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

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 to perform an analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15% acrylamide gel to check the progress of the purification. Mix enough denaturing Laemmli sample buffer with each aliquot, and heat the mixture at 95 °C. Freeze and store the tubes at -20 °C until analysis.

1. Expression of GST-C2_Lact de Escherichia coli
   1. Mix 20 µL of BL21 Gold competent cells with 18 µL of sterilized water. Then, mix 2 µL of pGEX-C2_Lact plasmid (at ~65 ng/µL) with the bacteria, and transform them by electroporation. Resuspend the bacteria with 150 µL of autoclaved Lennox Lysogeny-Broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl in deionized water, glucose-free). Let the bacteria grow at 37 °C for 1 h in a 2 mL snap-cap microcentrifuge tube.
   2. Inoculate 25 mL of LB medium, supplemented with 50 µg/mL ampicillin, with 150 µL of bacterial suspension in a 125 mL sterile Erlenmeyer flask. Place the flask in an orbital shaker at 37 °C, and let the bacteria grow overnight with agitation at 185 rpm.
   3. Fill two sterile 2 L Erlenmeyer flasks with 500 mL of LB medium supplemented with 50 µg/mL ampicillin, and add 5 mL of preculture suspension. Let the bacteria grow at 37 °C with agitation at 220 rpm.
   4. Periodically measure the optical density (OD) of the suspension at a wavelength (λ) of 600 nm. When the OD reaches a value of ~0.6-0.7, add 500 µL of a stock solution of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) to each flask to initiate the expression of GST-C2_Lact. Shake the flasks at 185 rpm for 4 h at 37 °C.
   5. Transfer the contents of each flask to a polypropylene centrifuge bottle. Centrifuge the two bottles for 30 min at 4600 × g at 4 °C to pellet the bacteria. Discard the supernatant, and resuspend each pellet in 50 mL of cold phosphate-buffered saline.
   6. Transfer the bacterial suspension contained in each bottle to a 50 mL conical centrifuge tube. Centrifuge the two tubes for 30 min at 2300 × g at 4 °C. Remove the supernatant, and store the tubes, each of which contains a bacterial pellet, at -20 °C.
2. Purification of C2_Lact
   1. On ice, fill two 50 mL conical centrifugal tubes with 50 mL of buffer containing 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl (hereafter called TN buffer), previously filtered and degassed by membrane vacuum filtration.
   2. To prepare the lysis buffer in each tube, dissolve a tablet of ethylenediamine tetraacetic acid-free protease inhibitor cocktail in the TN buffer by mild sonication or vortexing. Add other antiproteases, (10 µM bestatin, 1 µg/mL pepstatin A, and 10 µM phosphoramidon). Importantly, supplement the buffer with 2 mM DTT.
   3. Fill the two tubes that contain the bacterial pellets prepared in step 1.1.6, with lysis buffer to obtain a final volume of 30 mL in each tube, and slowly defrost the pellets on ice for 10 min. Crush each pellet with a stainless steel spatula, and resuspend them by vortexing the tubes and/or by pipetting the suspension back and forth with a pipette controller and a 25 mL pipette until a homogeneous suspension is obtained.
   4. Perform lysis using a precooled cell disruptor (see the Table of Materials) by loading 30 mL of the sample inside the reservoir and running a breaking cycle in continuous mode with a pressure of 1.6 bar. Collect the lysate in the same tube, keep the tube on ice, and immediately add 250 µL from a stock solution of 200 mM phenylmethylsulfonyl fluoride (PMSF) prepared in isopropanol.
   5. Lyse the other sample following the same procedure. Use the remainder of the lysis buffer to wash the cell disruptor, and collect the wash to adjust the volume of each lysate (~30 mL) to a final volume of 50 mL.
