Name: fluorescence-based measurements of phosphatidylserine/phosphatidylinositol 4-phosphate exchange between membranes
Uploaded: 2021-03-14T13:30:00
Description: Watch this Scientific Journal Video about Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes at JoVE.com

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.

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...

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...

The precise distribution of lipids between different membranes and within the membranes of eukaryotic cells1,2&#160;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, an in vitro, fluorescence-based strategy is presented that has been instrumental in establishing that several ORP/Osh proteins effect PS/PI(4)P exchange between cell membranes12 and thereby constitute a new class of LTPs. PS is an anionic glycerophospholipid that represents 2-10% of total membrane lipids in eukaryotic cells13,14,16. It is distributed along a gradient between the ER and the PM, where it represents 5-7% and up to 30% of glycerophospholipids, respectively17,18,19. Moreover, PS is essentially concentrated in the cytosolic leaflet of the PM. This build-up and the uneven partition of PS in the PM are critical for cellular signaling processes19. Owing to the negative charge of PS molecules, the cytosolic leaflet of the PM is much more anionic than the cytosolic leaflet of other organelles1,2,19,20. This enables the recruitment, via electrostatic forces, of signaling proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS)21, sarcoma (Src)22, Kirsten-rat sarcoma viral oncogene (K-Ras)23, and Ras-related C3 botulinum toxin substrate 1 (Rac1)24 that contain a stretch of positively charged amino acids and a lipidic tail.
PS is also recognized by conventional protein kinase C in a stereoselective manner via a C2 domain25. However, PS is synthesized in the ER26, indicating that it must be exported to the PM before it can play its role. It was not known how this was accomplished19 until the finding that, in yeast, Osh6p and Osh7p transfer PS from the ER to the PM27. These LTPs belong to an evolutionarily conserved family in eukaryotes whose founding member is OSBP and that contains proteins (ORPs in human, Osh proteins in yeast) integrating an OSBP-related domain (ORD) with a pocket to host a lipid molecule. Osh6p and Osh7p consist only of an ORD whose structural features are adapted to specifically bind PS and transfer it between membranes. Nevertheless, how these proteins directionally transferred PS from the ER to the PM was unclear. Osh6p and Osh7p can trap PI(4)P as an alternative lipid ligand12. In yeast, PI(4)P is synthesized from phosphatidylinositol (PI) in the Golgi and the PM&#160;by PI 4-kinases, Pik1p and Stt4p, respectively. In contrast, there is no PI(4)P in the ER membrane, as this lipid is hydrolyzed to PI by the Sac1p phosphatase. Hence, a PI(4)P gradient exists at both the ER/Golgi and ER/PM interfaces. Osh6p and Osh7p transfer PS from the ER to the PM via PS/PI(4)P exchange cycles using the PI(4)P gradient that exists between these two membranes12.
Within one cycle, Osh6p extracts PS from the ER, exchanges PS for PI(4)P at the PM and transfers PI(4)P back to the ER to extract another PS molecule. Osh6p/Osh7p interact with Ist2p28, one of the few proteins that connect and bring the ER membrane and the PM into close proximity with each other to create ER-PM contact sites in yeast29,30,31. In addition, the association of Osh6p with negatively charged membranes becomes weak as soon as the protein extracts one of its lipid ligands due to a conformational change that modifies its electrostatic features32. This aids Osh6p by shortening its membrane dwell time, thereby maintaining the efficiency of its lipid transfer activity. Combined with the binding to Ist2p, this mechanism could allow Osh6p/7p to both quickly and accurately execute lipid exchange at the ER/PM interface. In human cells, ORP5 and ORP8 proteins execute PS/PI(4)P exchange at ER-PM contact sites via distinct mechanisms33. They have a central ORD, akin to Osh6p, but are directly anchored to the ER via a C-terminal transmembrane segment33 and dock into the PM via an N-terminal Pleckstrin homology (PH) domain that recognizes PI(4)P and PI(4,5)P233,34,35. ORP5/8 use PI(4)P to transfer PS, and it has been shown that ORP5/8 additionally regulate PM PI(4,5)P2 levels and presumably modulate signaling pathways. In turn, a decrease in PI(4)P and PI(4,5)P2 levels lowers ORP5/ORP8 activity as these proteins associate with the PM in a PIP-dependent fashion. Abnormally high PS synthesis, which leads to Lenz-Majewski syndrome, impacts PI(4)P levels through ORP5/836. When the activity of both proteins is blocked, PS becomes less abundant at the PM, lowering the oncogenic capability of signaling proteins37.
