ROLE OF DROSOSPHIA PHOSPHOLIPASE D IN CELLULARIZATION

果蝇磷脂酶 D 在细胞化中的作用

• 批准号: 6032932
• 负责人: Michael A. Frohman
• 金额: $ 19.58万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6032932
• 关键词: Drosophilidae; G protein; alleles; autoradiography; biological signal transduction; cell migration; confocal scanning microscopy; early embryonic stage; electron microscopy; enzyme activity; gene expression; immunofluorescence technique; invertebrate embryology; membrane biogenesis; phospholipase D; protein structure function; protein tyrosine kinase; scintillation counter; thin layer chromatography; tissue /cell culture

During the past several years, a superfamily of phosphatidylcholine-hydrolyzing Phospholipase D (PLD) genes conserved from prokaryotes to mammals has been described. Mammalian PLDs are activated by G-protein-coupled receptor and tyrosine kinase receptor signal transduction pathways. The biochemical step mediated by PLD and the second messenger generated by it are fairly well characterized, but functional roles for PLDs although thought to be important are not well defined except in yeast, where intriguing results have been generated. These results, together with less definitive mammalian studies, suggest that PLD promotes specialized types of membrane biogenesis as it relates to vesicular trafficking from the Golgi and plasma membrane. This proposal addresses potential roles of PLD in Drosophila melanogaster embryogenesis in cellularization, which is a specialized form of membrane biogenesis originating from the Golgi. We have mapped and cloned a PLD gene from Drosophila (denoted dPLD). Similar to mammalian PLD, dPLD appears to be regulated by Protein Kinase C-stimulated pathways. Unlike mammalian PLD but similar to yeast PLD, dPLD is not stimulated by the small G-protein ARF. dPLD-specific antisera reveal that it is expressed in germ cells and in the periphery of the embryo during cellularization. We propose 1) to define the mechanisms that regulate dPLD; 2) to characterize dPLD spatial expression and subcellular localization at high resolution in wild type embryos and embryos with defects in cellularization; 3) to generate and characterize loss-of-function dPLD alleles; 4) to examine the consequence of overexpression of wild-type and mutant PLD during cellularization and germ cell migration. Ultimately, we seek to understand what functional roles dPLD mediates, to model the more complex cell biological and physiological roles undertaken by the mammalian PLDs, and to bridge the growing knowledge concerning PLD mechanism of action in yeast and mammals using technological approaches unique to Drosophila.

在过去的几年中，从原核生物到哺乳动物都保守的磷脂酰胆碱水解磷脂酶 D (PLD) 基因超家族已被描述。哺乳动物 PLD 通过 G 蛋白偶联受体和酪氨酸激酶受体信号转导途径激活。 PLD 介导的生化步骤及其产生的第二信使已得到很好的表征，但 PLD 的功能作用虽然被认为很重要，但除在酵母中产生了有趣的结果外，还没有得到很好的定义。 这些结果与不太确定的哺乳动物研究一起表明，PLD 促进特殊类型的膜生物发生，因为它与高尔基体和质膜的囊泡运输有关。该提案探讨了 PLD 在细胞化中果蝇胚胎发生中的潜在作用，细胞化是起源于高尔基体的膜生物发生的特殊形式。 我们已经定位并克隆了果蝇的 PLD 基因（表示为 dPLD）。 与哺乳动物 PLD 类似，dPLD 似乎受到蛋白激酶 C 刺激途径的调节。 与哺乳动物 PLD 不同，但与酵母 PLD 相似，dPLD 不受小 G 蛋白 ARF 的刺激。 dPLD 特异性抗血清表明，它在细胞化过程中在生殖细胞和胚胎周围表达。我们建议 1) 定义调节 dPLD 的机制； 2) 以高分辨率表征野生型胚胎和细胞化缺陷胚胎中的 dPLD 空间表达和亚细胞定位； 3) 生成和表征功能丧失的 dPLD 等位基因； 4) 检查细胞化和生殖细胞迁移过程中野生型和突变型PLD过度表达的结果。最终，我们试图了解 dPLD 介导的功能作用，模拟哺乳动物 PLD 所承担的更复杂的细胞生物学和生理作用，并利用果蝇独特的技术方法，弥合有关酵母和哺乳动物中 PLD 作用机制的不断增长的知识。