Materials and Reagents

Biological Materials

1. Triton X-100 extracted endoplasmic reticulum (ER) membrane proteins (TE) were prepared according to Chalat et al. (2012) and Verchère et al. (2021) resulting in ~1 µg µL^-1, in 10 mM HEPES/NaOH, pH 7.4, 100 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail (Calbiochem, Merck Millipore Sigma, Darmstadt, Germany, catalog number: 539131), 1% (weight/volume) Triton X-100, stored at -80°C.

   Note: The protocol can also be applied to purified protein preparations in combination with reconstitution procedures not based on Triton X-100, as described here. In all cases, samples should be prepared in phosphate-free buffers, to allow for lipid phosphate determination.

   
   

Materials and Reagents

1. Aluminium foil

2. Clamps (e.g., office paper clamps, Lomax, catalog number: 9801110)

3. Copper tape (cut to ~70 mm long, 50 mm wide, 0.07 mm thick, Conrad Electronik, catalog number: 545614-62)

4. Cover glass slides (26 × 76 mm, #1.5, Thermo Fisher Scientific, Life Technologies Corporation Eugene, OR, USA)

5. Detergent for cleaning the ITO glass slides

6. Disposable glass Pasteur pipettes (150 mm; VWR, catalog number: 612-1701)

7. Filters (SAMPLE DISCS, SS-033, ELITechGroup, BIOMEDICAL SYSTEMS, Logan, UT, USA)

8. Forceps (e.g., VWR, catalog number: 232-0032)

9. Glass jar(s) (e.g., Fisher Scientific, catalog number: 15603977)

10. Glass pipettes (e.g., graduated pipettes BLAUBRAND^® Type 3 Class AS, 10 mL, Graduation: 10 mL; Carl Roth, catalog number: HXT8.1)

11. Glass vials (Rotilabo^®-screw neck ND8 vials, Brown glass, 1.5 mL; Carl Roth, catalog number: KE30.1) with screw caps (without borehole, without septum, PP, black, ND8; Carl Roth, catalog number: KE39.1) for lipid aliquotation

12. Ice bucket (e.g., Magic Touch 2^TM ice bucket with lid; Sigma-Aldrich, catalog number: BAM168072002)

13. ITO-coated glass slides (50 × 60 mm, 1 mm thick; Präzisions Glas & Optik GmbH, Iserlohn, Germany)

14. Microcentrifuge tubes of 1.5 mL capacity (SARSTEDT AG & Co. KG, catalog number: 72.690.001)

15. Microcentrifuge tubes of 2 mL capacity (SARSTEDT AG & Co. KG, catalog number: 72.691)

16. Pyrex flask (Fisher Scientific, catalog number: 15446113)

17. Round bottom glass tubes (KIMBLE^® Disposable Screw Thread Culture Tube, Round Bottom, 16 × 100 mm, DWK Life Science, catalog number: 73770-16100) with screw caps (PP screw cap with seal, black, DURAN WHEATON KIMBLE, DWK Life Science, catalog number: 299901307)

18. Silicone rubber spacer (cut to 50 × 60 mm, 1 mm thick, Deutsch & Neumann GmbH, catalog number: 510 1001)

19. Sticky-slide 8 Well (Ibidi, catalog number: 80828)

20. Wipes (Precision Wipes, KIMTECH Science, Kimberly-Clark^® Professional, catalog number: 7552)

21. Acetic acid, glacial (VWR, catalog number: 20104)

22. Acetone (Sigma-Aldrich, catalog number: 904082)

23. Alexa Fluor 647 NHS-Ester (Succinimidyl Ester) (Thermo Fisher Scientific, Life Technologies, catalog number: A20006), dissolved in DMSO and stored at -20°C

24. Ammonium molybdate tetrahydrate (Carl Roth, catalog number: 3666.1)

25. (3-Aminopropyl)triethoxysilane (APTES, Sigma-Aldrich, catalog number: 440140)

26. L-ascorbic acid (Carl Roth, catalog number: 3525.2)

27. Avidin (Thermo Fisher Scientific, Life Technologies, catalog number: 434401), stored at -20°C

28. Bio-Beads SM-2 Resin (Bio-Rad Laboratories Inc., catalog number: 1523920)

29. Biotin-Poly (Ethylene Glycol)-Succinimidyl carbonate ((Ethylene Glycol)-(biotin-PEG-SC, 5,000 Da, Laysan Bio Inc., Alabama, USA), stored at -20°C

