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.

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    • "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. "
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    Molecular pharmacology 12/2013; 85(3). DOI:10.1124/mol.113.090837 · 4.13 Impact Factor
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    • "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 "
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    Journal of Cell Science 02/2013; 126(8). DOI:10.1242/jcs.118505 · 5.43 Impact Factor
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    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.
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