Adhesion dynamics in Drosophila border cell migration

描述(申请人提供)：细胞迁移是胚胎发育的一个吸引人的特征，不适当地调节细胞迁移会导致出生缺陷和肿瘤转移。我们已经开发了一个简单的模型系统，用于研究体内细胞运动的正向遗传学方法，即果蝇卵巢中被称为边缘细胞的毛囊细胞子集的迁移。我们已经确定，多种细胞外信号调节它们的运动。1)全球类固醇激素信号，蜕皮激素，通过蜕皮激素受体和转录辅助激活因子Taiman起作用；2)高度定位的细胞因子信号，激活JAK/STAT途径；以及3)几种生长因子，通过EGFR和PVR受体酪氨酸激酶发出信号，引导细胞到达目的地。此外，我们还研究了多种细胞骨架相关蛋白和细胞黏附分子，它们在边界细胞迁移中发挥作用。这项建议探索了在迁移的边缘细胞中细胞黏附被动态调节的机制，这是细胞运动的一个重要方面，对于任何细胞类型都不是很好地理解。我们提出了三个具体目标。第一个是研究控制E-钙粘蛋白运输和稳定性的机制，E-钙粘附素是一种亲水性细胞-细胞黏附分子，在边界细胞和它们迁移到的细胞中是必需的。为了测试E-钙粘素在边缘细胞中的翻转速度是否比在非迁移性毛囊细胞中更快，我们将使用一种先前描述的红色荧光蛋白的变体，该变体会随着时间的推移而改变颜色，并与E-钙粘蛋白融合。我们将研究EGFR和PVR信号是否通过磷酸化β-连环蛋白/Armadillo上的特定酪氨酸残基来破坏细胞黏附的稳定。我们还将确定内吞作用是否对迁移的边缘细胞中细胞表面E-钙粘附素的调节起重要作用。我们将测试果蝇Moesin是否对E-钙粘附素动力学有贡献。我们将研究肌球蛋白VI在边缘细胞中促进E-钙粘蛋白动力学的机制。在第二个特定目标中，我们建议通过研究RhoGAP93B的生化活性、表达模式、亚细胞定位、致死表型及其调控来详细研究RhoGAP93B促进边界细胞迁移的机制。最后，我们建议通过对突变的表型、Rho的上位性分析以及相互作用蛋白的鉴定和鉴定，来研究Rho在边界细胞中可能的下游靶标--Rh亲和素。

DESCRIPTION (provided by applicant): Cell migration is a fascinating feature of embryonic development, and improperly regulated cell migration contributes to birth defects and tumor metastasis. We have developed a simple model system for a forward genetic approach to the study of cell motility in vivo, the migration of a subset of follicle cells, known as border cells, in the Drosophila ovary. We have established that multiple extracellular signals regulate their movement. 1) a global steroid hormone signal, ecdysone, acting through the ecdysone receptor and a transcriptional coactivator called Taiman; 2) a highly localized cytokine signal, which activates the JAK/STAT pathway; and 3) several growth factors, which signal through the EGFR and PVR receptor tyrosine kinases to guide the cells to their destination. In addition we have studied a variety of cytoskeleton-associated proteins and cell adhesion molecules that function in border cell migration. This proposal explores the mechanisms by which cell adhesion is dynamically regulated in migrating border cells, an important aspect of cell motility that is not well understood for any cell type. We propose three specific aims. The first is to investigate the mechanisms that govern trafficking and stability of E-cadherin, a homophilic cell-cell adhesion molecule that is required in border cells and in the cells upon which they migrate. To test whether E-cadherin is turned over more rapidly in border cells than in non-migrating follicle cells, we will employ a previously characterized variant of the red fluorescent protein that changes color over time, fused to E-cadherin. We will investigate whether EGFR and PVR signaling destabilizes cell adhesion by phosphorylation of specific tyrosine residues on beta-catenin/Armadillo. We will also determine whether endocytosis is important for regulating cell surface E-cadherin in migrating border cells. We will test whether Drosophila moesin contributes to E-cadherin dynamics. And we will investigate the mechanisms by which Myosin VI contributes to E-cadherin dynamics in border cells. In the second specific aim we propose to investigate in detail the mechanisms by which RhoGAP93B contributes to border cell migration by studying its biochemical activity, its expression pattern, subcellular localization, lethal phenotype and its regulation. Finally we propose to study a putative downstream target of Rho in border cells, rhophilin by characterizing the mutant phenotype, epistasis analysis with Rho and by identifying and characterizing interacting proteins.