   6. Supplement each lysate with 5 mM MgCl₂,and add 20 µg/mL of DNAse I to fragment the DNA and thus reduce the viscosity of the sample. Incubate on ice for 30 min. Collect a sample for gel analysis.
   7. Transfer each 50 mL lysate to a prechilled polycarbonate ultracentrifuge tube (two in total, see the Table of Materials). Centrifuge at 186,000 × g at 4 °C for at least 1 h using an ultracentrifuge.
   8. In parallel to the centrifugation step, dispense 1.4 mL of a slurry containing glutathione coupled to 4% agarose beads into two 50 mL conical centrifugal tubes, add 20 mL of TN buffer supplemented with 1 mM DTT (TND buffer) to each tube, centrifuge at 1200 × g for 5 min, and discard the supernatant. Repeat this washing step twice.
   9. After the centrifugation of the bacterial lysate, remove a 30 µL sample from the supernatant, and transfer the supernatant from each ultracentrifuge tube to a corresponding 50 mL conical centrifuge tube that contains clean beads. For gel analysis, resuspend the debris pellet in one of the ultracentrifuge tubes with 50 mL of TND buffer, and collect a 30 µL sample.
   10. Place the tubes on a rotator for 3-4 h at 4 °C to obtain a homogeneous bead suspension. Pool the bead suspensions in an empty 25 mL chromatography column. Let the beads decant, and remove the buffer and unbound proteins by gravity flow. Take a sample from the eluate for analysis.
   11. Resuspend the beads with 20 mL of TND buffer, and collect the eluate by gravity flow. Repeat this step twice to completely wash the beads. Pool the collected eluates, and retain a 30 µL sample for further analysis.
       NOTE: After a short decantation, a volume of ~2 mL of bead suspension, to which GST-C2_Lact is attached, sediments at the bottom of the column.
   12. Add 1 mL of bead suspension to two 2 mL snap-cap microcentrifuge tubes. Fill each tube with TND buffer to a final volume of 1.970 mL. Take a 30 µL sample from one tube for further analysis (B1 sample). Add 10 µL of 10 mM CaCl₂ solution and 25 µL from a stock solution of human thrombin protease solution at 0.02 U/µL.
   13. Place the two tubes on a rotator at 4 °C overnight to allow thrombin to cleave off the GST tag from the C2_Lact domain. On the next day, in each tube, mix 10 µL of 200 mM PMSF solution with the bead suspension to inhibit the thrombin action.
   14. Centrifuge the tubes at 700 × g for 5 min, and collect the supernatant, which contains soluble C2_Lact domain, from each tube, without taking the beads. Pool the supernatants into a 2 mL snap-cap microcentrifuge tube (E1 eluate) that is kept on ice.
   15. Add 1 mL of TND buffer to each tube to resuspend the beads, and wash them; repeat step 1.2.14. Perform this step thrice more to recover a maximum amount of protein. Each time, pool the collected supernatants into a new 2 mL tube (E2, E3, E4, and E5 eluates), and take an aliquot for further analysis. At the end of washing steps, take an aliquot from the bead suspension (aliquot B2).
   16. Analyze the 30 µL samples that were collected at the different steps of the purification protocol by SDS-PAGE separation on a 15% acrylamide gel.
   17. Remove potential contaminating beads by pooling all the supernatants (i.e., ~10 mL) collected during steps 1.2.14 and 1.2.15 into a 10 mL chromatography column. Collect the eluate by gravity flow, and retain the beads at the bottom of the column.
   18. Concentrate the C2_Lact sample using a centrifugal filter unit with a molecular weight cut-off (MWCO) of 3 kDa and a centrifugation speed of 2300 × g. Stop the concentration procedure when the volume of the protein sample is ~1 mL.
