Molecular and genetic analysis of sanpodo

描述(申请人提供)：神经系统的细胞复杂性使其有别于其他组织。不对称细胞分裂是指前体细胞分裂产生两个命运不同的兄弟细胞，这是神经系统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簇Numb的相反活性。在前体分裂期间，Numb只分离成一个兄弟细胞，在那里它阻止Notch信号转导，以防止采用Notch依赖的命运。在另一个兄弟姐妹中没有Numb，允许Notch信令，从而采用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的活性。然而，对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表现出同等水平的Notch，并且在Notch介导的不对称分裂过程中，所有已知内吞基序删除的截短形式的Numb是起作用的。我们实验室对Sanpodo的研究提出了一个不同的模型，Sanpodo是一种仅在不对称分裂期间才需要Notch信号传递的新型跨膜蛋白。Sanpodo定位于兄弟细胞的细胞膜，其命运取决于Notch活性，而在另一个细胞中，Numb阻止Sanpodo定位于细胞膜。这些观察导致了Sanpodo作用于细胞膜促进Notch信号转导的模型，该数字通过使Sanpodo远离细胞膜来阻止Notch的活性。然而，Sanpodo促进Notch活性和Numb调节Sanpodo定位的分子机制尚不清楚。这项建议旨在阐明Sanpodo功能和调控的分子基础。具体地说，我们建议(I)通过结构/功能研究确定Sanpodo的功能结构域，(Ii)通过互补的生化和遗传学方法识别和表征与Sanpodo在遗传或物理上相互作用的因子，以及(Iii)通过计算搜索脊椎动物Sanpodo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。虽然目前还没有在脊椎动物中发现Sanpodo的同源物，但基于(I)Sanpodo的氨基酸序列在昆虫中的高度分化和(Ii)作为调节不对称分裂的基本机制的Notch/Numb分子机制的保守，我们假设存在一个初级序列同源性有限的功能性Sanpodo同源物。因此，识别脊椎动物Sanpodo基因并阐明Sanpodo功能和调控的分子基础将为理解不对称分裂的分子控制提供关键的见解。这种洞察力应该有助于我们理解这一过程被解除调控的疾病的病因，并设计出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.