Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.

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Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.Genome-wide RNAi screens have been performed in cultured cells 1 -4 or by ubiquitous gene silencing in worms 5 , 6 or planarians 7 . To study complicated developmental processes, however, genes need to be inactivated in a tissue-specific manner in intact animals. This has become possible through the creation of a transgenic RNAi library targeting 88% of the Drosophila 8 protein-coding genes. To test the feasibility of this new approach, we focused on the Notch pathway, one of the most important regulators of development 9 , 10 . Notch is activated by binding to its ligands Delta or Serrate. After ligand binding, Notch is cleaved by Presenilin and the intracellular domain acts in the nucleus as a transcriptional co-activator. Europe PMC Funders Group Genome-wide RNAi screenHairpin constructs in the RNAi library are expressed under UAS/GAL4 control 18 . We tested scabrous-GAL4 18 , pannier (pnr)-GAL4 19 and fzIII-GAL4 (also known as MS248-GAL4 or P{GawB}MS248) 20 using a set of 40 RNAi lines targeting 21 genes involved in external sensory organ development (Supplementary Table 1). Consistently, phenotypes were stronger and lethality lower with pnr-GAL4, and this line was selected for large-scale analysis.A total of 20,262 transgenic RNAi lines were screened; these are predicted to target 11,619 of the 14,139 protein-coding genes (82.2%) in release 5.7 of the Drosophila genome 21 . Ten flies each were analysed and phenotypic abnormalities were recorded in a database (http:// bristlescreen.imba.oeaw.ac.at). Because pnr-GAL4 is only expressed in a central region of the notum (Fig. 1b), lateral areas were unaffected and served as internal controls. Phenotypes were described using controlled vocabulary (Fig. 1b, Supplementary Table 2). Phenotypic strength (P x ) was expressed on a scale of 0 (not affected) to 10 (completely affected) as the fraction of the pnr-GAL4 expression a...