EMBRYONIC DORSOVENTRAL POLARITY IN DROSOPHILA

果蝇胚胎背腹极性

• 批准号: 2459489
• 负责人: CARL HASHIMOTO
• 金额: $ 28.75万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459489
• 关键词: Drosophilidae; affinity chromatography; binding proteins; biological signal transduction; cell membrane; cellular polarity; early embryonic stage; electron microscopy; enzyme activity; enzyme substrate; immunochemistry; immunoprecipitation; membrane proteins; oogenesis; polymerase chain reaction; protein structure function; serine proteinases; western blottings

Dorsoventral polarity of the Drosophila embryo is established by a transmembrane signaling pathway involving maternal molecules. Two outstanding questions about this pathway concern how the extracellular signal that induces polarity is asymmetrically generated and how it is transmitted to the interior of the embryo. The extracellular signal is the ligand for the transmembrane Toll protein, which is similar in its cytoplasmic domain to the mammalian interleukin l receptor. Activation of either protein causes, by an unclear mechanism, the nuclear targeting of a rel proto-oncogene transcription factor. Earlier studies suggested that localized activation of a protease cascade restricts production of Toll's ligand to the ventral side of the embryo, and that this localized activation is determined by a component asymmetrically laid down during oogenesis in the future extracellular compartment of the embryo. We recently uncovered a possible clue to how Toll's ligand is asymmetrically produced. We cloned and sequenced the nudel gene, which is expressed during oogenesis and is required to activate the proteases that produce Toll's ligand. The nudel gene product is predicted to be a large (greater than or equal to 300 kD) extracellular glycoprotein, modular like extracellular matrix molecules, with cysteine-rich motifs first seen in the mammalian LDL receptor. Nudel's most intriguing structural motif is a serine protease domain, so it may initiate the protease cascade producing Toll's ligand. Since the nuclei RNA appears to be ventrally enriched in the egg chamber during oogenesis, the nudel protein may also function as a spatial determinant of embryonic dorsoventral polarity. Our long-term goal is to understand in biochemical detail how the nudel protein activates and localizes the proteases that produce Toll's ligand, and how Toll activates intracellular signaling reactions. Specific aims are: l) Immunolocalize the nudel protein in egg chambers and embryos to determine if it is ventrally enriched; and perform fractionation experiments to determine if nudel is bound to the embryonic plasma membrane or the surrounding vitelline envelope, since it must be non- diffusible in order to localize protease activation. 2) Test nudel's serine protease and other functions in embryos using a combination of in vitro mutagenesis, germline transformation, and RNA injection methods. 3) Identify nudel's protease substrate and other molecules that bind to nudel using in vitro protease assays, immunoprecipitation methods, and affinity chromatography. 4) Identify proteins that bind to Toll's cytoplasmic domain through immunoisolation, affinity chromatography, and the yeast interaction trap screen. The proposed studies on nudel and Toll, and their interacting proteins, will increase our understanding of similar molecules involved in cancer and cardiovascular disorders.

果蝇胚胎的背腹极性是由 涉及母体分子的跨膜信号通路。两 关于这一途径的悬而未决的问题涉及细胞外 诱导极性的信号是不对称产生的，以及它是如何 传递到胚胎内部。细胞外信号是 跨膜Toll蛋白的配体，其在其 哺乳动物白细胞介素1受体的胞质结构域。激活 这两种蛋白质都通过一种不清楚的机制引起核靶向， rel原癌基因转录因子。早期的研究表明， 蛋白酶级联的局部激活限制了Toll's的产生 配体的腹侧胚胎，这本地化 激活是由一个组件不对称铺设期间， 卵子发生在胚胎未来的细胞外区室中。我们 最近发现了一个可能的线索， 制作。我们克隆并测序了nudel基因， 在卵子发生过程中，需要激活蛋白酶， 托尔的配体。预计裸基因产物很大（更大） 大于或等于300 kD）细胞外糖蛋白，模块样 细胞外基质分子，富含半胱氨酸的基序首次见于 哺乳动物LDL受体。Nudel最有趣的结构基序是 丝氨酸蛋白酶结构域，所以它可以启动蛋白酶级联产生 托尔的配体。由于细胞核RNA似乎在腹侧富集， 在卵子发生过程中，裸蛋白也可以起作用， 胚胎背腹极性的空间决定因素。 我们的长期目标是从生物化学的角度详细了解 蛋白质激活并定位产生Toll配体的蛋白酶， 以及Toll如何激活细胞内信号反应。具体目标 l）免疫定位卵室和胚胎中的裸蛋白， 确定其是否为腹侧富集;并进行分馏 确定裸蛋白是否与胚胎血浆结合的实验 膜或周围的卵黄被膜，因为它必须是非- 为了定位蛋白酶活化，可扩散。2)测试nudel的 丝氨酸蛋白酶和其他功能在胚胎中使用的组合， 体外诱变、种系转化和RNA注射方法。第三章 识别nudel的蛋白酶底物和其他与nudel结合的分子 使用体外蛋白酶测定、免疫沉淀方法和亲和性测定， 层析4)识别与Toll细胞质结合的蛋白质 结构域通过免疫分离，亲和层析，和酵母 交互陷阱屏幕。建议对nudel和Toll进行研究， 相互作用的蛋白质，将增加我们对类似分子的理解， 与癌症和心血管疾病有关。