Conversely, ORP5 overexpression seems to promote cancer cell invasion and metastatic processes38. Thus, alterations to ORP5/8 activity can severely modify cellular behavior through changes in lipid homeostasis. Further, ORP5 and ORP8 occupy ER-mitochondria contact sites and preserve some mitochondrial functions, possibly by supplying PS39. Additionally, ORP5 localizes to ER-lipid droplet contact sites to deliver PS to lipid droplets by PS/PI(4)P exchange40. The strategy described herein to measure (i) PS and PI(4)P extraction from liposomes and (ii) PS and PI(4)P transport between liposomes has been devised to establish and analyze the PS/PI(4)P exchange activity of Osh6p/Osh7p12,32 and used by other groups to analyze the activity of ORP5/ORP835 and other LTPs10,41. It is based on the use of a fluorescence plate reader, a standard L-format spectrofluorometer, and two fluorescent sensors, NBD-C2Lact and NBD-PHFAPP, that can detect PS and PI(4)P, respectively.
NBD-C2Lact corresponds to the C2 domain of the glycoprotein, lactadherin, that was reengineered to include a unique solvent-exposed cysteine near the presumed PS binding site; a polarity-sensitive NBD (7-nitrobenz-2-oxa-1,3-diazol) fluorophore is covalently linked to this residue (Figure 1A)12. To be more precise, the C2 domain of lactadherin (Bos taurus, UniProt: Q95114,residues 270-427) was cloned into a pGEX-4T3 vector to be expressed in fusion with glutathione S-transferase (GST) in Escherichia coli. The C2Lact sequence was then mutated to substitute two solvent-accessible cysteine residues (C270, C427) with alanine residues and to introduce a cysteine residue into a region near the putative PS-binding site (H352C mutation) that can be subsequently labeled with N,N&#39;-dimethyl-N-(iodoacetyl)-N&#39;-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (IANBD)&#160;12. A cleavage site for thrombin is present between the GST protein and the N-terminus of the C2 domain. A major advantage is that this domain selectively recognizes PS in a Ca2+-independent manner contrary to other known C2 domains or Annexin A542. NBD-PHFAPP is derived from the PH domain of the human&#160;four-phosphate-adaptor protein 1 (FAPP1), which was reengineered to include a single solvent-exposed cysteine that can be labeled with an NBD group near the PI(4)P binding site (Figure 1A)43. The nucleotide sequence of the PH domain of the human FAPP protein (UniProt: Q9HB20, segment [1-100]) has been cloned into a pGEX-4T3 vector to be expressed in tandem with a GST tag. The PHFAPP sequence has been modified to insert a unique cysteine residue within the membrane-binding interface of the protein43. Moreover, a nine-residue linker has been introduced between the thrombin cleavage site and the N-terminus of the PH domain to ensure accessibility to the protease.
To measure PS extraction from liposomes, NBD-C2Lact is mixed with liposomes made of phosphatidylcholine (PC) containing trace amounts of PS. Owing to its affinity for PS, this construct binds to the liposomes, and the NBD fluorophore experiences a change in polarity as it comes into contact with the hydrophobic environment of the membrane, which elicits a blue-shift and an increase in fluorescence. If PS is extracted almost completely by a stoichiometric amount of LTP, the probe does not associate with liposomes, and the NBD signal is lower (Figure 1B)32. This difference in signal is used to determine whether an LTP (e.g., Osh6p) extracts PS. A similar strategy is used with NBD-PHFAPP to measure PI(4)P extraction (Figure 1B), as described previously12,32. Two FRET-based assays were designed to (i) measure PS transport from LA to LB liposomes, which mimic the ER membrane and the PM, respectively, and (ii) PI(4)P transport in the reverse direction. These assays are performed under the same conditions (i.e., same buffer, temperature, and lipid concentration) to measure PS/PI(4)P exchange. To measure PS transport, NBD-C2Lact is mixed with LA liposomes composed of PC and doped with 5 mol% PS and 2 mol% of a fluorescent rhodamine-labelled phosphatidylethanolamine&#160;(Rhod-PE)-and LB liposomes incorporating 5 mol% PI(4)P.
At time zero, FRET with Rhod-PE quenches the NBD fluorescence. If PS is transported from LA to LB liposomes (e.g., upon injecting Osh6p), a fast dequenching occurs due to the translocation of NBD-C2Lact molecules from LA to LB liposomes (Figure 1C). Given the amount of accessible PS, NBD-C2Lact remains essentially in a membrane-bound state over the course of the experiment12. Thus, the intensity of the NBD signal directly correlates with the distribution of NBD-C2Lact between LA and LB liposomes and can be easily normalized to determine how much PS is transferred. To measure the transfer of PI(4)P in the opposite direction, NBD-PHFAPP is mixed with LA and LB liposomes; given that it only binds to LB liposomes that contain PI(4)P, but not Rhod-PE, its fluorescence is high. If PI(4)P is transferred to LA liposomes, it translocates to these liposomes, and the signal decreases due to FRET with Rhod-PE (Figure 1C). The signal is normalized to determine how much PI(4)P is transferred43.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;L-cysteine &#8805;97 % (FG)&#160;</td>
