New Tools to Probe Notch Signal Transduction

Abstract

This dissertation presents approaches to reconstitute and observe specific biochemical events fundamental to Notch signaling. The Notch pathway is an example of direct cell-cell communication that governs cell fate during development and homeostasis in metazoans (Artavanis-Tsakonas et al., 1995; Artavanis-Tsakonas et al., 1999). Recent work (Gordon et al., 2015; Langridge and Struhl, 2017) suggests that Notch receptors are activated in response to the application of mechanical force by bound ligand. This thesis focuses on new methods to deliver, modulate, and analyze the effect of applied force to Notch receptors, or their isolated mechanosensors. In Chapter 2, I develop a multiplexed single-molecule flow-extension sorting technique to identify singly-tethered substrates, and perform single-molecule enzymology with tobacco etch virus N1a protease and ADAM17. In Chapter 3, I carry out preliminary optical trapping experiments on the Notch1 mechanosensitive domain, and observe discontinuities in some of the force-extension curves. These features may be attributable to unfolding transitions, such as disengagement of the autoinhibitory interface that masks the metalloprotease cleavages site (S2), or unfolding of the HD domain. In Chapter 4, I describe the construction and functional testing of a Delta-like ligand 4 (DLL4) chimeric protein that acts as a building block for a two-component system (designed by David Baker and co-workers (Ben-sasson, 2019)) that self-assembles into hexagonal protein arrays. This DLL4 chimeric “A” component can bind to Notch1 on cells, and form polymeric arrays with its “B” component partner. Arrays formed with the DLL4 chimera suppress Notch1 receptor endocytosis, and interfere with entry of the DLL4 chimeric protein into Notch1-expressing cells.