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

Summary

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.

Abstract

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-C2_Lact and NBD-PH_FAPP, 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.

Introduction

The precise distribution of lipids between different membranes and within the membranes of eukaryotic cells^1,2 has profound biological implications. Decrypting how LTPs function is an important issue in cell biology^3,4,5,6, and in vitro approaches are of great value in addressing this issue^7,8,9,10,11. Here, ....

Protocol

1. Purification of NBD-C2_Lact

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 carried out on ice or in a cold room to avoid any protein degradation. Samples of 30 µL volume must be collected at different steps of the protocol ....

Results

Figure 1: Description of the fluorescent lipid sensors and in vitro assays. (A) Three-dimensional models of NBD-C2_Lact and NBD-PH_FAPP 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 .......

Discussion

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.......

Materials

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