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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Published: March 14th, 2021

DOI:

10.3791/62177

1Université Côte d’Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire
* These authors contributed equally

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

The precise distribution of lipids between different membranes and within the membranes of eukaryotic cells1,2 has profound biological implications. Decrypting how LTPs function is an important issue in cell biology3,4,5,6, and in vitro approaches are of great value in addressing this issue7,8,9,10,11. Here, ....

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1. Purification of NBD-C2Lact

NOTE: Although this protocol details the use of a cell disruptor to break bacteria, it can be modified to use other lysis strategies (e.g., a French press). At the beginning of the purification, it is mandatory to use buffer that is freshly degassed, filtered, and supplemented with 2 mM dithiothreitol (DTT) to prevent the oxidation of cysteine. However, for the protein labelling step, it is crucial to completely remove DTT. Many steps must be car.......

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Figure 1
Figure 1: Description of the fluorescent lipid sensors and in vitro assays. (A) Three-dimensional models of NBD-C2Lact and NBD-PHFAPP based on the crystal structure of the C2 domain of bovine lactadherin (PDB ID: 3BN648) and the NMR structure of the PH domain of the human FAPP1 .......

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The outcomes of these assays directly rely on the signals of the fluorescent lipid sensors. Thus, the purification of these probes labelled at a 1:1 ratio with NBD and without free NBD fluorophore contamination is a critical step in this protocol. It is also mandatory to check whether the LTP under examination is properly folded and not aggregated. The amount of LTP tested in the extraction assays must be equal to or higher than that of accessible PS or PI(4)P molecules to properly measure whether this LTP efficiently ex.......

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We are grateful to Dr. A. Cuttriss for her careful proofreading of the manuscript. This work is funded by the French National Research Agency grant ExCHANGE (ANR-16-CE13-0006) and by the CNRS.

