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.
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