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Química

Радиоактивной и Количественная клеточном уровнях фосфоинозитидов с помощью высокоэффективной жидкостной хроматографии в сочетании-Flow мерцаний

Published: January 06, 2016
doi:
10.3791/53529
, ,
1Department of Chemistry and Biology, Program in Molecular Science,Ryerson University

Summary

Фосфоинозитиды сигнализируют липиды, чья относительная численность быстро меняется в ответ на различные раздражители. Эта статья описывает метод измерения обилие фосфоинозитидов метаболически нумерующих клеток с 3 H-мио -inositol с последующей экстракцией и деацилирования. Извлеченные глицеро-inositides затем разделяют с помощью высокоэффективной жидкостной хроматографии и количественно с помощью проточной сцинтилляции.

Abstract

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Introduction

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 activity1-3. 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 membrane3,4. 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 PtdInsP3,4. Hence, misregulation of the PtdInsP regulatory machinery can perturb cell function, leading to diseases such as cancer and degenerative diseases2,5,6. 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 domain7-9. 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 imaging1,10. 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 endosomes10-13. 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 PtdInsP14-16. 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 PtdInsPs14,17. However, mass spectrometry is mostly semi-quantitative and it remains difficult to resolve and concurrently quantify PtdInsP species of the same molecular weight14,17. 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 PtdIns16,19,20. 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 mammals22-24. 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 mammals25,26. In VAC14-deleted yeast cells, Fab1 does not function efficiently leading to a 90% decrease in PtdIns(3,5)P2 levels27,28. 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 levels29,30. 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.

Protocol

Примечание: Приведенный ниже текст подробно описывает метод измерения PtdInsPs в дрожжах. Она обеспечивает экспериментальные данные для маркировки клеток дрожжей с 3 H-мио -inositol, добывающих и deacylating липидов и протокола ВЭЖХ-элюции для фракционирования и количественно деацилиров…

Representative Results

Используя этот метод, дрожжи PtdInsPs были метаболически метили с 3 H-мио -inositol. После маркировки, фосфолипиды осаждают хлорной кислотой, а затем фосфолипидов деацилирования и извлечения водорастворимого Gro-InsPs (фиг.1). На этом этапе важно, чтобы определит…

Discussion

Эта статья подробно описывает экспериментальный протокол, необходимый для количественного клеточные уровни PtdnsPs с помощью ВЭЖХ связью потока мерцаний от дрожжей. Методика дает возможность обмена веществ маркировки PtdInsPs с 3 H-мио -inositol с последующей обработкой липидов и извл…

Declarações

The authors have nothing to disclose.

Acknowledgements

C.Y.H. was supported by an Ontario Graduate Scholarship from the Government of Ontario. This article was made possible by funding held by R.J.B. from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and Ryerson University.

Materials

1-Butanol Biobasic BC1800 Reagent grade
Ammonium phosphate dibasic Bioshop APD001 ACS grade
Ammonium sulfate Biobasic ADB0060 Ultra Pure grade
Autosampler Agilent G1329B Agilent 1260 infinity series
Biotin Sigma B4501
Boric acid Biobasic BB0044 Molecular biology grade
Calcium Chloride Biobasic CT1330 Ahydrous, industrial grade
Calcium pantothenate Sigma C8731
Copper(II) sulfate Sigma 451657 Anhydrous
D-Glucose Biobasic GB0219 Anhydrous, biotech grade
Dulbecco's modification of Eagle's Medium Life 11995-065 With 4.5 g/L glucose, 110 mg/L pyruate, L-glutamine
Dulbecco's modification of Eagle's Medium MP biomedicals 0916429  With 4.5 g/L glucose, without L-gluatmine, without inositol
EDTA Biobasic EB0107 Acid free, ultra pure grade
Ethyl ether Caledon labs 1/10/4700 Anhydrous, reagent grade
Ethyl formate Sigma 112682 Reagent grade
Fetal bovine serum Wisent 080-450 US origin, premium quality, heat inactivated
Fetal Bovine Serum, Dialyzed Life 26400044 US origin
FlowLogic U LabLogic Systems Ltd SG-BXX-05 Scintillation fluid for flow scintillation 
Folic acid Biobasic FB0466 USP grade
HEPES buffer solution Life 15630080 1 M solution
Inositol, Myo-[2-3H(N)] Perkin Elmer NET114005MC 9:1 ethanol to water
Insulin-Transferrin-Selenium-Ethanolamine Life 51500056 100x solution
Iron(III) chloride Sigma 157740 Reagent grade
Laura – Chromatography data collection and analysis software LabLogic Systems Ltd Version 4.2.1.18 Flow scintillator software
L-glutamine Sigma G7513 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
Magnesium Chloride Sigma M8266 Anhydrous
Manganese sulfate Biobasic MB0334 Monohydrate, ACS grade
Methanol Caledon labs 6701-7-40 HPLC Grade
Methylamine solution Sigma 426466 40% (v/v)
Monopotassium phosphate Biobasic PB0445 Anhydrous, ACS grade
Nicotinic acid Biobasic NB0660 Reagent grade
OpenLAB CSD ChemStation  Agilent Rev. C.01.03  HPLC software
p-aminobenzoic acid (PABA) Bioshop PAB001.100 Free acid
Penicillin-Streptomycin Sigma P4333 100X, liquid, stabilized, sterile-filtered, cell culture tested
Perchloric acid Sigma 244252 ACS reagent, 70%
PhenoSpher SAX column Phenomenex 00G-315-E0 5µm, 80Å, 250 x 4.6 mm
Phosphoric acid Caledon labs 1/29/8425 Reagent grade
Potassium Chloride Biobasic PB0440 ACS grade
Potassium iodide Biobasic PB0443 ACS grade
Pyridoxine hydrochloride Sigma P9755
Quaternary pump Agilent G1311C Agilent 1260 infinity series
Riboflavin Bioshop RIB333.100 USP grade
Sodium Chloride Biobasic DB0483 Biotech grade
Sodium molybdate Sigma 243655
Thermostatted Column Compartment Agilent G1316A Agilent 1260 infinity series
Thiamine hydrochloride Sigma T4625 Reagent grade; make solution of 0.02% (w/v), forms a suspension. mix and freeze aliquots
Ultima Gold Perkin Elmer 6013321 Scintillation coctail for liquid scintillation counting
Zinc sulfate Biobasic ZB2906 Heptahydrate, reagent grade
β-RAM 4 IN/US systems Model 4 Flow scintillator – 500 µL flow cell; alternative Radiomatic Flow Scintillator Analyser by Perkin Elmer 
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Tags

Keywords: RadiolabelingPhosphoinositidesHPLCFlow ScintillationYeastMammalian CellsInositolPerchloric AcidEDTACentrifugationSonicationLiquid CultureCell GrowthCell Lysis
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Ho, C. Y., Choy, C. H., Botelho, R. J. Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation. J. Vis. Exp. (107), e53529, doi:10.3791/53529 (2016).
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