Rab11-mediated polarization of the Drosophilia oocyte

Rab11 介导的果蝇卵母细胞极化

• 批准号: 6605114
• 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/6605114
• 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; 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。该提案的广泛目标是了解奥斯卡mRNA位于果蝇卵母细胞后极的机制。该定位取决于细胞外信号，该信号偏振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（Rab 11），这是Rab raS like GTPases的成员，可介导所有检查的生物体的内核循环途径中介导YEAST YEAST YEAST Yeast to Humans to Humans的内部核循环途径中的囊泡流量。尽管微管和肌动蛋白在Oskar mRNA定位中的作用已经很好地确定 - 它们提供了沿着沿着Oskar mRNA运输的曲目和锚定的基板 - Rab 11和囊泡运输的作用知之甚少，并且是拟议的工作的重点。在Rab11突变体中，Oskar mRNA积聚在附近而不是在卵母细胞的后极，未翻译。 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复合物和C）RAB 11通过修复与活性内吞作用相关的损伤来维持皮质肌动蛋白丝的组织。为了检验这些假设，我们将确定OSKAR mRNA定位途径的哪些方面或方面，即细胞外信号传导/MT组织，Oskar mRNA传输和锚定和/或组织肌动蛋白细胞骨架的组织，对RAB11基因活性的减少最敏感（AIM 1）。这将通过在Oskar mRNA定位途径中的RAB11和其他基因的活性进行成对的降低来实现。此外，将进行域交换和定点诱变，以确定RAB 11的哪些部分是Oskar mRNA定位的（AIM 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（AIM 3）。拟议的研究的结果应阐明RAB 11介导OSKAR mRNA定位的机制，并通过对膜运输的更多了解，可能揭示了对患病细胞进行治疗干预的新靶标。