Pani Ariel M, Gibney Theresa V, Medwig-Kinney Taylor N, Matus David Q, Goldstein Bob
Department of Biology, University of Virginia, Charlottesville, VA, USA.
Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA, USA.
MicroPubl Biol. 2022 Jul 28;2022. doi: 10.17912/micropub.biology.000603. eCollection 2022.
Notch signaling mediates cell-cell interactions during development and homeostasis. Methods for visualizing and manipulating Notch activity are essential to elucidate how the Notch pathway functions. Here, we provide new tools for use in to visualize and perturb Notch signaling using endogenously tagged alleles of the Notch receptor . Tagging the endogenous LIN-12 intracellular domain with the fluorescent protein mNeonGreen (mNG) allowed for visualization of both its membrane-localized state and translocation of the Notch intracellular domain into the nucleus upon ligand activation. LIN-12::mNG localized to the nucleus in cells where and when Notch signaling is known to be active and provided a real-time readout of Notch activity that complements existing biosensors and transcriptional reporters. We also report an allele of endogenous that we tagged with both mNG and an auxin-inducible degron, to facilitate conditional LIN-12 protein degradation. This toolkit provides novel reagents for the research community to investigate mechanisms of Notch signaling and its functions .
Notch信号在发育和体内平衡过程中介导细胞间相互作用。可视化和操纵Notch活性的方法对于阐明Notch信号通路的功能至关重要。在这里，我们提供了新的工具，用于使用Notch受体内源标记的等位基因来可视化和干扰Notch信号。用荧光蛋白mNeonGreen（mNG）标记内源性LIN-12细胞内结构域，使得既能观察到其膜定位状态，又能观察到配体激活后Notch细胞内结构域向细胞核的转运。LIN-12::mNG在已知Notch信号活跃的细胞和时间定位于细胞核，并提供了Notch活性的实时读数，补充了现有的生物传感器和转录报告基因。我们还报告了一个内源性等位基因，我们用mNG和生长素诱导降解结构域对其进行了标记，以促进LIN-12蛋白的条件性降解。这个工具包为研究界提供了新的试剂，以研究Notch信号的机制及其功能。
