Dynamics of Notch Signaling

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Project Summary Signal transduction pathways underlie the very basis of life and are often critical targets for disease therapeutics. Yet, precise understanding of the intracellular dynamics, kinetics, and stoichiometry of how signals are transmitted – knowledge that is critical to capturing a holistic and more accurate view of signaling processes – has not been attainable for many pathways due to technological barriers to observing signaling events in real time in cells. Recent advances in technology, including proximity labeling approaches and light sheet microscopy, are finally now presenting solutions to knit together our fragmented view of signaling in vivo. Building on the expertise of the Blacklow laboratory in Notch signaling mechanism with the expertise of the Kirchhausen laboratory in advanced microscopy, we propose to use the Notch signaling system as a model to define precisely the series of events required for Notch signal activation in normal and pathogenic states, and in so doing, develop approaches and computational visualization tools that can be broadly applied to other pathways and systems. Notch signaling is an ideal model signaling system for this work because it exerts a critical influence on cell differentiation in all metazoans and its misregulation is associated with diverse diseases, including the pathogenesis of many human cancers. Moreover, fundamental facets of this signaling mechanism, including the dynamics of ligand and receptor molecules, the stoichiometry of ligand-receptor complexes at sites of activation, the timing and location of activating Notch proteolysis, and the path of Notch from plasma membrane to nucleus, remain incompletely understood. Here, we will address these gaps in knowledge by using APEX proximity labeling coupled with quantitative mass spectrometry to elucidate the pathway for passage of the Notch intracellular domain from plasma membrane to nucleus, and by implementing lattice light sheet microscopy to visualize the molecular events of Notch signal transduction. In preliminary work, we have used CRISPR/Cas9 genomic labeling in SVG-A immortalized astrocytes to create a Notch-APEX2 fusion protein for proximity labeling, and our preliminary analysis of a pilot experiment reveals dynamic changes in the labeling of proteins in different cellular compartments as a function of time, confirming the feasibility of this approach. We have also engineered fluorescently labeled receptor and ligand knockin proteins for LLSM. Now we will use this approach to quantify the number of receptor and ligand molecules that come together at the site of cell-cell contact as a function of time, and determine how many copies of each must be present to induce receptor cleavage and activate target gene expression. Successful completion of these aims will provide unprecedented resolution, in both space and time, of the fundamental events required for physiologic Notch signal transduction in living cells. We expect to not only answer long-standing questions about the molecular events involved in activation of Notch signals but to also open up a whole new realm of biology to mechanistic analysis.