3. Preparation and purification of NBD-C2_Lact
   1. Equilibrate a desalting column (see the Table of Materials) with TN buffer. Load the column with 1 mL of concentrated C2_Lact sample. Allow the sample to enter the gel bed completely, add 1.5 mL of freshly degassed DTT-free TN buffer to the column, and collect the eluate by gravity flow into a 2 mL snap-cap microcentrifuge tube.
   2. Dilute 50 µL of eluate in a final volume of 300 µL TN buffer, and record an absorbance spectrum from 230 to 450 nm using pure TN buffer as a blank. Determine the C2_Lact concentration based on the absorbance measured at 280 nm, considering an extinction coefficient ε equal to 44,920 M^-1.cm^-1.
   3. To label the C2_Lact construct with a NBD fluorophore, mix the protein with a ten-fold molar excess of N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD amide).
      1. Dissolve 1 mg of IANBD in anhydrous dimethylformamide (DMF), keeping in mind that the final volume of DMF used for labeling the C2_Lact construct must not exceed 5% (v/v) of the protein sample volume.
      2. To determine the volume of DMF (V_DMF) to dissolve IANBD, first calculate the required amount of IANBD (m, expressed in mg) to label the protein by using formula 1.
         m = 10,000 × C × V × MW_IANBD (1)
         ​where C is the concentration of C2_Lact measured in step 1.3.2, V is the volume of the C2_Lact sample, and MW_IANBD is the molecular weight of the fluorophore (420 g/mol).
      3. Calculate V_DMF by using formulae 2 (with m₀=1) and 3.
         V_DMF= (m₀/m) × V_IANBD (2)
         V_IANBD=0.05 × V (3)
         Where m₀ is the quantity of IANBD powder in mg, and V_IANBD is the volume of IANBD solution to be added to the C2_Lact sample.
      4. Add volume V_IANBD of the freshly-prepared IANBD solution to the C2_Lact sample, and shake the reaction mixture at 800-900 rpm for 30 min at 25 °C using a thermomixer protected from light. Let the reaction proceed for 90 min on ice. In the meantime, clean a centrifugal filter unit (MWCO= 3 kDa) with 10 mL of TN buffer.
   4. Add L-cysteine (in 10-fold molar excess to IANBD) to the reaction mixture to inactivate free IANBD. 
   5. Add 15 mL of TN buffer to the NBD-C2_Lact solution, and transfer the NBD-C2_Lact solution to the centrifugal filter unit. Concentrate the sample to 2 mL to separate most of the free NBD from the protein by centrifugation at 2300 × g. Repeat this washing step two times. Centrifuge the sample in a 2 mL snap-cap centrifuge tube for 10 min at 19,000 × g at 4 °C to pellet potential aggregates, and collect the supernatant.
   6. Dilute 50 µL of eluate in a final volume of 300 µL of TN buffer. Record the absorbance spectrum from 230 to 650 nm using the eluate collected during the concentration procedure as a blank. Determine the NBD-C2_Lact concentration using the maximal absorbance at λ=280 and 495 nm and extinction coefficients ε= 44,920 M^-1.cm^-1 (protein) and 25,000 M^-1.cm^-1 (NBD fluorophore).
      NOTE: If the two concentration values are alike, this indicates that the C2_Lact construct is labeled at a 1:1 ratio with the NBD group.
   7. If the NBD-C2_Lact concentration estimated from the measurement of NBD absorbance exceeds the concentration estimated from the absorbance of tryptophan (Trp) residues, repeat step 1.3.5 to further remove free NBD.
   8. Add glycerol to the sample to obtain a final concentration of 10% (v/v) to cryo-protect the NBD-C2_Lact construct during flash-freezing. Measure the final protein concentration.
   9. Prepare 50 µL aliquots of protein in 0.5 mL snap-cap microcentrifuge tubes. Flash-freeze the tubes in liquid nitrogen, and store them at -80 °C.

