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Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes
量化检测纯化Drp1与合成脂质体之间的相互作用   

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Abstract

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

Keywords: Mitochondria(线粒体), Organelle division(细胞器分裂), Dynamin superfamily(动力蛋白超家族), Phospholipids(磷脂)

Background

Interactions of proteins and membrane lipids are critical for the remodeling of biological membranes in cells such as organelle division. In mitochondrial division, Drp1 constricts the mitochondrial membranes and drives this membrane remodeling process. We have recently shown that a signaling phospholipid, phosphatidic acid, interacts with Drp1 and creates the priming step by restraining assembled division machinery on mitochondria (Adachi et al., 2016). Drp1 recognizes both the head group and acyl chains of phosphatidic acid. To analyze Drp1-phosphatidic acid binding, we set up several protein-lipid interaction assays, including a lipid dot blot assay, a competition assay and a liposome flotation assay. These assays allowed us to determine the function of the head group and acyl chain composition in protein-lipid interactions. In addition, we analyzed lipids under bilayer conditions in the liposome assay and lipids under non-bilayer conditions in dot blot and competition assays, which made it possible to examine the contribution of the membrane curvature and lipid packing to protein-lipid interactions. Below, we describe a liposome flotation assay that can be applied to many peripheral membrane proteins.

Materials and Reagents

  1. 0.45 µm Millex-HA syringe filter unit (EMD Millipore, catalog number: SLHA033SS )
  2. 15-ml column (Poly-prep chromatography column) (Bio-Rad Laboratories, catalog number: 7311550 )
  3. Amicon Ultra-15 centrifugal filter unit with Ultracel-10 membrane (10k filter for domains of Drp1) (EMD Millipore, catalog number: UFC901024 )
  4. Amicon Ultra-15 centrifugal filter unit with Ultracel-50 membrane (50k filter for full length Drp1) (EMD Millipore, catalog number: UFC905024 )
  5. Polycarbonate membranes 0.4 μm (Avanti Polar Lipids, catalog number: 610007 )
  6. 96 well assay plate (Corning, catalog number: 3915 )
  7. Extruder set with holder/heating block (Avanti Polar Lipids, catalog number: 610000 )
  8. Filter support (Avanti Polar Lipids, catalog number: 610014 )
  9. 2 ml amber glass sample vials (WHEATON, catalog number: 224981 )
  10. Disposable culture tubes (Fisher Scientific, catalog number: 14-961-27 )
  11. 50 Ulrta-Clear tubes (Beckman Coulter, catalog number: 344057 )
  12. RosettaTM 2(DE3)pLysS competent cells (EMD Millipore, catalog number: 71403 )
  13. pET15b vectors (EMD Millipore, Novagen)
  14. 50% Ni-NTA beads (EMD Millipore, catalog number: 70666 )
  15. 4-20% Tris-HCl precast gel (Bio-Rad Laboratories, catalog number: 3450034 )
  16. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  17. Ethanol (PHARMCO-AAPER, catalog number: 111000200 )
  18. DMSO (Fisher Scientific, catalog number: S369-500 )
  19. Liquid nitrogen
  20. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457 )
  21. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DPPE) (Avanti Polar Lipids, catalog number: 810158 )
  22. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) (Avanti Polar Lipids, catalog number: 850705 )
  23. 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) (Avanti Polar Lipids, catalog number: 830855 )
  24. 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (Avanti Polar Lipids, catalog number: 840875 )