30. Bio-Gel P-6 (Bio-Rad Laboratories Inc., catalog number: 1504130), dissolved in deionized water and stored at 4°C

31. Chloroform, ethanol-stabilized and certified for absence of phosgene and HCl (VWR, catalog number: 22711.290)

32. Deionized water

33. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine labeled with ATTO488 (ATTO488-PE) was purchased as powder (ATTO-TEC GmbH, catalog number: 67335), dissolved in chloroform, and stored at -20°C

34. DMSO (Sigma-Aldrich, catalog number: 276855)

35. Egg L-α-phosphatidylcholine (EggPC, catalog number: 840051) and 1,2-dioleoyl-sn-glycero-3-phosphoetholamine-N-(cap biotinyl, 18:1) (biotin-PE, catalog number: 870273) were purchased as powders (Avanti Polar Lipids, Birmingham, AL, USA), dissolved in chloroform:methanol (1:1, volume/volume), and stored at -20°C

36. 70% ethanol

37. Glucose (Fisher Scientific, catalog number: 10122730)

38. HEPES (Carl Roth, catalog number: 6763.3)

39. Ice

40. Methanol (VWR PROLABO^® CHEMICALS; Fontenay-sous-Bois, catalog number: 20834.291)

41. Methoxy-Poly (Ethylene Glycol)-Succinimidyl carbonate (mPEG-SC, 5,000 Da, Laysan Bio Inc., Alabama, USA), stored at -20°C

42. N₂ gas (ALPHAGAZ 1 N₂, 99.999%, Air Liquide, Düsseldorf, Germany)

43. NaOH (Sigma-Aldrich, catalog number: 30620)

44. NaCl (AnalaR NORMAPUR, VWR PROLABO^® CHEMICALS; Fontenay-sous-Bois, catalog number: 27810.295)

45. Norland Optical Adhesive 60 (Norland Products, Inc. catalog number: 6016)

46. Osmolarity standards of 290 mmol kg^-1 (OPTIMOLETM, 290 mmol kg^-1, ELITechGroup, BIOMEDICAL SYSTEMS, Logan, UT, USA)

47. Parafilm (PARAFILM^® M; Sigma-Aldrich, catalog number: P7793-1EA)

48. Perchloric acid (VWR, catalog number: 20589)

49. Pierce spin column (Bio-Rad Laboratories Inc., catalog number: 7326008)

50. Polypropylene tubes of 15 mL and 50 mL capacity (e.g., Falcon tubes, SARSTEDT AG & Co. KG, catalog numbers: 62.554.502 and 62.547.254)

51. Polyethersulfone membrane with a pore size of 0.2 μm (Filtropur, SARSTEDT AG & Co. KG, catalog number: 83.1826.001)

52. Potassium hydroxide (Fisher Scientific, catalog number: P250-1)

53. Sodium bicarbonate (Fisher Scientific, catalog number: S233-500)

54. Sodium dithionite (Fisher Scientific, catalog number: 10274490)

55. Di-Sodium hydrogenphosphate dihydrate (VWR, catalog number: 28029)

56. Sucrose (Fisher Scientific, catalog number: 10638403)

57. Triton X^® 100 (Carl Roth, catalog number: 3051.3)

58. Low ionic labeling buffer (see Recipes)

59. Bio-Gel P-6 in deionized water (see Recipes)

60. Low ionic buffer (see Recipes)

61. Low ionic buffer with Triton X-100 (see Recipes)

62. Phosphate standard solution (4 mM) (see Recipes)

63. Molybdate solution (12 mg mL^-1) (see Recipes)

64. Ascorbic acid solution (50 mg mL^-1) (see Recipes)

65. 250 mM sucrose solution (osmolality 250 mmol kg^-1) (see Recipes)

66. 250 mM glucose solution (osmolality 250 mmol kg^-1) (see Recipes)

67. Saturated sodium chloride solution (see Recipes)

68. 5.0 M potassium hydroxide (see Recipes)

