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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞粘附和极性对于正常发育至关重要，而粘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，神经祖细胞监测并调整其增殖速率，以产生预定大小的器官。祖先是高度两极化的。它们包含不同的顶膜结构域（衬在脑室内）和基底外侧膜结构域。一个突出的细胞-细胞粘附结构称为顶端连接复合物分隔这两个膜室。为了确定神经前体细胞中顶端连接复合物的作用，我们使用条件性删除必需的粘附连接基因α E-连环蛋白来消融胚胎前体细胞中的这些结构。我们发现突变动物表现出严重的脑发育不良和增生，并在出生时有效地使脑细胞总数增加一倍。α E-连环蛋白-/-脑的微阵列分析揭示了音刺猬（SHH）通路的激活。 抑制体内发育胚胎中的SHH信号传导并不能阻止增殖，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α E-连环蛋白介导的增生中的因果作用。因此，我们发现，α E-连环蛋白连接细胞密度依赖性粘附连接与发育刺猬途径，这种连接可能提供一个负反馈回路控制发育中的哺乳动物大脑的大小。为了揭示连接α-连环蛋白与SHH通路的分子机制，我们建议确定α-连环蛋白相互作用的伙伴，并分析这些蛋白质在刺猬信号传导中的活性。为此目的，我们将开发两步纯化含α-连环蛋白的蛋白质复合物和质谱鉴定该复合物中存在的蛋白质的方案。

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.