Monitoring Notch signaling dynamics in real time using advanced microscopy techniques.

点击翻译按钮获取中文摘要

The Notch signaling pathway regulates a wide spectrum of developmental and homeostatic processes and its dysregulation is often associated with the pathogenesis of various human cancers. The signaling is activated through ligand-induced proteolysis of the Notch receptor, eventually generating a transcriptional effector that participates in the regulation of Notch target gene expression. This work aims to establish a toolkit to directly visualize key components of the Notch signaling pathway in their endogenous context and to define with unprecedented spatiotemporal resolution the sequence of events that take place when physiologic Notch signals are generated. Endogenous Notch ligand, receptor, and accessory proteins will be labeled with fluorophores using CRISPR/Cas9 genome editing and tracked by advanced microscopy techniques (spinning disk, confocal, TIRF, AO-LLSM) in living cells in real time. We will apply this system to elucidate the precise timing and subcellular locations of the signaling events, the stoichiometry of ligand-receptor complexes, and the dynamics required to induce a successful Notch response. These findings will then be linked to the prevailing model of signal induction to broaden our understanding of Notch signaling and may in future be applied to study cell models of cancer caused by dysregulated Notch signaling.