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

使用先进的显微镜技术实时监测 Notch 信号动态。

• 批准号: 450757120
• 负责人: Dr. Lena Tveriakhina
• 金额: --
• 依托单位: Department of Biological Chemistry and Molecular Pharmacology
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/450757120?language=en
• 关键词: Monitoring; Notch; signaling; dynamics; real

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.

Notch信号通路调节广泛的发育和稳态过程，并且其失调通常与各种人类癌症的发病机制相关。通过配体诱导的Notch受体的蛋白水解激活信号传导，最终产生参与Notch靶基因表达调控的转录效应子。这项工作的目的是建立一个工具包，直接可视化的Notch信号通路的关键组成部分，在其内源性的背景下，并定义前所未有的时空分辨率的事件发生时，生理Notch信号产生的序列。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）在活细胞中真实的实时跟踪。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，以及诱导成功的Notch反应所需的动力学。然后，这些发现将与信号诱导的流行模型联系起来，以拓宽我们对Notch信号传导的理解，并可能在未来应用于研究Notch信号传导失调引起的癌症细胞模型。