses powerful synthetic utility, with the potential for realizing the promise of tissue engineering for biomedicine. Despite the sophistication of current DNA-encoded tool systems, the ability to direct pattern formation within synthetic multicellular assemblies remains limited. Indeed, in order to more closely recapitulate the intricate and complex assembly patterns that are observed in vivo, novel molecular tools that can be precisely actuated in space and time are required. In particular, tools that can be used to facilitate dynamic gene expression control, to encode the formation of cell-cell interactions, and to drive signal propagation across organized cellular assemblies, will be especially powerful in the realization of artificial tissue synthesis. In this thesis, I describe novel synthetic biology tools which have been designed in order to enable precise spatiotemporal control over gene expression programs within mammalian cells. By combining chemical, optical, and genetic techniques, a versatile light-inducible platform has been devised in which the activation of synthetic Notch (“SynNotch”) receptors can be controlled. In this approach, light is used to control the ability of our engineered SynNotch receptor to recognize, bind to, and subsequently activate in the presence of an optochemical ligand, fluorescein, and it’s photocaged derivatives. Herein, I describe the development, validation, and characterization of this system in order to achieve versatile optochemical control. In addition, I describe strategies by which chemogenetic control was integrated into the receptor platform in order to facilitate light-controlled activation of gene expression that can be subsequently down regulated upon administration of drug. Furthermore, through the incorporation of characterized split fluorescent protein components, we successfully rationally designed a SynNotch activation reporter system that depends on subcellular localization of proteins of interest and is capable of detecting SynNotch activation in under an hour. Overall, these systems represent a programable and tunable framework in which optogenetic and chemogenetic approaches are combined in order to provide the field with light-inducible molecular tools allowing for enhanced spatiotemporal control over proteins of interest. The future implementation and further adaptation of these tools will provide a powerful approach for dissecting how isogenic cells interact to propagate signals, coordinate protein expression patterns, and direct the formation of distinct multicellular phenotypes throughout development.2022-05-23T00:00:00