2. Purification of NBD-PH_FAPP

NOTE: The procedure to produce and label PH_FAPP is identical to that of NBD-C2_Lact until the transfer of NBD-C2_Lact solution to a centrifugal filter unit in step 1.3.4. From this step onwards, follow the protocol that is described below.

1. After the concentration step, keep 2 mL of NBD-PH_FAPP at 4 °C in the dark for not more than 1 day before performing size-exclusion chromatography. Prior to size-exclusion chromatography, verify that there is no orange deposit (aggregation during concentration) at the bottom of the tube. If this is the case, centrifuge the sample at 540,000 × g for 10 min at 4 °C, and purify the supernatant by size-exclusion chromatography.
   NOTE: Size-exclusion chromatography is performed on a column packed with crosslinked dextran-acrylamide copolymer (see the Table of Materials), previously equilibrated with TND buffer, using a fast protein liquid chromatography system (see the Table of Materials). The column must be protected from light. A flow rate of 1 mL/min was used, and the elution of the NBD-PH_FAPP construct was followed by recording the absorbance at λ=280 (protein) and 480 nm (NBD) at the column exit.
   1. Inject the NBD-PH_FAPP sample loaded in a 2 mL injection loop onto the column, and immediately collect 2.5 mL fractions of eluate.
2. Analyze all the fractions that correspond to the main peak detected at 280 and 480 nm on a 15% SDS-PAGE gel. Mix a 25 µL sample of each fraction with 15 µL of Laemmli sample buffer prior to heating and loading onto the gel.
   NOTE: A main peak, which is simultaneously detected at λ= 280 and 480 nm, appears once a volume of ~150 mL buffer is passed through the column.
3. Pool the fractions that exclusively contain NBD-PH_FAPP protein (~12.2 kDa), and add glycerol at a final concentration of 10% (v/v). Concentrate the sample using a centrifugal filter unit with a MWCO of 3 kDa to a final volume of 1 mL using a centrifugation speed of 2300 × g.
4. Prepare aliquots, and record an absorbance spectrum as described for NBD-C2_Lact. Use an extinction coefficient ε= 29,450 M^-1.cm^-1 to determine the concentration of the protein based on the absorbance measured at λ=280 nm.

3. Preparation of liposomes for PS and PI(4)P extraction or transfer assays

NOTE: Perform all the steps at room temperature unless otherwise specified. Handle organic solvents, rotavapor, and liquid nitrogen with caution.

1. Prepare fresh, filtered, and degassed 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-potassium hydroxide (KOH), pH 7.4, 120 mM potassium acetate (HK) buffer.
2. For each type of liposome, take precise amounts of different lipids from stock solutions, and mix them in a 25 mL pear-shaped glass flask (Table 1). Add pure chloroform to adjust the volume of each mixture to 1 mL. Label each flask with the liposome name. Wrap the flasks containing lipid mixture doped with Rhod-PE with aluminum foil.
3. Place the flask on a rotary evaporator. Dry the lipids under vacuum and at 25 °C for at least 30 min at a rotation speed of 500 rpm. For lipid films that contain PI(4)P, prewarm the flask at 32-34 °C for 5 min, under gentle rotation, to properly mix PI(4)P with the other lipids before creating vacuum in the flask to remove the solvent, which will leave behind a film of dry lipids on the flask wall.
4. Disconnect the flask from the evaporator, and place it in a vacuum chamber for 45 min to remove any remaining traces of solvent. Fill the flask with 2 mL of HK buffer, and add a few 4 mm-diameter glass beads to the solution. Gently vortex the flask for 2 min to resuspend the lipids and prepare multilamellar lipid vesicles (MLVs) with a lipid concentration of 4 mM. Prepare 0.5 mL aliquots of MLVs in 1.5 mL screw-cap microcentrifuge tubes.
5. Freeze-thaw the tubes 5x (using liquid nitrogen and a water bath at 37 °C, respectively). Extrude the liposomes or store them at -20 °C.
6. Use a mini extruder to prepare the liposomes (i.e., large unilamellar vesicles) from the MLVs according to the manufacturer's guidelines. Use a polycarbonate filter with uniform cylindrical pores of 200 nm diameter.
7. To prepare each type of liposome, extrude at least 250 µL of the corresponding suspension of MLVs. Store the extruded liposomes at 4 °C and in the dark if they contain Rhod-PE. Use the liposomes within 2 days.

