Rab11-mediated polarization of the Drosophilia oocyte

Rab11 介导的果蝇卵母细胞极化

• 批准号: 6745551
• 负责人: ROBERT S COHEN
• 金额: $ 23.28万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745551
• 关键词: Drosophilidae; SDS polyacrylamide gel electrophoresis; actins; binding proteins; biological signal transduction; cellular polarity; cytoskeleton; egg /ovum; endocytosis; gene interaction; guanine nucleotide binding protein; immunoprecipitation; laboratory rat; matrix assisted laser desorption ionization; messenger RNA; microtubules; nucleic acid quantitation /detection; protein localization; protein protein interaction; protein transport; site directed mutagenesis; transfection; yeast two hybrid system

DESCRIPTION (provided by applicant): Understanding the mechanisms that generate and maintain cell polarity is a fundamental challenge of cell biology. The broad goal of this proposal is to understand the mechanism by which oskar mRNA is localized to the posterior pole of the Drosophila oocyte. This localization is dependent on an extracellular signal that polarizes the oocyte's microtubule cytoskeleton, an intact actin cytoskeleton, and Rab 11, a member of the Rab family of Ras-like GTPases, which mediate vesicle traffic in the endocytic recycling pathways of all examined organisms from yeast to human. While the roles of microtubules and actin filaments in oskar mRNA localization are well established--they provide the tracks along which oskar mRNA is transported and a substrate for anchoring--the role of Rab 11 and vesicle trafficking is poorly understood and is the focus of the proposed work. In rab11 mutants, oskar mRNA accumulates near, rather than at, the oocyte's posterior pole and is not translated. The following hypotheses could explain the requirement for rab 11 in oskar mRNA localization: a) Rab 11 protein maintains extracellular signaling (and thus MT polarization) by recycling key receptor molecules back to the oocyte's plasma membrane, b) Rab 11 mediates short-range transport of oskar mRNA from MTs to actin filaments by recruiting actin-based motor proteins to oskar mRNA transport complexes, and c) Rab 11 maintains the organization of cortical actin filaments by repairing damage associated with active endocytosis. To test these hypotheses, we will determine which aspect or aspects of the oskar mRNA localization pathway, i.e., extracellular signaling/MT organization, oskar mRNA transport and anchoring, and/or organization of the actin cytoskeleton, are most sensitive to a reduction in rab11 gene activity (Aim 1). This will be accomplished through pair-wise reductions in the activity of rab11 and other genes in the oskar mRNA localization pathway. In addition, domain swapping and site-directed mutagenesis will be carried out to determine which parts of Rab 11 are required for oskar mRNA localization (Aim 2). Finally, genetic and biochemical strategies will be used to identify and characterize proteins that bind to Rab 11 (Aim 3). The results of the proposed studies should elucidate the mechanism by which Rab 11 mediates oskar mRNA localization and, through an increased understanding of membrane trafficking, may reveal new targets for therapeutic intervention in diseased cells.

描述（由申请人提供）：了解产生和维持细胞极性的机制是细胞生物学的基本挑战。该提案的主要目标是了解 oskar mRNA 定位于果蝇卵母细胞后极的机制。这种定位依赖于细胞外信号，该信号使卵母细胞的微管细胞骨架（完整的肌动蛋白细胞骨架）和 Rab 11 极化，Rab 11 是 Ras 样 GTPases 的 Rab 家族的成员，它介导来自所有受检生物体的内吞循环途径中的囊泡运输。酵母对人类。虽然微管和肌动蛋白丝在 oskar mRNA 定位中的作用已得到充分证实——它们提供了 oskar mRNA 运输的轨道和锚定的底物——但 Rab 11 和囊泡运输的作用却知之甚少，并且是研究的焦点。拟议的工作。在 rab11 突变体中，oskar mRNA 聚集在卵母细胞后极附近，而不是在卵母细胞后极处，并且不被翻译。以下假设可以解释 oskar mRNA 定位中对 rab 11 的需求：a) Rab 11 蛋白通过将关键受体分子循环回卵母细胞质膜来维持细胞外信号传导（从而维持 MT 极化），b) Rab 11 介导短程运输通过将基于肌动蛋白的运动蛋白招募到 oskar mRNA 转运复合物，将 oskar mRNA 从 MT 转移到肌动蛋白丝，并且 c) Rab 11 维持通过修复与主动内吞作用相关的损伤来组织皮质肌动蛋白丝。为了检验这些假设，我们将确定 oskar mRNA 定位途径的哪些方面，即细胞外信号/MT 组织、oskar mRNA 运输和锚定、和/或肌动蛋白细胞骨架的组织，对 rab11 的减少最敏感基因活性（目标 1）。这将通过成对减少 rab11 和 oskar mRNA 定位途径中其他基因的活性来实现。此外，将进行结构域交换和定点诱变以确定 oskar mRNA 定位需要 Rab 11 的哪些部分（目标 2）。最后，将使用遗传和生化策略来识别和表征与 Rab 11 结合的蛋白质（目标 3）。拟议研究的结果应阐明 Rab 11 介导 oskar mRNA 定位的机制，并通过加深对膜运输的了解，可能揭示病变细胞治疗干预的新靶点。