Genetic and cellular analysis of germ cell transepithelial migration

描述(申请人提供)：了解细胞迁移的调节对发育生物学、免疫学和癌症生物学领域至关重要。然而，我们对体内迁移细胞所采用的策略缺乏基本的了解。这项提议的长期目标是利用果蝇生殖细胞迁移作为体内系统来研究细胞迁移和侵袭行为。Tre1是一种G蛋白偶联受体(GPCR)，是生殖细胞通过后中肠原基迁移所必需的。我们的假设是，Tre1通过极化生殖细胞和调节DE-钙粘蛋白的定位来调节生殖细胞的跨上皮迁移。为了验证这一假设，我们将首先在野生型和Trel背景下使用超微结构分析和免疫组织化学来确定Tre1对生殖细胞极化的程度。我们还将确定极化是否是受限的Tre1定位和/或激活的直接结果。其次，我们将测试Tre1是否负责DE-钙粘蛋白的定向运输、靶向回收和/或靶向降解，从而导致限制DE-钙粘蛋白的定位。最后，我们将对DE-钙粘蛋白进行结构-功能分析，以确定该蛋白负责定位的区域。我们将利用这个区域作为诱饵来确定负责DE-钙粘蛋白定位的Tre1下游效应子。通过这项工作，我们将确定Tre1调节生殖细胞极化和DE-钙粘蛋白定位的机制，使我们能够表征GPCRs在侵袭性迁移中的新角色。生殖细胞是一种特殊的细胞群体，负责产生下一代细胞，因此它们具有特殊的特性，例如，它们能够在胚胎早期通过组织从一个部位迁移到另一个部位。在癌症期间，来自原发肿瘤的一些细胞能够从原始肿瘤中迁移出来，并在新的部位形成更多的肿瘤。通过研究生殖细胞中发生的允许它们迁移的过程，我们将能够揭示癌细胞的突变如何导致它们的运动行为。

DESCRIPTION (provided by applicant): Understanding the regulation of cell migration is essential to the fields of developmental biology, immunology and cancer biology. However we lack a fundamental understanding1 of the strategies employed by migrating cells in vivo. The long-term goal of this proposal is to use Drosophila germ cell migration as an in vivo system to study cell migration and invasive behavior. Tre1 is a G-protein coupled receptor (GPCR) that is required specifically for germ cells to migrate through the posterior midgut primordium. Our hypothesis is that Tre1 regulates germ cell transepithelial migration by polarizing germ cells and regulating the localization of DE-cadherin. To test this hypothesis we will first determine the extent of germ cell polarization by Tre1 using ultra structural analysis and immunohistochemistry in wild type and trel backgrounds. We will also determine if polarization is a direct consequence of restricted Tre1 localization and/or activation. Secondly, we will test whether Tre1 is responsible for directed transport, targeted recycling and/or targeted degradation of DE-cadherin, leading to restricted DE-cadherin localization. Lastly, we will perform structure-function analysis of DE-cadherin to identify regions of the protein responsible for localization. We will use this region as bait to identify downstream effectors of Tre1 that are responsible for DE-cadherin localization. Through this work we will determine the mechanism by which Tre1 regulates germ cell polarization and DE-cadherin localization, allowing us to characterize a novel role for GPCRs in invasive migration. Germ cells are a special population of cells that are responsible for producing the next generation and as such they have special properties, for instance they are able to migrate through the tissue from one site to another early in the embryo. During cancer, some cells from the primary tumor gain the ability to migrate away from the original tumor and form additional tumors at new sites. By studying the processes occurring in germ cells that allow them to migrate we will be able to uncover how mutations in cancer cells lead to their motile behavior.