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Name Company Catalog Number Comments
 L-cysteine ≥97 % (FG)  Sigma W326305-100G Prepare a 10 mM L-cysteine stock solution in water. Aliquots are stored at -20 °C
2 mL Amber Vial, PTFE/Rub Lnr, for lipids storage in CHCL3 Wheaton W224681
4 mm-diameter glass beads Sigma Z265934-1EA
50 mL conical centrifuge tube Falcon
ÄKTA purifier GE healthcare FPLC
Aluminium foil
Amicon Ultra-15 with a MWCO of 3 and 10 kDa Merck UFC900324, UFC901024
Amicon Ultra-4 with a MWCO of 3 and 10 kDa Merck UFC800324, UFC801024
Ampicillin Prepare a 50 mg/mL stock solution with filtered and sterilized water and store it at -20 °C.
Bestatin Sigma B8385-10mg
BL21 Gold Competent Cells Agilent
C16:0 Liss  (Rhod-PE) in CHCl3 (1 mg/mL) Avanti Polar Lipids 810158C-5MG
C16:0/C16:0-PI(4)P   Echelon Lipids P-4016-3 Dissolve 1 mg of C16:0/C16:0-PI(4)P powder in 250 µL of MeOH and 250 µL of CHCl3. Then complete with CHCl3 to 1 mL. The solution must become clear.
C16:0/C18:1-PS (POPS) in CHCl3 (10 mg/mL) Avanti Polar Lipids 840034C-25mg
C18:1/C18:1-PC (DOPC) in CHCl3 (25 mg/mL) Avanti Polar Lipids 850375C-500mg
CaCl2  Sigma Prepare 10 mM CaCl2 stock solution in water.
Cell Disruptor Constant Dynamics
Chloroform (CHCl3) RPE-ISO Carlo Erba 438601
Complete EDTA-free protease inhibitor cocktail Roche 5056489001
Deionized (Milli-Q) water
Dimethylformamide (DMF), anhydrous, >99% pure
DNAse I Recombinant, RNAse free, in powder Roche 10104159001
DTT Euromedex EU0006-B Prepare 1 M DTT stock solution in Milli-Q water.  Prepare 1 mL aliquots and store them at -20 °C. 
Econo-Pac chromatography columns (1.5 × 12 cm). Biorad 7321010
Electroporation cuvette 2 mm Ozyme EP102
Electroporator Eppendorf 2510 Eppendorf
Fixed-Angle Rotor Ti45 and Ti45 tubes Beckman Spinning the batcerial lysates
Glass-syringes (10, 25, and 50 µL) for fluorescence experiment Hamilton
Glass-syringes (25 , 100, 250, 500, and 1000 µL) to handle lipid stock solutions Hamilton 1702RNR, 1710RNR, 1725RNR, 1750RN type3, 1001RN
Glutathione Sepharose 4B beads GE Healthcare 17-0756-05
Glycerol (99% pure) Sigma G5516-500ML
Hemolysis tubes with a cap
HEPES , >99 % pure Sigma H3375-500G
Illustra NAP 10 desalting column GE healthcare GE17-0854-02
Isopropyl β-D-1-thiogalactopyranoside (IPTG)  Euromedex EU0008-B Prepare 1 M IPTG stock solution in Milli-Qwater. Prepare 1 mL aliquots and store them at -20 °C. 
K-Acetate Prolabo 26664.293
Lennox LB Broth medium without glucose Prepared with milli-Q water and autoclaved.
Liquid nitrogen Linde
Methanol (MeOH) ≥99.8% VWR 20847.24
MgCl2 Sigma Prepare a 2 M MgCl2 solution. Filter the solution using a 0.45 µm filter. 
Microplate 96 Well PS F-Botom Black Non-Binding Greiner Bio-one 655900
Mini-Extruder with two 1 mL gas-tight Hamilton syringes Avanti Polar Lipids 610023
Monochromator-based fluorescence plate reader TECAN M1000 Pro
N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine) (IANBD Amide) Molecular Probes Dissolve 25 mg of IANBD in 2.5 mL of dimethylsulfoxide (DMSO) and prepare 25 aliquot of 100 µL in 1.5 mL screw-cap tubes. Do not completely screw the cap. Then, remove DMSO in a freeze-dryer to obtain 1 mg of dry IANBD per tube. Tubes are closed and stored at -20 °C in the dark.  
NaCl Sigma S3014-1KG
PBS 137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, autoclaved and stored at 4 °C.
Pear-shaped glass flasks (25 mL, 14/23, Duran glass) Duran Group
Pepstatin Sigma p5318-25mg
pGEX-C2LACT  plasmid Available on request from our lab
pGEX-PHFAPP plasmid Available on request from our lab
Phenylmethylsulfonyl fluoride (PMSF) ≥98.5% (GC) Sigma P7626-25g Prepare a 200 mM PMSF stock solution in isopropanol
Phosphoramidon Sigma R7385-10mg
Polycarbonate filters (19 mm in diameter) with pore size of 0.2 µm Avanti Polar Lipids 610006
Poly-Prep chromatography column (with a 0-2 mL bed volume and a 10 mL reservoir) Biorad 7311550
Prefilters (10 mm in diameter). Avanti Polar Lipids 610014
PyMOL http://pymol.org/ Construction of the 3D models of the proteins (Figure 1A)
Quartz cuvette for UV/visible fluorescence (minimum volume of 600 µL) Hellma
Quartz cuvettes Hellma
Refrigerated centrifuge  Eppendorf 5427R Eppendorf
Rotary evaporator Buchi B-100
Screw-cap microcentriguge tubes (1.5 mL) Sarsted
Small magnetic PFTE stirring bar (5 × 2 mm)
Snap-cap microcentriguge tubes (0.5, 1, and 2 mL) Eppendorf
SYPRO orange fluorescent stain to detect protein in SDS-PAGE gel
Thermomixer Starlab
THROMBIN, FROM HUMAN PLASMA Sigma 10602400001 Dissolve 20 units in 1 mL of milli-Q water and prepare 25 µL aliquots in 0.5 mL Eppendorf tubes. Then freeze and store at -80 °C.
Tris, ultra pure MP 819623
Ultracentrifuge L90K Beckman
UV/Visible absorbance spectrophotometer SAFAS
UV/visible spectrofluorometer with a temperature-controlled cell holder and stirring device Jasco or Shimadzu Jasco FP-8300 or Shimadzu RF-5301PC
Vacuum chamber
Water bath Julabo
XK 16/70 column packed with Sephacryl S200HR GE healthcare

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