69. 1.0 M potassium hydroxide (see Recipes)

70. Sodium bicarbonate (10 mM, pH 8.5) (see Recipes)

71. Avidin 0.2 mg mL^-1 in deionized water (see Recipes)

Equipment

1. Analytical balance (e.g., Sartorius Entris-I II, 220 g/0.1 mg; Buch Holm, catalog number: 4669128)

2. Confocal laser scanning microscope. For this protocol, a Leica TCS SP8 (Leitz, Wetzlar, Germany) equipped with 63×/1.20, NA water objective was used. Images were acquired using a 400 Hz unidirectional scanner, a pixel size of 246.27 × 246.27 μm, a pinhole of 100 μm (1 AU) with Leica HyD detectors.

3. End-over-end rotator (INTELLI-MIXER, neoLab^®, Heidelberg, Germany, catalog number: 7-0045)

4. Flow cabinet to work with organic solvents

5. Freezer (-20°C)

6. Freezer (-80°C)

7. Function generator (e.g., TG315 Function generator 3 MHz; Telemeter Electronic, catalog number: 29788)

8. Glass desiccator (Boro 3.3 with socket in lid, 20 cm, including stopcock; BRAND GmbH, catalog number: 65238)

9. Glass beaker (e.g., Aldrich^® Essentials beaker, Griffin; Sigma-Aldrich, catalog number: Z740572)

10. Glass tubes (Carl Roth, catalog number: DURAN C208.1)

11. Glass marbles (e.g., Fisher Scientific, catalog number: S04581)

12. Hamilton 700 Series Syringes of 10 µL, 100 µL, 250 µL, 500 µL, and 1,000 µL (Hamilton^® syringe, 700 and 1000 Series, Nevada, USA)

13. Heating block (Rotilabo^®-Block-Heater H 250; CARL ROTH, catalog number: Y264.1)

14. Ice machine

15. Magnetic stirrer (e.g., IKAMAG^®, DREHZAHL ELECTRONIC, IKA, Staufen im Breisgau, Germany)

16. Magnets

17. pH-meter (pH-Meter 761 Calimatic, Knick, Berlin, Germany)

18. Pipettes P20, P200, P1000 (GILSON^®, catalog numbers: FD10001, FD10005, and FD10006)

19. Pipette tips 2 µL, 20 µL, 200 µL, and 1,000 µL (SARSTEDT AG & Co. KG, catalog numbers: 70.1130.212, 70.3021, 70.760.002, and 70.3050.020)

20. Polystyrene cuvettes (SARSTEDT AG & Co. KG, catalog number: 67.742)

21. Quartz cuvette (10 mm wide, QS, High Precision Cell, Quartz SUPRASIL^®; Hellma^® Analytics, catalog number: HL109004F-10-40)

