Understanding cell biology of Delta-Notch interactions

描述（由申请人提供）：Notch信号调节多种哺乳动物细胞命运和过程。Notch信号通路是通过直接的细胞间相互作用激活的，这种相互作用促进了信号细胞上的跨膜配体Delta与应答细胞上的Notch受体之间的结合。该信号通路的激活依赖于Delta诱导Notch蛋白水解，从而释放调节Notch靶基因表达的具有生物活性的Notch胞内结构域（NICD）。尽管NICD的产生和活性已被深入研究，但对于delta诱导的Notch信号传导过程中产生的Notch细胞外结构域（NECD）的作用和命运知之甚少。研究表明NECD的去除是Notch蛋白水解发生的必要条件；然而，NECD释放的机制尚不明确。有人提出NECD是通过ADAM脱落释放的，但没有报道直接证明配体诱导的NECD脱落。另外，也有人提出NECD可以通过内化到Delta细胞中，通过一个特征不明确的反式内吞作用过程被移除。然而，到目前为止，只有在果蝇细胞中获得了NECD转移到Delta细胞的证据。本提案的主要目的是开发一种系统，使我们能够利用哺乳动物细胞共培养试验对NECD向相互作用的Delta细胞的转移进行成像和表征。在这些研究中，我们将使用荧光团共轭抗体和标记形式的Delta和Notch结合使用共聚焦显微镜来定位Notch向共培养的Delta细胞的转移。考虑到Notch细胞外结构域在CADASIL患者的大脑中积累，了解NECD的命运与这种遗传性血管性痴呆有关。任何关于NECD在Notch信号传导过程中的命运和作用的信息，都有望对了解CADASIL中卒中和痴呆的分子基础具有生物学和健康相关的意义。与NINDS探索性/发展性资助（R21）计划一致，实验被提议开始研究内吞作用在Delta诱导的Notch信号传导中的作用，为长期的、特定项目的资助提供必要的数据（R01）。

DESCRIPTION (provided by applicant): Notch signaling regulates a wide variety of mammalian cell fates and processes. The Notch signaling pathway is activated through direct cell-cell interactions that facilitate binding between the transmembrane ligand Delta on a signaling cell and the Notch receptor on a responding cell. Activation of this signaling pathway relies on Delta to induce Notch proteolysis for release of the biologically active Notch intracellular domain (NICD) that regulates expression of Notch target genes. Although the generation and activity of NICD has been intensively studied, little is known regarding the role and fate of the Notch extracellular domain (NECD) generated during Delta-induced Notch signaling. Studies have indicated that NECD removal is necessary for Notch proteolysis to occur; however, the mechanism of NECD release is not well defined. It has been proposed that NECD is released by ADAM shedding, but direct demonstration of ligand-induced NECD shedding has not been reported. Alternatively, NECD has been proposed to be removed through internalization into Delta cells, through a poorly characterized process of trans-endocytosis. However, evidence for transfer of NECD into Delta cells has so far only been obtained for Drosophila cells. The major aim of this proposal is to develop a system that will allow us to image and characterize transfer of NECD to interacting Delta cells using a mammalian cell co-culture assay. For these studies, we will use a combination of fluorophore-conjugated antibodies and tagged forms of Delta and Notch to localize transfer of Notch to co-cultured Delta cells using confocal microscopy. Given that the Notch extracellular domain accumulates in brains of CADASIL patients, understanding the fate of NECD is relevant to this inherited vascular dementia. Any information gained regarding the fate and role of the NECD during Notch signaling is expected to have biological and health related significance for understanding the molecular basis of stroke and dementia in CADASIL. Consistent with the NINDS Exploratory/Developmental Grant (R21) Program, experiments are proposed to initiate studies on the role of endocytosis in Delta induced Notch signaling to provide requisite data for longer-term, program specific funding (R01).