  25. 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (Palmitoyl lysoPA) (Avanti Polar Lipids, catalog number: 857123 )
  26. 1,2-dihexanoyl-sn-glycero-3-phosphate (Dihexanoyl PA) (Avanti Polar Lipids, catalog number: 830841 )
  27. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Avanti Polar Lipids, catalog number: 850355 )
  28. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375 )
  29. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, catalog number: 850725 )
  30. 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG) (Avanti Polar Lipids, catalog number: 840455 )
  31. 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) (Avanti Polar Lipids, catalog number: 840475 )
  32. 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) (Avanti Polar Lipids, catalog number: 840037 )
  33. 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) (Avanti Polar Lipids, catalog number: 840035 )
  34. 1’,3’-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL) (Avanti Polar Lipids, catalog number: 710335 )
  35. 1’,3’-bis[1,2-dipalmitoyl-sn-glycero-3-phospho]-sn-glycerol TPCL (Echelon Biosciences, catalog number: L-C160 )
  36. Gas nitrogen
  37. SilverQuestTM Silver Staining Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: LC6070 )
  38. BactoTM tryptone (BD, BactoTM, catalog number: 211705 )
  39. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  40. BactoTM yeast extract (BD, BactoTM, catalog number: 212750 )
  41. Agar (Alfa Aesar, catalog number: J10906 )
  42. Ultrapure water (e.g., MilliQ)
  43. Ampicillin, sodium salt (Alfa Aesar, catalog number: J11259 )
  44. Chloramphenicol (Mediatech, catalog number: 61-239-RI )
  45. beta-D-Galactopyranoside, Isopropyl-beta-D-thiogalactopyranoside (IPTG) (AMRESCO, catalog number: 0487 )
  46. HEPES (Fisher Scientific, catalog number: BP310-1 )
  47. Potassium hydroxide (KOH) (Avantor® Performance Materials, J.T. Baker, catalog number: 3116-01 )
  48. Magnesium chloride hydrate (MgCl2·6H2O) (Avantor® Performance Materials, J.T. Baker, catalog number: 2444 )
  49. Imidazole (Sigma-Aldrich, catalog number: I0125 or I202 )
    Note: The product “ I0125 ” has been discontinue
  50. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  51. Sodium phosphate monobasic (NaH2PO4·H2O) (Fisher Scientific, catalog number: S369-500 )
  52. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373-3 )
  53. Sodium hydroxide (NaOH) (Avantor® Performance Materials, J.T. Baker, catalog number: 3722 )
  54. Coomassie Brilliant Blue R-250 (AMRESCO, catalog number: M128-50G )
  55. Acetic acid (Fisher Scientific, catalog number: A38-212 )
  56. Methanol (Fisher Scientific, catalog number: A412 )
  57. MES (2-morpholinoethanesulfonic acid, mono hydrate) (Sigma-Aldrich, catalog number: M8250 )
  58. Sucrose (Fisher Scientific, catalog number: S5-3 )
  59. LB plate containing ampicillin and chloramphenicol (see Recipes)
  60. LB with ampicillin and chloramphenicol (see Recipes)
  61. 0.5 M IPTG
  62. 0.5 M HEPES (pH 7.4) (see Recipes)
  63. Lysis buffer (see Recipes)
  64. Wash buffer (see Recipes)
  65. Elution buffer (see Recipes)
  66. 10x PBS (see Recipes)
  67. Coomassie Brilliant Blue solution (see Recipes)
  68. 100 mM MES (pH 7.0) (see Recipes)
  69. 20 mM MES (pH 7.0) (see Recipes)
  70. 20 mM MES (pH 7.0)/100 mM NaCl (see Recipes)
  71. 2.7 M sucrose/20 mM MES/100 mM NaCl (see Recipes)
  72. 1.25 M sucrose/20 mM MES/100 mM NaCl (see Recipes)
  73. 0.25 M sucrose/20 mM MES/100 mM NaCl (see Recipes)

