Transport, processing and regulation of the Notch receptor

Notch受体参与重要的发育和分化决定。Notch激活后，胞内结构域（NICD）被γ-分泌酶切割并转运到细胞核中。在那里，它与共激活因子RBP-J一起激活基因转录。过度活跃的Notch可导致癌症，例如T-ALL白血病。在第一个资助期内，使用基于图像的高通量筛选，我们筛选了一个化合物文库以及一个全基因组siRNA文库，以寻找参与Notch转运和加工的因子。我们确定了新的γ-分泌酶抑制剂和分泌抑制剂与一种新的行动模式。在下一个资助期内，我们希望专注于siRNA筛选的三个命中，Nup 214，核孔蛋白，输出蛋白4（XPO 4），核输出因子和磷酸酶，MTMR 14。我们以前的工作表明，Nup 214通过将RBP-J输出到细胞核外来负调节Notch信号传导。我们现在要讨论以下问题。Nup 214在Notch信号传导中的分子作用是什么？Nup 214是直接与RBP-J相互作用，还是与转运蛋白RITA相互作用？RBP-J的核输出是否受到调节，例如在某些分化阶段，在应激下的Notch激活时？初步数据显示，引起Set-Nup融合的T-ALL在细胞核中聚集并隔离其他核孔蛋白。这可能激活Notch信号传导并有助于肿瘤发生。我们想研究几种导致Nup 214融合的T-ALL对Notch信号的影响，并分析XPO 4在Notch信号中的作用。为此，我们希望在细胞培养和体外研究其在NICD核输入/输出中的作用。在斑马鱼中，我们希望确定Nup 214和XPO 4的时空表达模式，并希望研究针对这两种mRNA的特定Morpholinos的表型。为了研究Nup 214融合蛋白的病理功能，我们还想建立一个T-ALL融合的斑马鱼模型。利用我们建立的ER输出和分泌途径中转运的检测方法，我们计划阐明MTMR 14在Notch信号传导中的功能。最后，我们希望通过改进的分割和分类方法重新评估siRNA筛选的图像，以发现更多的表型。所有计划的实验都使用了已建立的方法，如免疫沉淀，GST-下拉，荧光显微镜，siRNA技术，体外检测，吗啉代注射和原位杂交。

The Notch receptor is involved in important developmental and differentiation decisions. After activation of Notch the intracellular domain (NICD) is cleaved by gamma-secretase and transported into the nucleus. There it activates gene transcription, together with the co-activator RBP-J. Hyperactive Notch can cause cancer, for example T-ALL leukemia. In the first funding period using image-based high throughput screens we screened a compound library as well as a genome-wide siRNA library for factors involved in transport and processing of Notch. We identified novel gamma-secretase inhibitors and a secretion inhibitor with a novel mode of action. In the next funding period we want to focus on three hits from the siRNA screen, Nup214, a nuclear pore protein, exportin 4 (XPO4), a nuclear export factor and a phosphatase, MTMR14. Our previous work showed that Nup214 negatively regulates Notch signaling by exporting RBP-J out of the nucleus. We now want to address the following questions. What is the molecular role of Nup214 in Notch signaling? Is Nup214 directly interacting with RBP-J, or with the transport protein RITA? Is the nuclear export of RBP-J regulated, for example upon Notch activation, in certain differentiation stages, under stress?Preliminary data show that a T-ALL causing Set-Nup fusion aggregates in the nucleus and sequesters other nucleoporins. This might activate Notch signaling and contribute to tumorgenesis. We want to study the effects of several T-ALL causing Nup214 fusions on Notch signaling.We also want to analyze the function of XPO4 in Notch signaling. To this end we want to study its role in nuclear import/export of NICD in cell culture and in vitro.In zebrafish we want to determine the spatial and temporal expression pattern of Nup214 and XPO4 and want to study the phenotypes of specific Morpholinos against both mRNAs. To study the pathologic functions of Nup214 fusion proteins, we also want to establish a zebrafish model for T-ALL-causing fusions.The phosphatase MTMR14 seems to play a role in transport of Notch. Using our established assays for ER-export and transport in the secretory pathway we plan to elucidate the function of MTMR14 in Notch signaling.Finally we want to reassess the images from the siRNA screen by improved segmentation and classification approaches, to find additional phenotypes.All planned experiments make use of established methods like immuno-precipitation, GST-pull down, fluorescence microscopy, siRNA technology, in vitro assays, morpholino injections and in situ hybridization.