ROLE OF DROSOSPHIA PHOSPHOLIPASE D IN CELLULARIZATION

在过去的几年里，一个超家族的磷脂酰胆碱水解型磷脂酶D(PLD)基因已被描述，该基因从原核生物到哺乳动物都是保守的。哺乳动物的PLD由G蛋白偶联受体和酪氨酸激酶受体信号转导通路激活。PLD介导的生化步骤和由其产生的第二信使已被很好地描述，但PLD的功能角色尽管被认为是重要的，但除了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，加上不太确定的哺乳动物研究，表明PLD促进特定类型的膜生物发生，因为它与来自高尔基体和质膜的囊泡运输有关。这项建议阐述了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。我们定位并克隆了果蝇的PLD基因(记为dPLD)。与哺乳动物的PLD相似，dPLD似乎受蛋白激酶C刺激的通路的调节。与哺乳动物的PLD不同，但类似于酵母的PLD，dPLD不受小G蛋白ARF的刺激。DPLD特异性抗血清表明，在细胞化过程中，dPLD在生殖细胞和胚胎外围表达。我们建议1)确定dPLD的调控机制；2)在野生型胚胎和细胞化缺陷的胚胎中高分辨率地研究dPLD的空间表达和亚细胞定位；3)产生并鉴定功能丧失的dPLD等位基因；4)检测野生型和突变型PLD在细胞化和生殖细胞迁移过程中过度表达的后果。最终，我们试图了解dPLD介导的功能角色，对哺乳动物PLD所承担的更复杂的细胞生物学和生理角色进行建模，并利用果蝇特有的技术方法，弥合关于PLD在酵母和哺乳动物中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.