Equipment

  1. 1 ml syringe (Hamilton, catalog number: 81365 )
  2. 500 µl syringe (Hamilton, catalog number: 81265 )
  3. 100 µl syringe (Hamilton, catalog number: 81065 )
  4. 50 µl syringe (Hamilton, catalog number: 80500 )
  5. 25 µl syringe (Hamilton, catalog number: 80400 )
  6. Incubator shaker (37 °C) (Eppendorf, New BrunswickTM, model: Series 25 )
  7. Incubator shaker (16 °C) (Lab Companion, model: IS-971R )
  8. 1,000 ml bottle (Beckman Coulter, catalog number: A98814 )
  9. JLA8.1000 rotor (Beckman Coulter, catalog number: 969329 )
  10. GH3.8 rotor (Beckman Coulter, catalog number: 360581 )
  11. Fisher Scientific Sonic dismembrator (Fisher Scientific, model: 100 )
  12. CS-6R centrifuge (Beckman Coulter, model: CS-6R )
  13. Avanti J-E (Beckman Coulter, model: Avanti J-E , catalog number: 369001)
  14. SpeedVac SVC100 (Savant System, model: SpeedVac SVC100 )
  15. Heat block (37 °C, 42 °C, 95 °C) (Fisher Scientific, model: Fisher ScientificTM IsotempTM Digital and Analog Dry Bath Incubators , catalog number: 11-718)
  16. Motorized upright fluorescence microscope (Olympus, model: BX61 )
  17. POLARstar Omega (BMG LABTECH, model: POLARstar Omega )
  18. Labquake Shaker Rotisserie (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4152110 )
  19. SW55Ti rotor (Beckman Coulter, catalog number: 342196 )
  20. Avanti J-26XP (Beckman Coulter, model: Avanti® J-26 XP , catalog number: 393124)
  21. Optima L-100XP ultracentrifuge (Beckman Coulter, model: OptimaTM L-100XP , catalog number: 392050)
  22. Scanmaker 8700 (Microtek, model: Scanmaker 8700 )
  23. Power supply (Bio-Rad Laboratories, model: PowerPacTM HC )
  24. Multi-Purpose rotator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2309 )
  25. Vortex mixer (Fisher Scientific, model: Fisher ScientificTM Analog Vortex Mixer , catalog number: 02215365)

Software

  1. ImageJ (Version 1.48, http://imagej.net/)

Procedure

  1. Protein expression and purification
    1. Transform the pET15b vectors carrying Drp1 to Rosetta 2(DE3) pLysS competent cells.
    2. Plate the cells on an LB plate containing ampicillin and chloramphenicol (see Recipes) and incubate overnight at 37 °C.
    3. Select and place a colony into a 10 ml LB plate supplemented with ampicillin and chloramphenicol (see Recipes). Shake the bacteria culture overnight at 37 °C at 200 rpm (New Brunswick Scientific, series 25).
    4. Dilute the 5 ml of the culture into 1 L of the same growth medium and continue to grow the culture for 3-5 h at 37 °C using an incubator shaker at 200 rpm (New Brunswick Scientific, series 25).
    5. Cool down the cultured cells on ice, and add 0.2 ml of 0.5 M IPTG (see Recipes) to 1 L of LB buffer. Final IPTG concentration is 0.1 mM.
    6. Shake the culture overnight at 16 °C using a refrigerated incubator shaker (The Lab Companion, IS-971R) at 200 rpm with cooling system.
    7. After incubation, move the culture medium to a 1,000 ml bottle (Beckman Coulter) and centrifuge at 5,000 x g for 15 min at 4 °C in a JLA8.1000 rotor (Avanti J-26XP).
    8. Wash the bacteria twice with 40 ml PBS via centrifugation at 2,400 x g in a GH3.8 rotor using a CS-6R centrifuge (Beckman Coulter) for 15 min and freeze cell pellets at -80 °C.
    9. Resuspend the frozen cell pellets in 40 ml of lysis buffer (see Recipes).
    10. Sonicate the bacteria on ice for 10 x 5 sec at setting 5 and then 10 x 5 sec at setting 2 using a Fisher Scientific Sonic dismembrator model 100.
    11. Centrifuge the cell homogenate at 2,400 x g for 15 min at 4 °C in a GH3.8 rotor using a CS-6R centrifuge.
    12. Centrifuge the supernatant at 20,100 x g for 15 min at 4 °C in JA-20X (Avanti J-E).
    13. Pass the supernatant through a Millex-HA syringe filter with a pore size of 0.45 µm.
    14. Incubate the lysate with 2 ml of pre-washed 50% Ni-NTA beads (His-Bind Resin, Novagen) overnight at 4 °C using a Labquake Shaker Rotisserie.
    15. Load the sample in a 15-ml column (Poly-prep chromatography column, Bio-Rad Laboratories) in a cold room.
    16. Wash the beads with 3 ml of lysis buffer and then 15 ml of lysis buffer.
    17. Wash the beads with 3 ml of wash buffer (see Recipes) and then 15 ml of wash buffer.
    18. Elute His6-tagged recombinant Drp1 proteins from the column using 0.9 ml of elution buffer (see Recipes) three times and collect eluents into nine 300-µl fractions.
    19. Identify fractions containing Drp1 proteins using SDS-PAGE and Coomassie Brilliant Blue staining. Combine peak fractions (typically fractions #3 and 4) (Figure 1) and mix them with 15 ml of lysis buffer without imidazole.