			<td>Sigma</td>
			<td>W326305-100G</td>
			<td>Prepare a 10 mM L-cysteine stock solution in water. Aliquots are stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>2 mL Amber Vial, PTFE/Rub Lnr, for lipids storage in CHCL3</td>
			<td>Wheaton</td>
			<td>W224681</td>
			<td></td>
		</tr>
		<tr>
			<td>4 mm-diameter glass beads</td>
			<td>Sigma</td>
			<td>Z265934-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>Falcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTA purifier</td>
			<td>GE healthcare</td>
			<td></td>
			<td>FPLC</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 with a MWCO of 3 and 10 kDa</td>
			<td>Merck</td>
			<td>UFC900324, UFC901024</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 with a MWCO of 3 and 10 kDa</td>
			<td>Merck</td>
			<td>UFC800324, UFC801024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td></td>
			<td></td>
			<td>Prepare a 50 mg/mL stock solution with filtered and sterilized water and store it at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Bestatin</td>
			<td>Sigma</td>
			<td>B8385-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Gold Competent Cells</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C16:0 Liss&#160; (Rhod-PE) in CHCl3 (1 mg/mL)</td>
			<td>Avanti Polar Lipids</td>
			<td>810158C-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>C16:0/C16:0-PI(4)P&#160;&#160;</td>
			<td>Echelon Lipids</td>
			<td>P-4016-3</td>
			<td>Dissolve 1 mg of C16:0/C16:0-PI(4)P powder in 250 &#181;L of MeOH and 250 &#181;L of CHCl3. Then complete with CHCl3 to 1 mL. The solution must become clear.</td>
		</tr>
		<tr>
			<td>C16:0/C18:1-PS (POPS) in CHCl3 (10 mg/mL)</td>
			<td>Avanti Polar Lipids</td>
			<td>840034C-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>C18:1/C18:1-PC (DOPC) in CHCl3 (25 mg/mL)</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C-500mg</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td>Prepare 10 mM CaCl2 stock solution in water.</td>
		</tr>
		<tr>
			<td>Cell Disruptor</td>
			<td>Constant Dynamics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform (CHCl3) RPE-ISO</td>
			<td>Carlo Erba</td>
			<td>438601</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized (Milli-Q) water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF), anhydrous, &#62;99% pure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I Recombinant, RNAse free, in powder</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Euromedex</td>
			<td>EU0006-B</td>
			<td>Prepare 1 M DTT stock solution in Milli-Q water.&#160; Prepare 1 mL aliquots and store them at -20 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>Econo-Pac chromatography columns (1.5 &#215; 12 cm).</td>
			<td>Biorad</td>
			<td>7321010</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvette 2 mm</td>
			<td>Ozyme</td>
			<td>EP102</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator Eppendorf 2510</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed-Angle Rotor Ti45 and Ti45 tubes</td>
			<td>Beckman</td>
			<td>Spinning the batcerial lysates</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-syringes (10, 25, and 50 &#181;L) for fluorescence experiment</td>
			<td>Hamilton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-syringes (25 , 100, 250, 500, and 1000 &#181;L) to handle lipid stock solutions</td>
			<td>Hamilton</td>
			<td>1702RNR, 1710RNR, 1725RNR, 1750RN type3, 1001RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose 4B beads</td>
			<td>GE Healthcare</td>
			<td>17-0756-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol (99% pure)</td>
			<td>Sigma</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemolysis tubes with a cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES , &#62;99 % pure</td>
			<td>Sigma</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Illustra NAP 10 desalting column</td>
			<td>GE healthcare</td>
			<td>GE17-0854-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)&#160;</td>
			<td>Euromedex</td>
			<td>EU0008-B</td>
			<td>Prepare 1 M IPTG stock solution in Milli-Qwater. Prepare 1 mL aliquots and store them at -20 &#176;C.&#160;</td>
		</tr>
		<tr>
			<td>K-Acetate</td>
			<td>Prolabo</td>
			<td>26664.293</td>
			<td></td>
		</tr>
		<tr>
			<td>Lennox LB Broth medium without glucose</td>
			<td></td>
			<td></td>
			<td>Prepared with milli-Q water and autoclaved.</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Linde</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (MeOH) &#8805;99.8%</td>
			<td>VWR</td>
