Characterization of genes required for border cell migration in Drosophila

描述（由申请人提供）：在生物体的正常发育过程中，细胞从其原始位置迁移以形成特定器官，填充干细胞龛并形成复杂的神经系统。在浸润性癌症情况下也观察到异常细胞迁移。为了揭示细胞迁移的潜在机制，模型系统如果蝇卵巢在过去一直很重要。然而，许多关于细胞迁移的调节问题仍然没有得到解决，这一建议解决了一个新的因素，佝偻病，我们已经发现，在果蝇卵室的正常边缘细胞迁移是重要的作用。佝偻病编码G蛋白偶联受体，我们已经观察到，边缘细胞突变的rk往往无法达到其目的地在前端的卵母细胞。该建议旨在研究迁移缺陷的潜在分子原因，并了解Rk促进细胞迁移的途径。在目标1中，我将确定突变细胞中改变的细胞结构。这将涉及检测突变细胞中重要的细胞粘附调节因子（如E-钙粘蛋白和β-连环蛋白）和极性标记物（如PAR-1和PAR-3）的水平和分布。我还将测试Rk是否影响JAK/STAT信号传导。在目标2中，我将使用实时成像方法来精确确定突变细胞中细胞迁移的哪些方面发生了变化。我将重点介绍边缘细胞从上皮形态到迁移形态的转变，细胞从上皮上的分离，迁移过程中细胞的速度和实际运动（翻滚与奔跑）以及迁移细胞中富含肌动蛋白的突起的延伸。我还将检测PKA和cAMP是否在边缘细胞中作用于Rk的下游。在目标3中，我将测试Rk对迁移的影响是否也可以在哺乳动物组织培养中观察到。为此，我将使用一个已建立的乳腺癌衍生细胞系统，该系统可以通过RNAi方法进行操作。先前描述的划痕试验将使我能够确定是否与Rk敲低水平的哺乳动物细胞表现出相同的缺陷，在果蝇的突变边缘细胞迁移。因此，拟议的工作将定义果蝇细胞迁移的新输入，并为这一与人类发育和疾病相关的重要发育机制提供新的见解。

DESCRIPTION (provided by applicant): During normal development of an organism, cells migrate from their original location to form specific organs, to populate stem cell niches and to form the complex nervous system. Abnormal cell migration is also observed in invasive cancer situations. In order to reveal the underlying mechanisms of cell migration, model systems such as the Drosophila ovary have been important in the past. Nevertheless, many questions about the regulation of cell migration are still unresolved, and this proposal addresses the role of a novel factor, Rickets, that we have found to be important for normal border cell migration in the Drosophila egg chamber. Rickets encodes a G-protein coupled receptor, and we have observed that border cells mutant for rk often fail to reach their destination at the anterior end of the oocyte. This proposal aims to investigate the underlying molecular causes of the migration defects and to understand the pathway in which Rk acts to promote cell migration. In Aim 1, I will identify the cellular architecture that is altered in the mutant cells. This will involve testng the level and distribution of important cell adhesion regulators, such as E-cadherin and Beta-Catenin, and polarity markers such as PAR-1 and PAR-3 in the mutant cells. I will also test whether Rk affects JAK/STAT signaling. In Aim 2, I will perform live imaging approaches to determine precisely what aspect of cell migration is altered in the mutant cells. I will focus on te transition of the border cells from epithelial to migratory morphology, on the detachment of the cells from the epithelium, on the speed and actual motion of the cells during migration (tumbling versus running) and on the extension of actin rich protrusions from the migrating cells. I will als test whether PKA and cAMP are acting downstream of Rk in the border cells. In Aim 3, I will test whether the effects or Rk on migration can also be observed in mammalian tissue culture. For this I will use an established system of breast cancer-derived cells that can be manipulated by RNAi approaches. A previously described scratch assay will allow me to determine whether mammalian cells with knockdown levels of Rk exhibit the same defects in migration as the mutant border cells in Drosophila. The proposed work will therefore define a new input into cell migration in Drosophila, and provide novel insights into this important developmental mechanism that are relevant to human development and disease.