      Figure 1. Analysis of purified Drp1 by SDS-PAGE and Coomassie Brilliant Blue staining

    20. Pass the samples through an Amicon Ultra Centrifugation Filter (50k filter for full length Drp1 and 10k filter for domains) three times using lysis buffer without imidazole. The final volume is typically 1 ml.
    21. Mix the sample with final 20% DMSO, aliquoted, snap frozen in liquid nitrogen, and store at -80 °C. Typically concentrations of full-length Drp1 were 30 µM.

  2. Flotation assay
    1. Unless otherwise noted, mix the lipids in a POPC:rhodamine-DPPE:variable lipid ratio of 84:1:15 (% mol).
    2. Dry up the lipids using nitrogen gas for 5 min and additionally dried in a SpeedVac overnight.
    3. Add 100 µl/one sample of 20 mM MES (pH 7.0)/100 mM NaCl (see Recipes) in lipid film and vortex for 1 h.
    4. Conduct five freeze-thaw cycles using dry ice and a 42 °C heat block.
    5. Generate the unilamellar liposomes via extrusion through a nanopore membrane with a pore size of 400 nm. This process is repeated 21 times.
    6. Confirm that the liposome is formed using fluorescence microscopy (Figure 2).


      Figure 2. Confocal fluorescence microscopy of liposomes

    7. Check the fluorescence intensity of rhodamine-PE by POLARstar Omega (Wavelength; Ex 544 nm, Em 590 nm) and then adjust the concentration of lipids based on the rhodamine intensity using 20 mM MES (pH 7.0)/100 mM NaCl.
    8. Mix the liposomes (5 mM lipids) with His6-Drp1 (5 μM) (final volume: 200 μl) and incubate at 4 °C with gentle mixing using a Labquake Shaker Rotisserie for 1 h in 20 mM MES (pH 7.0)/100 mM NaCl (Figure 3).


      Figure 3. Drp1 and liposomes are incubated and analyzed by sucrose gradient centrifugation. Amounts of Drp1 that co-migrate with liposomes into fractions 1 and 2 are quantified.

    9. Dilute the Drp1-liposome mixture in final 1.73 M sucrose/20 mM MES (pH 7.0) (final volume: 1.25 ml) placed at the bottom of tubes. Then overlay 2.9 ml of 1.25 M sucrose/20 mM MES (pH 7.0) and 0.85 ml of 0.25 M sucrose/20 mM MES (pH 7.0) (Figure 3).
    10. Centrifuge the sucrose gradient at 287,000 x g for 2 h at 4 °C in an SW55Ti rotor (Beckman Coulter).
    11. Collect the five fractions (0.5, 0.75, 1.75, 1.75 and 0.25 ml) from the top.
    12. The majority of the liposomes typically floats to the top two fractions.
    13. Measure the rhodamine intensity using a POLARstar Omega. Analyze each fraction (equal volume) using SDS-PAGE and silver staining (SilverQuestTM silver staining kit) according to the manufacturer’s instructions.

