Endocytic control of Notch-mediated cell fate decisions in neurogenesis

 描述（由申请人提供）：本提案的目标是确定不对称细胞分裂期间关键Notch途径组分的膜运输如何控制神经系统细胞中Notch介导的细胞命运转换。Notch功能在多种正常干细胞和祖细胞中是必需的，包括果蝇和脊椎动物中的神经干细胞、血细胞前体和肠道祖细胞。干细胞中异常的Notch调节导致发育事件中的严重缺陷，并导致人类疾病，如CADASIL综合征和癌症，如T细胞急性淋巴细胞白血病。为了详细了解控制Notch功能的细胞生物学机制，该提案将重点放在果蝇成体外周神经系统的感觉器官祖细胞（SOP）上，其中细胞命运决定取决于Notch活性的激活或抑制。这项工作是 基于过去五年的发现，确定了Notch信号在感觉器官细胞中的关键调节因子Sanpodo的功能作用。在上一个资助周期中，研究表明，sanpodo促进Notch活性，以在成人外周神经系统不对称细胞分裂后赋予正确的细胞命运。通过生物化学研究，体内成像和救援实验，已确定Sanpodo的功能域介导两个关键功能。首先，Sanpodo与g-分泌酶复合物结合，以促进具有高Notch活性的细胞中的Notch活化。其次，Sanpodo控制Notch在细胞质膜上的再循环，具有低Notch活性，在这种情况下充当Notch信号传导的阻遏物。该提案旨在确定这些拮抗功能是如何在囊泡贩运水平进行调节的，并定义保守基序的作用，这些保守基序决定了Sanpodo与Notch监管机构（如Numb）的相互作用。将评估保守的囊泡运输调节剂在控制不对称分裂的神经祖细胞中的Notch水平方面的作用，使用将完整动物中祖细胞行为的分子建模、生物化学分析和活细胞成像相结合的独特能力，以泰特组成性水平的内吞作用在神经祖细胞中建立稳态Notch水平的假设。 上皮细胞，而Sanpodo与Numb协同作用，以在不对称潜水神经祖细胞中从质膜中耗尽Notch。因此，这项建议将提供一个更完整的了解进化保守的机制，在这种情况下，Notch贩运。

 DESCRIPTION (provided by applicant): The goal of this proposal is to determine how membrane trafficking of critical Notch pathway components during asymmetric cell division controls a Notch-mediated cell fate switch in cells of the nervous system. Notch function is required in a variety of normal stem and progenitor cells, including neural stem cells, blood cell precursors, and gut progenitors in both Drosophila and vertebrates. Aberrant Notch regulation in stem cells leads to severe defects in developmental events and contributes to human diseases, such as CADASIL syndrome, and cancers, such T-cell acute lymphoblastic leukemia. To understand in detail the cell biological mechanisms that control Notch function, the proposal focuses on the sensory organ progenitor (SOP) cells of the Drosophila adult peripheral nervous system, where cell fate decisions depend on activation or inhibition of Notch activity. The work is based on the findings over the last five years that establish the functional role of a key regulato of Notch signaling in sensory organ cells, Sanpodo. In the previous funding cycle, studies demonstrated that sanpodo promotes Notch activity to confer correct cell fates after asymmetric cell division in the adult peripheral nervous system. Through biochemical studies, in vivo imaging, and rescue experiments, functional domains of Sanpodo have been identified that mediate two critical functions. First, Sanpodo associates with the g-secretase complex to promote Notch activation in the cell with high Notch activity. Second, Sanpodo controls Notch recycling at the plasma membrane in the cell with low Notch activity, acting as a repressor of Notch signaling in this context. This proposal aims to determine how these antagonistic functions are regulated at the level of vesicle trafficking and to define the role of conserved motifs that determine Sanpodo's interaction with Notch regulators such as Numb. The role of conserved vesicle trafficking regulators on controlling Notch levels in asymmetrically dividing neural progenitors will be assessed, using the unique ability to combine molecular modeling, biochemical analysis, and live cell imaging of progenitor cell behavior in the intact animal, to tet the hypothesis that a constitutive level of endocytosis establishes steady state levels of Notch in epithelial cells, whereas Sanpodo acts synergistically with Numb to deplete Notch from the plasma membrane in asymmetrically diving neural progenitors. This proposal will therefore provide a more complete understanding of the evolutionarily conserved mechanisms governing Notch trafficking in this context.