Table 1: Volumes of lipid stock solutions to be mixed for liposome preparation. Abbreviations: PS= phosphatidylserine; PC = phosphatidylcholine; PI(4)P = phosphatidylinositol 4-phosphate; Rhod-PE = rhodamine-labelled phosphatidylethanolamine; DOPC = dioleoylphosphatidylcholine; POPS= 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; 16:0 Liss Rhod-PE = 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl).

4. Measurement of PS or PI(4)P extraction

NOTE: Measurements must be conducted using a black 96-well plate and a fluorescence plate reader equipped with monochromators: one for fluorescence excitation and one for emission, with a variable bandwidth.

1. Prepare fresh, filtered, and degassed HK buffer supplemented with 1 mM MgCl₂ (HKM buffer). Prepare pure PC liposomes and PC liposomes doped with 2 mol% PS or 2 mol% PI(4)P (4 mM final lipid concentration, see Table 1).
   NOTE: Keep the tubes filled with the suspension of extruded liposomes at room temperature throughout the experiment, and keep the proteins on ice. Additionally, protect the lipid sensors from light.
2. For the PS extraction assay, in one well, mix liposomes containing 2 mol% PS (80 µM final lipid concentration, 0.8 µM accessible PS concentration) with NBD-C2_Lact (250 nM final concentration) in a final volume of 100 µL. Fill a second well with the same amount of liposome (80 µM, 2 mol% PS) and NBD-C2_Lact (250 nM) mixed with 3 µM LTP (Osh6p as a positive control or a protein of interest).
   NOTE: An incubation time of 5 min is sufficient for Osh6p to achieve lipid extraction.
3. Fill a third well with NBD-C2_Lact (250 nM) mixed with pure PC liposomes (80 µM). Fill a fourth well only with pure PC liposomes (80 µM). Repeat steps 4.2-4.3 to prepare three additional series of four wells.
4. For each well, record an NBD spectrum from 505 to 650 nm (bandwidth 5 nm) upon excitation at 490 nm (bandwidth 5 nm) at 25 °C. For each series, subtract the spectrum recorded with only liposomes from the other spectra.
   NOTE: F and F_max correspond to the intensity at 536 nm measured with PS-containing liposomes in the presence or absence of LTP, respectively, whereas F₀ is the intensity at the same wavelength with pure PC liposomes. For each series, the percentage of accessible PS that is extracted by the protein is given by using the following formula.
   100 × (1-((F-F₀)/(F_max-F₀))) (4)
5. For the PI(4)P extraction assay, prepare liposomes doped with 2 mol% PI(4)P, and carry out measurements with the NBD-PH_FAPP probe. Perform control experiments and determine the extraction percentage in the same way as described above.
   NOTE: Liposome and protein concentration are identical to those used in the PS extraction assay.

5. Real-time measurement of PS transport

NOTE: A standard fluorimeter (90° format) equipped with a temperature-controlled cell holder and a magnetic stirrer is used to record lipid transfer kinetics. To accurately acquire data, it is key to permanently maintain the sample at the same temperature (set between 25 and 37 °C depending on the origin of the protein (e.g., yeast or human)) and to constantly stir it. The protocol described below is for the measurement of lipid transport in a 600 µL sample contained in a cylindrical quartz cell.

