Radiomarcatura e quantificazione dei livelli cellulari di fosfoinositidi da alte prestazioni cromatografia liquida accoppiata flusso scintillazione

Phosphoinositides (PtdInsPs) are important signaling phospholipids that help regulate a variety of cellular functions, including signal transduction, membrane trafficking and gene expression, which then modulate higher-order cell behavior such as cell division, organelle identity and metabolic activity^1-3. There are seven species of PtdInsPs that are derived from the phosphorylation of the 3, 4, and/or 5 positions of the inositol head group of phosphatidylinositol (PtdIns), the parent phospholipid. Importantly, the seven PtdInsPs are unequally distributed and the local concentration of each PtdInsP species can increase or decrease at specific subcellular sites where they bind to a distinct set of protein effectors, which together permits each PtdInsP to control the identity and function of its host membrane^3,4. In addition, the levels of each PtdInsP need to be tightly controlled since this can significantly impact the signal intensityproduced by a PtdInsP. The localization and levels of each PtdInsP depends on the targeting and activity of numerous lipid kinases, phosphatases and phospholipases that mediate the synthesis and turnover of each PtdInsP^3,4. Hence, misregulation of the PtdInsP regulatory machinery can perturb cell function, leading to diseases such as cancer and degenerative diseases^2,5,6. To fully understand the roles and functions of PtdInsPs and their regulatory machinery, both microscopy-based and biochemical-based techniques have been developed to track and quantify PtdInsPs.

In many cases, PtdInsPs bind to their protein effectors via a specific protein domain^7-9. These protein modules often retain their proper fold and lipid recognition properties when expressed separately from the entire protein. This gave rise to PtdInsP probes by fusing a specific PtdInsP-binding protein domain to a fluorescent protein (FP) like green fluorescent protein (GFP) for the subcellular detection of PtdInsPs by microscopy. Indeed, many studies have used FP-fused PtdInsP-binding protein modules to identify the localization and dynamics of specific PtdInsP species by live-cell imaging^1,10. For example, the Pleckstrin homology (PH) domain of phospholipase C δ1 (PLCδ1) fused to GFP specifically recognizes the phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] on the plasma membrane, whereas tandem copies of the FYVE domain of early endosome antigen 1 (EEA1) has been employed to track phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes^10-13. Overall, microscopy-based techniques are great to visualize PtdInsP localization and dynamics, but there are several caveats including that PtdInsP-binding domains may also interact with additional factors other than the target PtdInsP species and that they cannot detect changes below cytosolic fluorescence of the FP-probes.

Biochemical techniques including thin-layer chromatography, mass spectrometry and radioisotope labeling can also be used to characterize and quantify the levels of each PtdInsP^14-16. These methods require the isolation of lipids for the detection of cellular levels of PtdInsPs. Mass spectrometry can be used to characterize phospholipids from lipid extracts and is invaluable for determining the acyl chain composition of PtdInsPs^14,17. However, mass spectrometry is mostly semi-quantitative and it remains difficult to resolve and concurrently quantify PtdInsP species of the same molecular weight^14,17. In comparison, radioisotope labeling of PtdInsPs followed by high performance liquid chromatography (HPLC)-coupled flow scintillation is useful for the separation and concurrent quantification of all seven species of PtdInsPs¹⁸. The use of HPLC with a strong anion exchange (SAX) column achieves separation based on molecular weight, charge and shape, thus fractionating deacylated PtdInsPs (Gro-InsPs) even of the same molecular weight and charge. Coupling the HPLC eluent to a flow scintillator then generates radioactive-based signal peaks for each Gro-InsPs species relative to the original parent glycerol-inositol (Gro-Ins)¹⁸. This ultimately corresponds to relative levels of PtdInsPs in cells.

Radiolabeling of PtdInsPs and HPLC-coupled flow scintillation is a useful tool to investigate the regulation and function of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂], a PtdInsP that only constitutes ~0.1-0.3% of PtdIns^16,19,20. Synthesis of PtdIns(3,5)P₂ is performed by the PtdIns3P 5-kinase called Fab1 in yeast and PIKfyve in mammals²¹. This reaction is counteracted by a PtdIns(3,5)P₂ 5-phosphatase called Fig4 in yeast or mFig4/Sac3 in mammals^22-24. Interestingly, both PIKfyve/Fab1 and mFig4/Fig4/Sac3 exist in a single complex and are regulated by the scaffolding adaptor protein, Vac14 in yeast or mVac14/ArPIKfyve in mammals^25,26. In VAC14-deleted yeast cells, Fab1 does not function efficiently leading to a 90% decrease in PtdIns(3,5)P₂ levels^27,28. On the other hand, Atg18 is a PtdIns(3,5)P₂ effector protein that may control vacuolar fission ²⁹. Atg18 is also a negative regulator of PtdIns(3,5)P₂ since the deletion of its gene, ATG18, causes a 10 to 20-fold increase in PtdIns(3,5)P₂ levels^29,30. Overall, changes in the levels of PtdIns(3,5)P₂ severely impacts the function of the yeast vacuole and the mammalian lysosomes, consequently affecting processes such as membrane trafficking, phagosome maturation, autophagy and ion transport ^6,19,21,31.

This article describes the process of radioisotope labeling of PtdInsPs with ³H-myo-inositol in yeast to detect the relative levels of PtdIns(3,5)P₂ in wild-type, vac14Δ and atg18Δ yeast strains. Using this as an example, the resolving capabilities of HPLC for the separation of individual PtdInsPs as well as the sensitivity of flow scintillation for detection of trace amount of ³H-myo-inositol is shown. We also elaborate on how one might optimize the methodology for labeling and separating ³H-labelled PtdInsPs from mammalian cells, whose samples tend to be more complex since these cells possess all seven PtdInsP species.