22. Refrigerator (4°C)

23. Rotoevaporator (BUCHI, Vacuum Pump V-100, Interface I-100, Rotavapor R-100, Flawil, Switzerland)

24. Scissors

25. Sonicator (130 W, ULTRASONIC CLEANER; ALLPAX, catalog number: 10015895;0)

26. Spectrophotometer (e.g., UV-VIS Spectrophotometer, UV-1280, SHIMADZU, Kyoto, Japan)

27. Tabletop centrifuge (Eppendorf 5810 R, rotor A-4-62, Hamburg, Germany)

28. Ultra-Violet/Ozone Probe and Surface Decontamination Unit (Novascan Technologies Inc., Boone, IA, USA)

29. Vapor Pressure Osmometer (WESCOR^®, Model 5600, ELITechGroup, BIOMEDICAL SYSTEMS, Logan, UT, USA)

30. Vortexer (Vortex Genie 2 TM, BENDER & HOBEIN AG, Zurich, Switzerland)

Software

1. GraphPad Prism version 9.1.0 for Windows (GraphPad Software, 2021, La Jolla California, USA, www.graphpad.com)

2. ImageJ (Wayne, Rasband, S., U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/index.html)

3. Inkscape (Inkscape Project., 2020, Inkscape, Team, 2021, https://inkscape.org)

4. Leica Application Suite X (LAS X, Leitz, Wetzlar, Germany)

5. Microsoft Excel (Microsoft Corporation, 2018, https://office.microsoft.com/excel)

Considerations before starting

1. Choice of lipid reporter

   The fluorescent lipids employed in the microscopy-based assay need to fulfill three requirements: they should be (i) substrates for the ER scramblases under low salt conditions, (ii) rendered non-fluorescent by chemical reduction with the membrane-impermeant dianion dithionite, and (iii) be photostable for microscopy. These requirements are met by lipid analogues tagged with fluorescent ATTO488 (Andra et al., 2018; Mathiassen et al., 2021). Phospholipid scramblases display a relatively low specificity and transport ceramide-based lipids and glycerophospholipids, including the ATTO488-modified phospholipids, equally well within the limits of the time resolution of the assay (Herrmann et al., 1990; Buton et al., 2002; Vishwakarma et al., 2005; Sahu and Gummadi, 2008; Sanyal et al., 2008; Chalat et al., 2012; Wang et al., 2018; Bushell et al. 2019; Mathiassen et al., 2021). We verified requirements (i) and (ii) for each scramblase-containing LUV reconstitution prior to GUV formation. Requirement (iii) was verified by a series of confocal fluorescence imaging of individual GUVs containing ATTO488-PE, and by quantifying the membrane ATTO488 fluorescence intensity at the first image versus the subsequent images (Mathiassen et al., 2021). Note that the majority of ATP-dependent lipid transporters show headgroup specificity for phospholipid transport, which in this case excludes the use of headgroup-labeled lipid analogues (Theorin et al., 2019).

   
   

2. Formation of giant unilamellar vesicles

   We reconstituted TE proteins into LUVs under low salt conditions to facilitate their subsequent use in electroformation for GUV formation by applying an oscillating electric field in an ITO coated chamber. The electroformation technique under low salt conditions was effective in incorporating and maintaining the activity of the sarcoplasmic reticulum Ca²⁺-ATPase and the H⁺-pump bacteriorhodopsin in GUVs (Girard et al., 2004). However, activity may not be preserved for all membrane transporters under these conditions. In this case, we note that GUVs can be prepared in solutions containing physiological salt concentrations, by applying a voltage with a higher frequency (~500 Hz) (Pott et al., 2008; Méléard et al., 2009; Shaklee et al., 2010; Stein et al., 2017). Under these conditions, the yield of GUVs is lower and protein incorporation into GUVs is more heterogeneous, mainly because of the electric field being shielded by the ions of the solution (Girard et al., 2004; Politano et al., 2010; Aimon et al., 2011; Garten et al., 2015). In addition, negatively charged lipids can give rise to an asymmetric lipid distribution over the bilayer directly after electroformation. Under such conditions, the GUV suspension should be stored at room temperature for several hours and the lipid distribution tested, by using dithionite-reactive fluorescent lipid probes for example (Steinkühler et al., 2018). Furthermore, various dimensions of ITO-coated glass slides are available, which may alter the amount, morphology, and size distribution of the GUVs formed. Hence, the GUV output should be analyzed to ensure reproducibility. We note that studies of other transporters may require other reconstitution procedures into LUVs for subsequent generation of protein-containing GUVs. We recommend verification of protein incorporation into the GUVs (e.g., by using fluorescently labeled proteins), and assaying the ATTO488-PE distribution over the bilayer in protein-free/empty GUVs using dithionite, as described in the next sections.

   
   

3. Handling of giant unilamellar vesicles

   GUVs are fragile and rupture easily on glass. Thus, it is important to passivate the cover glass slides with e.g. β-Casein, to prevent bursting/attachment when observing free-floating GUVs (Streicher et al., 2009). In this protocol, we aimed to immobilize a fraction of the biotinylated GUVs to ease single vesicle tracking over time. The cover glass slides were cleaned using a protocol adapted from Chandradoss et al. (2014) , followed by aminosilanization using (3-Aminopropyl)triethoxysilane (APTES). Surface PEGylation was performed using a protocol adapted from Lamichhane et al. (2010) , which employed a mixture of functionalized PEG (mPEG-SC and biotin-PEG-SC), introducing biotinylated PEG molecules. To increase GUV immobilization using avidin as the immobilization-linker, adjustments to the protocol may be required, as most GUVs settled rather than immobilized in our hands.

Procedure