Molecular and genetic analysis of sanpodo

描述（由申请人提供）：神经系统的细胞复杂性使其有别于其他组织。不对称细胞分裂，即一个前体细胞分裂产生两个命运不同的兄弟细胞，是神经系统中细胞多样性产生的核心。果蝇和脊椎动物神经系统中的大多数不对称分裂依赖于Notch信号通路和细胞质决定因子Numb的相反活动。在前体分裂过程中，Numb只在一个兄弟细胞中分离，在那里它阻断Notch信号以防止Notch依赖性命运的采用。另一个兄弟姐妹中Numb的缺失允许Notch信号，因此，采用Notch依赖的命运。目前的模型表明，Numb通过促进Notch受体的内吞作用来阻断Notch活性。然而，这种模式有一些重要的警告。例如，两个兄弟细胞在细胞膜上都表现出相同水平的Notch，并且在Notch介导的不对称分裂中，所有已知的内吞基序中缺失的Numb的截断形式是有效的。Sanpodo是一种新型跨膜蛋白，仅在不对称分裂时才需要Notch信号传导，我们实验室的研究表明了一种不同的模型。Sanpodo定位在兄弟细胞的细胞膜上，而兄弟细胞的命运取决于Notch活性，而在另一个细胞中，Numb阻止Sanpodo定位到细胞膜上。这些观察结果导致了Sanpodo在细胞膜上促进Notch信号传导的模型，而Numb通过使Sanpodo远离细胞膜来阻止Notch活性。然而，Sanpodo促进Notch活性和Numb调控Sanpodo定位的分子机制尚不清楚。本提案旨在阐明三坡多功能和调控的分子基础。具体来说，我们建议(i)通过结构/功能研究确定三足病的功能域，（ii）通过互补的生化和遗传方法确定和表征与三足病在遗传或物理上相互作用的因素，以及（iii）通过计算搜索脊椎动物三足病基因。Notch活性缺陷与越来越多的疾病有关，包括多种类型的脑癌。尽管在脊椎动物中尚未发现Sanpodo同源基因，但基于(i)昆虫中Sanpodo氨基酸序列的高度分化以及（ii） Notch/numb分子机制作为调节不对称分裂的基本机制的保守性，我们假设存在具有有限初级序列同源性的功能性Sanpodo同源基因。因此，鉴定脊椎动物三坡多基因并阐明三坡多功能和调控的分子基础将为非对称分裂的分子控制提供关键的见解。这种见解应该有助于我们理解这种过程被解除调控的疾病的病因学，并设计治疗这些疾病的新方法。

DESCRIPTION (provided by applicant): The cellular complexity of the nervous system sets it apart from other tissues. Asymmetric cell divisions, in which a precursor cell divides to produce two sibling cells of different fates, are central to the generation of cell diversity in nervous systems. Most asymmetric divisions in Drosophila and vertebrate nervous systems depend on the opposing activities of the Notch signaling pathway and the cytoplasmic determinant Numb. During precursor divisions Numb segregates exclusively into one sibling cell where it blocks Notch signaling to prevent adoption of the Notch-dependent fate. The absence of Numb in the other sibling allows Notch signaling, and thus, adoption of the Notch-dependent fate. Present models suggest that Numb blocks Notch activity by promoting endocytosis of the Notch receptor. However, there are significant caveats to this model. For example, both sibling cells exhibit equivalent levels of Notch at the cell membrane and a truncated form of Numb deleted for all known endocytic motifs is functional during Notch-mediated asymmetric divisions. Work from our lab on Sanpodo, a novel transmembrane protein required for Notch signaling only during asymmetric divisions, suggests a different model. Sanpodo localizes to the cell membrane of the sibling cell whose fate depends on Notch activity, while in the other cell Numb blocks Sanpodo from localizing to the cell membrane. These observations led to the model that Sanpodo acts at the cell membrane to promote Notch signaling, and that Numb blocks Notch activity by keeping Sanpodo off of the cell membrane. However, the molecular mechanisms by which Sanpodo promotes Notch activity arid Numb regulates Sanpodo localization remain unknown. This proposal seeks to elucidate the molecular basis of Sanpodo function and regulation. Specifically, we propose to (i) identify the functional domains of Sanpodo via structure/function studies, (ii) identify and characterize factors that interact genetically or physically with sanpodo via complementary biochemical and genetic approaches and (iii) search computationally for vertebrate sanpodo genes. Defects in Notch activity are being implicated in a growing number of diseases, including multiple types of brain cancer. And while no homolog of Sanpodo has yet been identified in vertebrates, based on (i) the high degree of divergence between the amino acid sequences of Sanpodo in insects and (ii) the conservation of the Notch/numb molecular machinery as a fundamental mechanism regulating asymmetric divisions, we hypothesize the existence of a functional Sanpodo homolog with limited primary sequence homology. Thus, identifying vertebrate sanpodo genes and elucidating the molecular basis of Sanpodo function and regulation will provide key insight into the molecular control of asymmetric divisions. Such insight should help us understand the etiology of diseases in which this process is de-regulated and design new methods to treat these diseases.