Data analysis

  1. Scan the stained gels using Scanmaker 8700, and quantify the band intensity using NIH ImageJ. Detect the liposomes based on the fluorescence intensity of rhodamine-PE. Normalize the amounts of Drp1 and liposomes relative to the volume of each fraction.
  2. For statistical analysis, P values are calculated using the Student’s t-test.

Notes

When loading the sample on SDS-page, please warm the sample with a heat block at 37 °C. Because the sample contains sucrose, the fluidity of the sample changes depending on the temperature.

Recipes

  1. LB plate containing ampicillin and chloramphenicol
    10 g BactoTM tryptone
    10 g NaCl
    5 g BactoTM yeast extract
    15 g agar
    Fill up to 1 L with ultrapure water and autoclave at 121 °C for 30 min. After autoclaving, allow the agar solution to cool to 55 °C. Add final 100 µg/ml ampicillin and 25 µg/ml chloramphenicol
  2. LB with ampicillin and chloramphenicol
    10 g BactoTM tryptone
    10 g NaCl
    5 g BactoTM yeast extract
    Fill up to 1 L with ultrapure water and autoclave at 121 °C for 30 min. After autoclaving, allow the solution to cool to 55 °C. Add final 100 µg/ml ampicillin and 25 µg/ml chloramphenicol
  3. 0.5 M IPTG
    1.19 g IPTG
    Fill up to 10 ml with ultrapure water
  4. 0.5 M HEPES (pH 7.4)
    238.4 g/L HEPES
    Adjust pH to 7.4 with KOH and fill up to 1 L with ultrapure water
  5. Lysis buffer (10 mM imidazole, 1 mM MgCl2, 500 mM NaCl, 2 mM 2-mercaptoethanol, 20 mM HEPES, pH 7.4)
    40 ml/L 0.5 M HEPES (pH 7.4)
    500 ml/L 1 M NaCl
    1 ml/L 1 M MgCl2
    5 ml/L 2 M imidazole
    140 µl/L 2-mercaptoethanol
    Fill up to 1 L with ultrapure water
  6. Wash buffer (40 mM imidazole, 1 mM MgCl2, 500 mM NaCl, 2 mM 2-mercaptoethanol, 20 mM HEPES, pH 7.4)
    40 ml/L 0.5 M HEPES (pH 7.4)
    500 ml/L 1 M NaCl
    1 ml/L 1 M MgCl2
    20 ml/L 2 M imidazole
    140 µl/L 2-mercaptoethanol
    Fill up to 1 L with ultrapure water
  7. Elution buffer (250 mM imidazole, 1 mM MgCl2, 500 mM NaCl, 2 mM 2-mercaptoethanol, 20 mM HEPES, pH 7.4)
    40 ml/L 0.5 M HEPES (pH 7.4)
    500 ml/L 1 M NaCl
    1 ml/L 1 M MgCl2
    50 ml/L 2 M imidazole
    140 µl/L 2-mercaptoethanol
    Fill up to 1 L with ultrapure water
  8. 10x PBS (pH 7.4)
    2.56 g/L NaH2PO4·H2O
    22.5 g/L Na2HPO4·7H2O
    87.66 g/L NaCl
    Fill up to 1 L with ultrapure water. Adjust pH to 7.0 with NaOH
  9. Coomassie Brilliant Blue solution
    2.5 g/L Coomassie Brilliant Blue R-250
    100 ml/L acetic acid
    450 ml/L MeOH
    450 ml/L ultrapure water
  10. 100 mM MES (pH 7.0)
    21.538 g/L MES
    3.068 g/L NaOH
    Fill up to 1 L with ultrapure water
  11. 20 mM MES (pH 7.0)
    200 ml/L 100 mM MES (pH 7.0)
    Fill up to 1 L with ultrapure water. Adjust pH to 7.0 with NaOH
  12. 20 mM MES (pH 7.0)/100 mM NaCl
    200 ml/L 100 mM MES (pH 7.0)
    5.844 g/L NaCl
    140 µl/L 2-mercaptoethanol (final 2 mM)
    Fill up to 1 L with ultrapure water. Adjust pH to 7.0 with NaOH, as necessary
  13. 2.7 M sucrose/20 mM MES/100 mM NaCl
    924.21 g/L sucrose
    Fill up to 1 L with 20 mM MES/100 mM NaCl
  14. 1.25 M sucrose/20 mM MES/100 mM NaCl
    427.875 g/L sucrose
    Fill up to 1 L with 20 mM MES/100 mM NaCl
  15. 0.25 M sucrose/20 mM MES/100 mM NaCl
    85.575 g/L sucrose
    Fill up to 1 L with 20 mM MES/100 mM NaCl

