Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry.

The activation of phospholipase Cß (PLCß) is an essential step during sensory transduction in Drosophila photoreceptors. PLCß activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLCß also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Septins tune lipid kinase activity and PI(4,5)P2 turnover during G-protein-coupled PLC signalling in vivo.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P2, its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In Drosophila photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P2 is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P2 hydrolysis is unknown. In this study, we identify a unique isoform dPIP5KL, that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5KL activity in vitro and PI(4,5)P2 resynthesis in vivo; co-depletion of dPIP5KL reverses the enhanced rate of PI(4,5)P2 resynthesis in vivo. Thus, our work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P2 hydrolysis.