Highlights d Drosophila PIP4K mutant larvae have increased PIP 3 levels in cells d Cells show enhanced sensitivity to insulin in the absence of PIP4K d PIP4K regulates enzymes involved in PIP 3 turnover at the plasma membrane d Loss of PIP4K suppresses insulin resistance phenotypes
Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.
The activation of phospholipase C (PLC) is a conserved mechanism of receptor-activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P], and continued signaling requires the resynthesis and availability of PI(4,5)P at the plasma membrane. PI(4,5)P is synthesized by the phosphorylation of phosphatidylinositol 4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified as performing this function in cultured mammalian cells, its function in the context of an physiological model has not been established. In this study, we show that, in photoreceptors, PI4KIIIα activity is required to support signaling during G-protein-coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light, and reduced plasma membrane levels of PI4P and PI(4,5)P Depletion of the conserved proteins Efr3 and TTC7 [also known as StmA and L(2)k14710, respectively, in flies], which assemble PI4KIIIα at the plasma membrane, also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P levels. Thus, PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P levels during receptor activated PLC signaling.
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