Acknowledgments

We thank past and present members of the Iijima and Sesaki labs for helpful discussions and technical assistance. This work was supported by NIH grants to M.I. (GM084015) and H.S. (GM089853).

References

  1. Adachi, Y., Itoh, K., Yamada, T., Cerveny, K. L., Suzuki, T. L., Macdonald, P., Frohman, M. A., Ramachandran, R., Iijima, M. and Sesaki, H. (2016). Coincident phosphatidic acid interaction restrains Drp1 in mitochondrial division. Mol Cell 63(6): 1034-1043.
  2. Bustillo-Zabalbeitia, I., Montessuit, S., Raemy, E., Basanez, G., Terrones, O. and Martinou, J. C. (2014). Specific interaction with cardiolipin triggers functional activation of Dynamin-Related Protein 1. PLoS One 9(7): e102738.
  3. Macdonald, P. J., Stepanyants, N., Mehrotra, N., Mears, J. A., Qi, X., Sesaki, H. and Ramachandran, R. (2014). A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission. Mol Biol Cell 25(12): 1905-1915.
  4. Montessuit, S., Somasekharan, S. P., Terrones, O., Lucken-Ardjomande, S., Herzig, S., Schwarzenbacher, R., Manstein, D. J., Bossy-Wetzel, E., Basanez, G., Meda, P. and Martinou, J. C. (2010). Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization. Cell 142(6): 889-901.
  5. Roy, M., Reddy, P. H., Iijima, M. and Sesaki, H. (2015). Mitochondrial division and fusion in metabolism. Curr Opin Cell Biol 33: 111-118.
  6. Stepanyants, N., Macdonald, P. J., Francy, C. A., Mears, J. A., Qi, X. and Ramachandran, R. (2015). Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission. Mol Biol Cell 26(17): 3104-3116.
  7. Tamura, Y., Itoh, K. and Sesaki, H. (2011). SnapShot: Mitochondrial dynamics. Cell 145(7): 1158, 1158 e1151.

简介

线粒体是一种动态的细胞内细胞器,主动分裂和融合以控制细胞的大小,数量和形状。线粒体分裂和融合之间的调节平衡是线粒体功能,分布和周转的基础(Roy等,2015)。线粒体分化是由动力蛋白相关蛋白1(Drp1)介导的,其是限制线粒体膜的机械化学GTP酶(Tamura等人,2011)。线粒体膜脂质如磷脂酸和心磷脂结合Drp1,并且Drp1磷脂相互作用提供线粒体分裂的关键调控机制(Montessuit等人,2010; Bustillo-Zabalbeitia等人2014年; Macdonald等人,2014年; Stepanyants等人,2015; Adachi等人,2016)。在这里,我们描述了使用纯化的重组Drp1和具有定义的一组磷脂的合成脂质体定量测量Drp1与脂质的相互作用的生物化学实验。该测定使得可以定义蛋白质 - 脂质相互作用的特异性以及头基和酰基链的作用。

