Mechanistic Insights into the Notch signalling pathway through Molecular Condensation

Abstract

The ability of a cell to undergo cellular decisions is ingrained in the integration of cues from its local environments. One such cue is relaying information based on local cell density, neighbouring cell identity, and cell positional information. A key signalling pathway in interpreting this information is the Notch signalling pathway. It is currently poorly understood how Notch is able to play a titratable role in signal transduction as our current model does not effectively translate how fluctuations in Notch signalling lead to dynamic changes in Notch target gene expression. One potential mechanism that has recently been shown to play a role in titratable gene expression of several signalling pathways is the ability for proteins to undergo molecular condensation within living cells. To date, Notch signalling has been difficult to study because of the lack of live cell systems that allow us to study both Notch protein localization and Notch target gene expression in real-time. Using PlayBack, a system I developed that allows for cost-effective plasmid cloning, I was able to develop both a light controlled Notch construct, OptoNotch, and a Live Notch target gene promoter reporter, called Hes1-Live-RNA, to allow for both the visualization and quantification of Notch activity within living cells. Using these tools, I discovered that the Notch 1 intracellular domain forms molecular condensates which in turn positively facilitates both Notch1 target gene expression as well as facilitating Notch’s ability to regulate enhancer looping. This is the first instance of N1ICD undergoing condensation within live cells and offers a mechanism by which Notch can have a titratable effect on target gene expression.