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.

Endocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME.

Mantle cell lymphoma (MCL) is a distinct non-Hodgkin’s lymphoma type that commonly affects extra nodal sites. The most often affected sites are bone marrow, gastrointestinal tract and Waldeyer’s ring, being the skin rarely involved. We report a case of 56 year-old man with MCL, exhibiting multiple large maculopapular skin rashes and skin ulcers. Histopathological examination had not shown direct infiltration by any atypical cells. He had significant improvement of skin lesions with combination chemotherapy and debridement. Awareness of skin manifestations of MCL is crucial for dermatologists and haematologists to establish the early diagnosis and timely administration of appropriate treatment.

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP3, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing Drosophila melanogaster eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: Pi3K68D, Vps34 and fwd that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.