A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples

*Inserm, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
Biochemical Journal (Impact Factor: 4.4). 07/2012; 447(1):17-23. DOI: 10.1042/BJ20120945
Source: PubMed


PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

Download full-text


Available from: Hélène Tronchère, Oct 13, 2015
  • Source
    • "All other compounds used were from Sigma-Aldrich. Recombinant GST-PIKfyve protein corresponding to the short form of murine PIKfyve was produced in SF9 infected cells, purified, and its activity was measured as described previously (Chicanne et al., 2012). Briefly, increasing amounts of L41 or YM201636 were added to the samples containing recombinant PIKfyve (0.2 mg), and PtdIns3P (50 pmol)/PE (20 nmol) vesicles, and lipid kinase buffer. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Leucettines, a family of pharmacological inhibitors of DYRKs (dual-specificity tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases), are currently under investigation for their potential therapeutic application to Down syndrome and Alzheimer's disease. We here report that Leucettine L41 triggers bona fide autophagy in osteosarcoma U-2 OS cells and immortalized mouse hippocampal HT22 cells, characterized by LC3 membrane translocation and foci formation. Leucettine L41-triggered autophagy requires the ULK1 (Unc-51-like kinase) kinase and is sensitive to the PI3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and 3-methyladenine, suggesting that it acts through the mTOR/PI3K-dependent pathway. Leucettine L41 does not act by modifying the autophagic flux of vesicles. Leucettine L41-induced autophagy correlates best with inhibition of CLKs. Leucettine L41 modestly inhibited PIKfyve (phosphatidylinositol-3-phosphate 5-kinase) activity as tested both in vitro and in vivo, which may also contribute to autophagy induction. Altogether these results demonstrate that Leucettines can activate the autophagic mTOR/PI3K pathway, a characteristic that may turn advantageous in the context of Alzheimer's disease treatment.
    Full-text · Article · Dec 2013 · Molecular pharmacology
  • Source
    • "As active-phosphatase MTM1-WT induced stacking of flat membranes while the dead-phosphatase mutant MTM1-C375S generated highly curved cubic membranes, we hypothesized that the MTM1 substrate, PtdIns3P, could be determinant for the cubic 1 1 symmetry and more generally for SR curvature. As muscle tissues from living mice cannot be metabolically labeled, we used a novel sensitive mass assay (Chicanne et al., 2012) to measure the level of PtdIns3P in WT skeletal muscles injected with AAV-empty, AAV- Mtm1-WT and AAV-Mtm1-C375S and compared with Mtm1-null muscles. As expected, the "
    [Show abstract] [Hide abstract]
    ABSTRACT: The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads and Mtm1 deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated to flat membrane stacks, while dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Over-expression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.
    Full-text · Article · Feb 2013 · Journal of Cell Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1(C375S) and MTM1(S376N)) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1(C375S) mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.
    Full-text · Article · Oct 2012 · PLoS Genetics
Show more