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执行的。实验系统是果蝇卵子发生，在此过程中，哺育细胞喂养生长中的卵母细胞，包括mRNA、蛋白质和细胞器。细胞质从哺乳细胞到卵母细胞的运输需要肌动蛋白细胞骨架的精确定时重排和哺育细胞的程序性细胞死亡(凋亡)。在卵子发生的后期，在哺育细胞中形成致密的细胞质肌动蛋白束，正好在哺育细胞凋亡开始时。几个编码肌动蛋白结合蛋白的基因将结合遗传学、生物化学和细胞生物学进行研究，以阐明它们在卵子发生过程中的功能。此外，还将研究与肌动蛋白组织有关的调节蛋白。这项工作对于理解动态肌动蛋白细胞骨架在正常细胞和转移等疾病进展过程中对分子的运动性、细胞形状或亚细胞定位的贡献是相关的。该提案的具体目的是：(1)研究Profilin在体内的功能，Profilin是一种小的肌动蛋白单体结合蛋白，对于调节肌动蛋白细丝聚合的速度很重要；(2)对Profilin与Arp2/3复合体和FH结构域蛋白的相互作用进行遗传和生化分析；(3)对将肌动蛋白细丝交联成细胞质内高度有序的束的蛋白质进行功能分析；以及(4)研究类固醇激素蜕皮激素在调节哺育细胞凋亡中的作用。在果蝇哺育细胞中，Profilin是卵子发生第10阶段形成的细胞质肌动蛋白细丝聚合所必需的。将利用生殖系表达载体和P元件介导的转化，在护士细胞中制造和测试编码Profilin和Profilin结合蛋白的基因的特定突变。类似的实验策略将被用来测试果蝇Fasin的功能，这是一种在第10阶段也需要的肌动细丝捆绑蛋白。将通过检测两者的磷酸化和结合Armadillo(β-catenin)的能力来研究Fascin和一种类似于Villin的肌动蛋白捆绑蛋白Quail的可能的协调调节。Profilin、Quail和Fasin蛋白在经历细胞凋亡的细胞中发挥作用。将通过在蜕皮激素反应途径中使用突变体，并克隆一种名为Midway的新基因来研究类固醇激素蜕皮激素在信号传递护士细胞凋亡中的作用。

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.