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

Notch信号通路调节广泛的发育和体内平衡过程，其失调通常与各种人类癌症的发病机制有关。该信号通过配体诱导的Notch受体蛋白水解激活，最终产生参与调控Notch靶基因表达的转录效应物。这项工作旨在建立一个工具包，直接可视化Notch信号通路的关键组成部分在其内源性背景下，并以前所未有的时空分辨率定义生理Notch信号产生时发生的事件序列。内源性Notch配体、受体和辅助蛋白将使用CRISPR/Cas9基因组编辑技术用荧光团标记，并通过先进的显微镜技术（旋转盘、共聚焦、TIRF、AO-LLSM）在活细胞中实时跟踪。我们将应用该系统来阐明信号事件的精确时间和亚细胞位置，配体-受体复合物的化学计量学，以及诱导成功Notch反应所需的动力学。这些发现将与主流的信号诱导模型联系起来，以扩大我们对Notch信号的理解，并可能在未来应用于研究由Notch信号失调引起的癌症细胞模型。

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.