Control of tissue growth and architecture by Drosophila Tsg101

描述(申请人提供)：顶基底部极性因子的表达改变与脊椎动物的癌症和模型无脊椎动物的组织过度生长有关，但这些因素影响生长调节途径的机制尚未很好地确定。我们之前已经确定果蝇黑腹果蝇基因tsg101(肿瘤易感基因-101)是一个同时维持组织极性和抑制幼虫想象盘肿瘤样过度生长所必需的因子，幼虫想象盘是产生大多数成体结构的简单上皮性器官。我们已经证明，tsg101的丢失阻止了关键的顶膜决定因素Crumbs的内体周转，并且这种多余的Crumbs蛋白积累在顶膜，异位扩散到基侧膜上，并在晚期的内体中积累。当Crumbs形成一个与PAR/aPKC和Scribble/DLG极性复合体在物理和功能上相互作用的顶端膜相关复合体时，Crumbs调控中的这些缺陷为tsg101丢失对上皮极性和结构的影响提供了一个容易的解释。值得注意的是，过度表达的Crumbs也推动了实质上的假想盘过度生长，这表明这种极性因素在tsg101细胞中具有第二个、更直接的致癌作用。在已发表的工作中，我们发现Crumbs的细胞质尾部包含一个先前未知的生长调节基序，通过它与扩展蛋白相互作用并调节扩展蛋白，扩展蛋白是保守的Hippo/Mst2肿瘤抑制通路的关键组成部分。这些数据表明，Crumbs是一种多功能蛋白质，能够将连接极性信号与已建立的Hippo/Mst2生长调控途径整合起来，而那些扰乱Crumbs运输和定位的损伤可能对这一途径的活性有非常直接的影响。我们在这项建议中的目标是测试这个新发现的Crumbs-Hpo/Mst2链接在tsg101突变肿瘤过度生长中的作用，并从我们已经确定为CRumbs驱动的椎间盘过度生长的显性修饰因子的一小部分突变中识别Crumbs-Hpo/Mst2途径的新成分。特别是，我们将重点分析taiman基因，它编码人类乳腺癌-1(AIB-1)癌基因的苍蝇同源基因，我们认为它是Crumbs-Hpo/Mst2途径的转录辅助激活因子。我们将在果蝇器官和培养细胞中使用标准的遗传和分子技术来开展拟议的研究。

DESCRIPTION (provided by applicant): Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in model invertebrates, yet mechanisms by which these factors affect growth regulatory pathways are not well defined. We have previously identified the Drosophila melanogaster gene tsg101 (tumor susceptibility gene-101) as a factor that is required to simultaneously maintain tissue polarity and suppress tumor-like overgrowth of the larval imaginal discs, which are simple epithelial organs that give rise to most adult structures. We have shown that loss of tsg101 blocks endolysosomal turnover of the key apical membrane determinant Crumbs, and that this excess Crumbs protein accumulates at the apical membrane, spreads ectopically onto the basolateral membrane, and accumulates in late endosomes. As Crumbs nucleates an apical membrane- associated complex that interacts physically and functionally with the Par/aPKC and Scribble/Dlg polarity complexes, these defects in Crumbs regulation provide a ready explanation of the effect of tsg101 loss on epithelial polarity and architecture. Notably, over expressed Crumbs also drives substantial imaginal disc overgrowth, suggesting that this polarity factor has a second, more direct oncogenic role in tsg101 cells. In published work, we have found that the cytoplasmic tail of Crumbs contains a previously unrecognized growth-regulatory motif through which it interacts with and regulates the expanded protein, which is a key component of the conserved Hippo/Mst2 tumor suppressor pathway. These data show that Crumbs is a multi-functional protein capable of integrating junctional polarity signals with the well-established Hippo/Mst2 growth-regulatory pathway, and those lesions that disrupt Crumbs trafficking and localization may have very direct effects on the activity of this pathway. Our objectives in this proposal are to test the role of this newly discovered Crumbs-Hpo/Mst2 link in the excessive growth of tsg101 mutant tumors, and to identify novel components of the Crumbs-Hpo/Mst2 pathway from among a small collection of mutations we have identified as dominant-modifiers of Crumbs driven disc overgrowth. In particular, we will focus our analysis on the taiman gene, which encodes the fly homolog of the human Amplified in Breast Cancer-1 (AIB-1) oncogene, and that we believe acts as a transcriptional coactivator for the Crumbs-Hpo/Mst2 pathway. We will use standard genetic and molecular techniques in Drosophila organs and cultured cells to carry out the proposed studies.