To develop a PtdIns3P mass assay capable of determining accurately the amount of this lipid in cell extracts, we used recombinant PIKfyve and [γ-32P]ATP to produce and quantify radiolabelled PtdIns(3,5)P2 formed from PtdIns3P. Recombinant GST–PIKfyve protein was produced in the baculovirus-SF9 insect cell system and the purified enzyme (A) was stable for months at −80°C. Its substrate specificity was first evaluated using pure C16:0/C16:0-PtdIns3P, PtdIns4P purified from brain extracts and C16:0/C16:0-PtdIns5P. PtdIns3P was efficiently phosphorylated into PtdIns(3,5)P2 by recombinant PIKfyve as demonstrated by the HPLC analysis (B, panel 1). As expected, PtdIns5P was not phosphorylated by PIKfyve. However, when PtdIns4P purified from brain extract was used as a substrate, we observed the production of radiolabelled PtdIns(3,5)P2 (B, panel 2), suggesting that some PtdIns3P was present in the samples, since the procedure used to purify PtdIns4P from the brain does not preclude contamination with other PtdInsPs. Recombinant PIKfyve also phosphorylated small amounts of PtdIns4P into PtdIns(4,5)P2, as shown by the HPLC analysis (B, panel 2). When a mixture of PtdInsPs containing 80% PtdIns4P, 10% PtdIns3P and 10% PtdIns5P was used to reproduce the ratio classically found in resting mammalian cells, PtdIns3P appeared as the preferred substrate of recombinant PIKfyve (B, panel 4). However, to further decrease the residual phosphorylation of PtdIns4P, which could cause a bias of quantification since it is much more abundant in mammalian cells than PtdIns3P, we optimized the assay by testing different conditions, especially variation of ions concentrations and lipid vesicle composition. Interestingly, in the presence of 0.4 μM PE and 2.5 mM MgCl2, recombinant PIKfyve selectively phosphorylated PtdIns3P (C). Under these conditions, even with a large excess of PtdIns4P (80% PtdIns4P, 10% PtdIns3P and 10% PtdIns5P), PIKfyve produced 50-fold more PtdIns(3,5)P2 than PtdIns(4,5)P2 (C, panel 4).
Purification and substrate specificity of recombinant PIKfyve
The assay was found to be linear with pure PtdIns3P quantities varying from a few pmol up to 100 pmol (A). To assay the linearity in the context of a cellular extract, increasing amounts of pure PtdIns3P were then mixed with HeLa cell lysates (B). Again, the assay was linear over a relatively large range of concentrations, indicating that the method displayed good sensitivity. However, it is noteworthy that the yield of lipid extraction is different from one biological sample to another (i.e. cultured cells, yeasts or biopsies such as muscle extracts), mostly because of the difference in mechanical resistance of tissues compared with cultured cells, and must therefore be optimized to accurately quantify PtdIns3P.
Sensitivity and linearity of the PtdIns3P mass assay
As a proof of principle, we tested our assay by measuring the amount of PtdIns3P
in yeast, where the only PI3K present is encoded by the vps34
gene. In this model, a previous study demonstrated that PtdIns3P
could hardly be detected by [3
H]inositol labelling and HPLC analysis in the vps34
-deleted strain [4
]. Accordingly, lipid extraction and mass assay showed a dramatic decrease of PtdIns3P
in the vps34
-deleted strain (A), demonstrating that the mass assay is accurate in quantifying PtdIns3P
in yeasts. To further validate the assay, we used mouse platelets stimulated by thrombin. As shown in (B), the mass of PtdIns3P
increased following stimulation, whereas inhibition of PI3Ks by wortmannin impaired this elevation. Importantly, the increase in PtdIns3P
measured by the mass assay correlated with the increase in 32
detected by HPLC after metabolic labelling in a parallel experiment (C). We also observed an increase in the mass of PtdIns3P
in human platelets stimulated through the thrombin receptor PAR1 (protease-activated receptor 1) with the agonist peptide (TRAP) in the presence of fibrinogen to fully engage the integrin GpIIbIIIa (D). Again, preincubation of platelets with the PI3K inhibitor wortmannin abolished the PtdIns3P
production measured after 8 min of TRAP stimulation (D). These results are consistent with a previous report showing an increase in 32
upon stimulation under similar conditions using HPLC analysis, after metabolic labelling of human platelets [17
]. Thus, in this case, the incorporation of the radiolabel did reflect an increase in the mass of this lipid. The role of PtdIns3P
in stimulated platelets is still unknown and the development of mouse models with deficiencies in enzymes involved in its metabolism might bring new light on the functions of this lipid in the near future.
Application of the PtdIns3P mass assay to yeast and mammalian cells
To test whether our assay would be efficient to quantify PtdIns3P
in isolated subcellular compartments, we then separated early and late endosomal fractions from BHK cells following a well-established procedure [34
]. We observed an enrichment of the relative amount of PtdIns3P
in early endosomes (10-fold increase compared with the post-nuclear fraction) and to a lesser extent in the late endosomal fraction when compared with post-nuclear fractions (6-fold increase) (E). Subcellular fractionation can induce artifactual changes in lipid levels due to potential transfer of material or to modification of their metabolism during the isolation procedure. However, the results of our mass assay are consistent with the imaging data showing that the GFP–2×FYVE probe used in living cells to visualize PtdIns3P
pools preferentially decorates early endosomes [6
Overall, these results validate the application and the sensitivity of the mass assay we developed in various biological samples (i.e. yeast, primary human cells and isolated subcellular fractions). This assay is relatively simple (), robust and should be useful to quantify PtdIns3P in various tissues from mice deficient for PtdIns3P-metabolizing enzymes (i.e. Vps34, PIKfyve, MTMs, inositol polyphosphate 4-phosphatase type I and II) or models exhibiting knockin forms of these enzymes that are under development. Accordingly, we already validated this assay by measuring the amount of PtdIns3P in muscle samples from mice deficient for myotubularin 1 and infected with wild-type or inactive MTM1-expressing adeno-associated virus (L. Amoasii, K. Hnia, G. Chicanne, A. Brech, B. S. Cowling, M. Mueller, Y. Scwab, P. Koebel, A. Ferry, B. Payrastre and J. Laporte, unpublished work).
Schematic representation of the major steps of the mass assay
In conclusion, we propose a new sensitive mass assay for PtdIns3P quantification in various samples with significant advantages compared with the method requiring radiolabelling of cultured cells followed by HPLC analysis.