Engineered molecular tools for mapping and manipulating intracellular and intercellular contacts

This thesis presents engineered molecular tools that have been developed for detecting, mapping, and manipulating both intracellular protein-protein and organelle-organelle interactions as well as intercellular cell-cell interactions. The first half of this thesis discusses work on the development and application of a new split proximity labeling enzyme for proteomic mapping with high spatial specificity. Most of the thousands of proteins that comprise a human cell have specific subcellular localization patterns essential for their function. Proximity labeling is a method for mapping the localization of endogenous cellular proteins on a proteome-wide scale. Proximity labeling catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current proximity labeling methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. We targeted split-TurboID to the endoplasmic reticulum (ER) and outer mitochondrial membranes for proteomic mapping of ER-mitochondria contacts. At ER-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of > 100 endogenous proteins, including many not previously linked to ER-mitochondria contacts, demonstrating the utility of applying split-TurboID for proteomic mapping in previously inaccessible subcellular regions. The second half of this thesis discusses work on developing synthetic molecular circuits that detect cell-cell contacts and either activate a transcription factor or toxin in response. Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. In Chapter 3, we report TRACC, a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. We incorporate light gating using the LOV domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC activation is specific and sensitive in mammalian cell culture and that it can be applied for detecting interactions between neurons and glioma cells. In Chapter 4, we design a molecular tool that incorporates a split tetanus neurotoxin pair, in which cell-cell interactions drive the reconstitution of the toxin, enabling cleavage of endogenous SNARE proteins for synaptic silencing. We develop and optimize a split tetanus pair that is only active when driven together by a protein-protein interaction. We also optimized gain-of-signal assays for detecting reconstituted tetanus activity in both HEK293T and primary neuron cultures. With additional optimization and development, this neurotoxin-based tool may be used to systematically correlate defined synaptic connections to behavioral phenotypes, enabling a wide range of potential applications in neuroscience

Advisor: Ting Alice Y.
Frydman Judith
Monje-Deisseroth Michelle
Zuchero J.