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，其中前体细胞分裂产生两个不同命运的兄弟细胞，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子Numb的相反活性。在前体分裂期间，Numb仅分离到一个兄弟细胞中，在那里它阻断Notch信号传导以防止采用Notch依赖性命运。另一个兄弟姐妹中Numb的缺失允许Notch信号传导，从而采用Notch依赖性命运。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，这种模式也有一些重要的警告。例如，两种同胞细胞在细胞膜处表现出相等水平的Notch，并且所有已知的内吞基序缺失的Numb的截短形式在Notch介导的不对称分裂期间是功能性的。我们实验室对Sanpodo的研究表明了一种不同的模型，Sanpodo是一种仅在不对称分裂期间Notch信号传导所需的新型跨膜蛋白。Sanpodo定位于其命运取决于Notch活性的同胞细胞的细胞膜，而在另一个细胞中，Numb阻止Sanpodo定位于细胞膜。这些观察结果导致了Sanpodo作用于细胞膜以促进Notch信号传导的模型，并且Numb通过使Sanpodo远离细胞膜来阻断Notch活性。然而，Sanpodo促进Notch活性和Numb调节Sanpodo定位的分子机制仍然未知。该建议旨在阐明三宝岛功能和调节的分子基础。具体而言，我们建议（一）通过结构/功能的研究，（二）确定和表征因素，通过互补的生物化学和遗传学方法与sanpodo的遗传或物理相互作用的sanpodo的功能域和（iii）搜索计算脊椎动物sanpodo基因。Notch活性的缺陷与越来越多的疾病有关，包括多种类型的脑癌。虽然尚未在脊椎动物中鉴定出Sanpodo的同源物，但基于（i）昆虫中Sanpodo的氨基酸序列之间的高度分歧和（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的一级序列同源性的功能性Sanpodo同源物。因此，确定脊椎动物sanpodo基因和阐明Sanpodo功能和调节的分子基础将提供关键的洞察不对称分裂的分子控制。这样的洞察力应该有助于我们了解疾病的病因，其中这一过程是失调，并设计新的方法来治疗这些疾病。

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.