1. Prepare freshly degassed and filtered HKM buffer. Keep the tubes containing extruded liposomes at room temperature. Wrap tubes containing liposomes with Rhod-PE in aluminum foil, and/or store them in an opaque box to prevent any photobleaching.
2. Adjust the excitation and emission monochromators at λ = 460 nm (with a short bandwidth (1-3 nm)) and at λ = 530 nm (with a large bandwidth (≥ 10 nm)), respectively. Set the acquisition time at 25 min with a time resolution ≤ 1 s.
3. In the quartz cuvette, dilute 30 µL of the L_A liposome suspension and a volume of NBD-C2_Lact stock solution in prewarmed HKM buffer to prepare a 570 µL sample that contains 200 µM total lipids and 250 nM NBD-C2_Lact. Add a small magnetic stirring bar, and position the cuvette in the fluorometer holder.
4. Once the sample is thermally equilibrated (after 3-5 min), trigger the measurement. After 1 min, add 30 µL of L_B liposome suspension (final concentration of 200 µM total lipids) to the sample. After 3 min, inject LTP into the sample so that the final concentration of the LTP is 200 nM, and acquire the signal for the remaining 21 min.
5. Carry out a parallel experiment to normalize the NBD signal. Mix 30 µL of L_A-Eq liposome suspension with 250 nM NBD-C2_Lact in HKM buffer (final volume of 570 µL). After 1 min, inject 30 µL of L_B-Eq liposome suspension.
   NOTE: The lipid composition of L_A-Eq and L_B-Eq liposomes are similar to that of L_A and L_B liposomes used in the transfer assay, except that each of them contains 2.5 mol% PS and 2.5 mol% PI(4)P. As a result, the NBD signal that is measured, referred to as F_Eq, corresponds to the signal that should be measured if PS was fully equilibrated between L_A and L_B liposomes by a transfer process.
6. Convert the kinetic curves measured with an LTP of interest to determine the amount of PS (in µM) transferred from L_A to L_B liposomes over time. Normalize each data point (F) of the curve by using the following formula.
   F_Norm = (F-F₀)/(F_Eq-F₀) (5)
   in which F₀ corresponds to the NBD signal just before the addition of an LTP, and F_Eq is the signal measured in step 5.5.
   NOTE: The amount of PS (in µM) transferred from L_A to L_B liposomes corresponds to 2.5 × F_Norm, considering that the equilibrium corresponds to a situation where one half of accessible PS molecules, contained in the outer leaflet of the L_A liposomes, (i.e., corresponding to 5 mol% of 0.5 × 200 µM total lipids) has been transferred into L_B liposomes.

6. Real-time measurement of PI(4)P transport

1. Set the fluorimeter (excitation and emission wavelength, bandwidth, acquisition time, time resolution) as done for the PS transfer assay. Likewise, use the same buffer, cuvette, and liposomes to perform the experiments under constant stirring at the same temperature.
2. In the cuvette, mix 30 µL of L_B liposome suspension and NBD-PH_FAPP with prewarmed HKM buffer to obtain a final volume of 570 µL (200 µM total lipids, 250 nM NBD- PH_FAPP). Once the thermal equilibration of the sample is reached, start the measurement, and after 1 min, inject 30 µL of L_A liposome suspension. After 3 min, inject the LTP of interest (final concentration of 200 nM), and record the signal.
3. Perform a second experiment to normalize the NBD signal. Mix 30 µL of L_B-Eq liposome suspension with 250 nM NBD-PH_FAPP in 570 µL of HKM buffer. After 1 min, inject 30 µL of L_A-Eq liposome suspension.
   NOTE: Here, the NBD signal, referred to as F_Eq, corresponds to the one which should be measured if PI(4)P was fully equilibrated between L_A and L_B liposomes.
4. Convert the kinetic curves to determine the amount of PI(4)P (in µM) transferred from L_B to L_A liposomes over time. Each data point (F) is normalized by using formula 5 in which F₀ corresponds to the NBD signal prior to the addition of an LTP, and F_Eq is the signal measured in step 6.3.
   NOTE: The amount of PI(4)P (in µM) transferred from L_B to L_A liposomes corresponds to 2.5 × F_Norm, considering that the equilibrium corresponds to a situation where one half of the PI(4)P contained in the outer leaflet of the L_B liposomes (i.e., 0.5 × 5 µM) has been transferred in L_A liposomes.

7. Analysis of kinetics curves

1. Quantify the extent to which an LTP is efficient by determining the speed at which this LTP transfers lipids from one liposome population to the other one in the first few seconds following its injection to the cuvette.
   1. Perform a linear regression of the first data points of the transfer kinetics to obtain a slope. Divide the slope value by the LTP concentration in the reaction mixture to determine the number of lipid molecules transferred per protein per time unit (min or s).