ROLE OF ALPHAE-CATENIN IN REGULATION OF BRAIN SIZE AND BRAIN CANCER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞黏附和极性是正常发育的关键，而黏附和极性的丧失是癌症的标志。在哺乳动物大脑发育过程中，神经前体细胞监测并调整其增殖速度，以产生预定大小的器官。祖先是高度两极分化的。它们包含不同的顶膜域和基侧膜域，顶膜域排列在脑室内。一种显著的细胞-细胞黏附结构称为顶端连接复合体，将这两个膜室隔开。为了确定顶端连接复合体在神经前体细胞中的作用，我们通过有条件地删除必要的黏附连接基因αE-catenin来去除胚胎前体细胞中的这些结构。我们发现，突变的动物表现出严重的脑部发育不良和增殖，并在出生时有效地将脑细胞总数翻了一番。对αE-连环蛋白-/-脑的微阵列分析显示，sonic hedgehog(SHH)通路激活。在体内发育的胚胎中抑制SHH信号并没有阻止增殖，但消除了野生型和突变型细胞之间的增殖差异，揭示了SHH激活在αE-catenin介导的增殖中的因果作用。因此，我们发现αE-catenin将依赖细胞密度的黏附连接与发育中的刺猬途径联系在一起，这种联系可能提供了一个控制哺乳动物发育中大脑大小的负反馈环。为了揭示α-连环蛋白与SHH途径之间的分子机制，我们建议确定α-连环蛋白相互作用的伙伴，并分析这些蛋白在Hedgehog信号中的活性。为此，我们将制定两步纯化含有α-连环蛋白的蛋白质复合体的方案，并对存在于该复合体中的蛋白质进行质谱学鉴定。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell adhesion and polarity are critical for normal development and loss of adhesion and polarity are the hallmarks of cancer. During mammalian brain development neural progenitor cells monitor and adjust their rates of proliferation to produce organ of predetermined size. Progenitors are highly polarized. They contain distinct apical membrane domains, which are lining the brain ventricles, and basolateral membrane domains. A prominent cell-cell adhesion structure called apical junctional complex separates these two membrane compartments. To determine the role of apical junctional complexes in neural progenitor cells we ablated these structures in embryonic progenitors using conditional deletion of essential adherens junction gene, alphaE-catenin. We found that mutant animals show severe brain dysplasia and hyperplasia and efficiently double total brain cell numbers at the time of birth. Microarray analysis of the alphaE-catenin-/- brains revealed activation of the sonic hedgehog (SHH) pathway. Inhibition of SHH signaling in developing embryos in vivo did not stop proliferation but eliminated the differences in proliferation between the wildtype and mutant cells revealing causal role of SHH activation in alphaE-catenin-mediated hyperplasia. Thus, we found that alphaE-catenin connects cell-density-dependent adherens junctions with the developmental hedgehog pathway and that this connection may provide a negative feedback loop controlling the size of developing mammalian brain. To reveal the molecular mechanisms connecting alpha-catenin with the SHH pathway we propose to determine alpha-catenin interacting partners and analyze the activity of these proteins in the hedgehog signaling. For this purpose we will develop the protocol for two-step purification of alpha-catenin-containing protein complexes and Mass Spec identification of proteins present in this complex.