OOCYTE DEVELOPMENT IN DROSOPHILA

这项工作的长期目标是了解细胞骨架介导的细胞反应是如何在发育过程中调节和进行的。实验系统是果蝇卵子发生，在此过程中，营养细胞喂养生长的卵母细胞母体成分，包括mRNA，蛋白质和细胞器。从保育细胞到卵母细胞的细胞质运输需要精确定时的肌动蛋白细胞骨架重排和保育细胞中程序性细胞死亡（凋亡）的执行。在卵子发生后期，致密的细胞质肌动蛋白束在保育细胞中形成，就在保育细胞凋亡开始时。编码肌动蛋白结合蛋白的几个基因将使用遗传学，生物化学和细胞生物学的组合进行研究，以阐明它们在卵子发生过程中的功能。此外，调节蛋白参与肌动蛋白组织将进行调查。这项工作是相关的了解动态肌动蛋白细胞骨架的运动，细胞形状或亚细胞定位的分子在正常细胞和疾病进展，如转移的贡献。该计划的具体目标是：（1）研究Profilin的体内功能，Profilin是一种小的肌动蛋白单体结合蛋白，对调节肌动蛋白丝聚合速率很重要;（2）对Profilin与Arp 2/3复合物和FH结构域蛋白的相互作用进行遗传和生物化学分析;（3）对在细胞质内将肌动蛋白丝交联成高度组织化的纤维束的蛋白质进行功能分析;（4）研究甾体激素蜕皮激素对滋养细胞凋亡的调控作用。在果蝇的保育细胞中，Profilin是卵子发生第10阶段形成的细胞质肌动蛋白丝聚合所必需的。将使用生殖系表达载体和P元件介导的转化在保育细胞中制备和测试编码前纤维蛋白原和前纤维蛋白原结合蛋白的基因中的特异性突变。一个类似的实验策略将被用来测试果蝇Fascin的功能，肌动蛋白丝捆绑蛋白也需要在第10阶段。Fascin和一种名为Quail的Villin样肌动蛋白捆绑蛋白可能的协调调节将通过检查它们的磷酸化和结合Armadillo（β-连环蛋白）的能力来研究。Profilin、Quail和Fascin蛋白在经历凋亡的细胞中起作用。将通过使用蜕皮激素反应途径中的突变体和克隆一个新的称为midway的基因来研究类固醇激素蜕皮激素在信号传导护理细胞凋亡中的作用。

The long-term goal of this work is to understand how cellular responses mediated by the cytoskeleton are regulated and carried out during development. The experimental system is Drosophila oogenesis, during which nurse cells feed growing oocytes maternal components including mRNA, protein and organelles. The transport of cytoplasm from nurse cell to the oocyte requires precisely timed rearrangements of the actin cytoskeleton and the execution of programmed cell death (apoptosis) in nurse cells. Late in oogenesis, dense cytoplasmic actin bundles form in nurse cells, just at the onset of nurse cell apoptosis. Several genes encoding actin binding proteins will be studied using a combination of genetics, biochemistry and cell biology to elucidate their function during oogenesis. In addition, regulatory proteins involved in actin organization will be investigated. This work is relevant to understanding the contribution of a dynamic actin cytoskeleton to motility, cell shape or subcellular localization of molecules in both normal cells and during disease progression such as metastasis. The specific aims of the proposal are: (1) to study the function in vivo of Profilin, a small actin monomer binding protein important for modulating the rate of actin filament polymerization; (2) to perform genetic and biochemical analyses of Profilin interactions with the Arp2/3 complex and FH domain proteins; (3) to carry out functional analyses on proteins that crosslink actin filaments into highly organized bundles within cytoplasm; and (4) to study the role of the steroid hormone ecdysone in regulating nurse cell apoptosis. In Drosophila nurse cells, Profilin is required for the polymerization of the cytoplasmic actin filaments that form during stage 10 of oogenesis. Specific mutations in the genes encoding Profilin and Profilin binding proteins will be made and tested in nurse cells using a germline expression vector and P element- mediated transformation. A similar experimental strategy will be used to test the function of Drosophila Fascin, an actin filament bundling protein also required at stage 10. The possible coordinate regulation of both Fascin and a Villin-like actin bundling protein called Quail will be studied by examining their phosphorylation and ability to bind Armadillo (beta-catenin). Profilin, Quail and Fascin proteins function in cells that are undergoing apoptosis. The role of the steroid hormone ecdysone in signaling nurse cell apoptosis will be studied by using mutants in the ecdysone response pathway, and cloning a new gene called midway.