ROLE OF DROSOSPHIA PHOSPHOLIPASE D IN CELLULARIZATION

在过去的几年里，磷脂酰胆碱水解磷脂酶D （PLD）基因的超家族从原核生物保守到哺乳动物已经被描述。哺乳动物pld是通过g蛋白偶联受体和酪氨酸激酶受体信号转导途径激活的。由PLD及其产生的第二信使介导的生化步骤已经得到了很好的表征，但PLD的功能作用虽然被认为是重要的，但除了在酵母中，还没有很好地定义，在酵母中已经产生了有趣的结果。这些结果以及不太确定的哺乳动物研究表明，PLD促进了特殊类型的膜生物发生，因为它与高尔基体膜和质膜的囊泡运输有关。这一提议提出了PLD在果蝇胚胎发生中的潜在作用，细胞化是起源于高尔基体的一种特殊形式的膜生物发生。我们从果蝇中克隆了一个PLD基因（标记为dPLD）。与哺乳动物PLD类似，dPLD似乎受蛋白激酶c刺激通路的调节。与哺乳动物PLD不同，与酵母PLD相似，dPLD不受小g蛋白ARF的刺激。dpld特异性抗血清显示，在细胞化过程中，它在生殖细胞和胚胎周围表达。我们建议1)定义调控dPLD的机制；2)高分辨率表征野生型胚胎和细胞化缺陷胚胎中dPLD的空间表达和亚细胞定位；3)生成和表征功能缺失的dPLD等位基因；4)研究野生型和突变型PLD在细胞化和生殖细胞迁移过程中过表达的后果。最终，我们试图了解dPLD介导的功能作用，模拟哺乳动物PLD承担的更复杂的细胞生物学和生理学作用，并利用果蝇独特的技术方法弥合酵母和哺乳动物中PLD作用机制的日益增长的知识。

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.