			<td>20847.24</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td></td>
			<td>Prepare a 2 M MgCl2 solution. Filter the solution using a 0.45 &#181;m filter.&#160;</td>
		</tr>
		<tr>
			<td>Microplate 96 Well PS F-Botom Black Non-Binding</td>
			<td>Greiner Bio-one</td>
			<td>655900</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Extruder with two 1 mL gas-tight Hamilton syringes</td>
			<td>Avanti Polar Lipids</td>
			<td>610023</td>
			<td></td>
		</tr>
		<tr>
			<td>Monochromator-based fluorescence plate reader</td>
			<td>TECAN</td>
			<td>M1000 Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-Dimethyl-N-(Iodoacetyl)-N&#39;-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine) (IANBD Amide)</td>
			<td>Molecular Probes</td>
			<td></td>
			<td>Dissolve 25 mg of IANBD in 2.5 mL of dimethylsulfoxide (DMSO) and prepare 25 aliquot of 100 &#181;L 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 &#176;C in the dark.&#160;&#160;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td>137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, autoclaved and stored at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Pear-shaped glass flasks (25 mL, 14/23, Duran glass)</td>
			<td>Duran Group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Sigma</td>
			<td>p5318-25mg</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX-C2LACT&#160; plasmid</td>
			<td></td>
			<td></td>
			<td>Available on request from our lab</td>
		</tr>
		<tr>
			<td>pGEX-PHFAPP plasmid</td>
			<td></td>
			<td></td>
			<td>Available on request from our lab</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF) &#8805;98.5% (GC)</td>
			<td>Sigma</td>
			<td>P7626-25g</td>
			<td>Prepare a 200 mM PMSF stock solution in isopropanol</td>
		</tr>
		<tr>
			<td>Phosphoramidon</td>
			<td>Sigma</td>
			<td>R7385-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filters (19 mm in diameter) with pore size of 0.2 &#181;m</td>
			<td>Avanti Polar Lipids</td>
			<td>610006</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Prep chromatography column (with a 0-2 mL bed volume and a 10 mL reservoir)</td>
			<td>Biorad</td>
			<td>7311550</td>
			<td></td>
		</tr>
		<tr>
			<td>Prefilters (10 mm in diameter).</td>
			<td>Avanti Polar Lipids</td>
			<td>610014</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>http://pymol.org/</td>
			<td></td>
			<td>Construction of the 3D models of the proteins (Figure 1A)</td>
		</tr>
		<tr>
			<td>Quartz cuvette for UV/visible fluorescence (minimum volume of 600 &#181;L)</td>
			<td>Hellma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz cuvettes</td>
			<td>Hellma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge&#160; Eppendorf 5427R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Buchi</td>
			<td>B-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw-cap microcentriguge tubes (1.5 mL)</td>
			<td>Sarsted</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small magnetic PFTE stirring bar (5 &#215; 2 mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Snap-cap microcentriguge tubes (0.5, 1, and 2 mL)</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYPRO orange</td>
			<td></td>
			<td></td>
			<td>fluorescent stain to detect protein in SDS-PAGE gel</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Starlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THROMBIN, FROM HUMAN PLASMA</td>
			<td>Sigma</td>
			<td>10602400001</td>
			<td>Dissolve 20 units in 1 mL of milli-Q water and prepare 25 &#181;L aliquots in 0.5 mL Eppendorf tubes. Then freeze and store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Tris, ultra pure</td>
			<td>MP</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge L90K</td>
			<td>Beckman</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Visible absorbance spectrophotometer</td>
			<td>SAFAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV/visible spectrofluorometer with a temperature-controlled cell holder and stirring device</td>
			<td>Jasco or Shimadzu</td>
			<td>Jasco FP-8300 or Shimadzu RF-5301PC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Julabo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XK 16/70 column packed with Sephacryl S200HR</td>
			<td>GE healthcare</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence-based measurements of phosphatidylserine/phosphatidylinositol 4-phosphate exchange between membranes

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.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62177/62177fig01.jpg" /> 
Figure 1:&#160;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 ...

Watch this Scientific Journal Video about Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes at JoVE.com

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

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 ...

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