背景 蛋白质和膜脂质的相互作用对于细胞如细胞器分裂中生物膜的重塑至关重要。在线粒体分裂中,Drp1限制线粒体膜并驱动该膜重塑过程。我们最近显示,信号磷脂,磷脂酸与Drp1相互作用,并通过限制线粒体上的组装分裂机制(Adachi等人,2016)产生启动步骤。 Drp1识别磷脂酸的头基和酰基链。为了分析Drp1-磷脂酸结合,我们建立了几种蛋白质 - 脂质相互作用测定,包括脂质斑点印迹测定,竞争测定和脂质体浮选测定。这些测定使我们能够确定蛋白 - 脂质相互作用中头基和酰基链组成的功能。此外,我们分析脂质体测定中的双层条件下的脂质和非双层条件下的脂质在斑点印迹和竞争测定中,这使得有可能检查膜曲率和脂质包装对蛋白质 - 脂质相互作用的贡献。下面,我们描述一种可应用于许多外周膜蛋白的脂质体浮选测定。

关键字:线粒体, 细胞器分裂, 动力蛋白超家族, 磷脂

材料和试剂

  1. 0.45μmMillex-HA注射器过滤器(EMD Millipore,目录号:SLHA033SS)
  2. (Poly-prep色谱柱)(Bio-Rad Laboratories,目录号:7311550)
  3. 具有Ultracel-10膜的Amicon Ultra-15离心过滤器单元(用于Drp1结构域的10k过滤器)(EMD Millipore,目录号:UFC901024)
  4. 带有Ultracel-50膜的Amicon Ultra-15离心过滤器(50k全长Drp1过滤器)(EMD Millipore,目录号:UFC905024)
  5. 聚碳酸酯膜0.4μm(Avanti Polar Lipids,目录号:610007)
  6. 96孔分析板(Corning,目录号:3915)
  7. 挤压机配有支架/加热块(Avanti Polar Lipids,目录号:610000)
  8. 过滤器支持(Avanti Polar Lipids,目录号:610014)
  9. 2毫升琥珀色玻璃样品瓶(WHEATON,目录号:224981)
  10. 一次性培养管(Fisher Scientific,目录号:14-961-27)
  11. 50 Ulrta-Clear管(Beckman Coulter,目录号:344057)
  12. Rosetta TM 2(DE3)pLysS感受态细胞(EMD Millipore,目录号:71403)
  13. pET15b载体(EMD Millipore,Novagen)
  14. 50%Ni-NTA珠(EMD Millipore,目录号:70666)
  15. 4-20%Tris-HCl预制凝胶(Bio-Rad Laboratories,目录号:3450034)
  16. 氯仿(Sigma-Aldrich,目录号:C2432)
  17. 乙醇(PHARMCO-AAPER,目录号:111000200)
  18. DMSO(Fisher Scientific,目录号:S369-500)
  19. 液氮
  20. 1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱(POPC)(Avanti Polar Lipids,目录号:850457)
  21. 1,2-二棕榈酰-sn-甘油基-3-磷酸乙醇胺-N-(赖斯汀罗丹明B磺酰基)(罗丹明DPPE)(Avanti Polar Lipids,目录号:810158)
  22. 1,2-二棕榈酰-sn-甘油基-3-磷酸乙醇胺(DPPE)(Avanti Polar Lipids,目录号:850705)
  23. 1,2-二棕榈酰-sn-甘油-3-磷酸酯(DPPA)(Avanti Polar Lipids,目录号:830855)
  24. 1,2-二油酰-sn-甘油-3-磷酸(DOPA)(Avanti Polar Lipids,目录号:840875)
  25. 1-棕榈酰基-2-羟基-sn-甘油基-3-磷酸酯(Palmitoyl lysoPA)(Avanti Polar Lipids,目录号:857123)
  26. 1,2-二己酰基-sn-甘油基-3-磷酸(Dihexanoyl PA)(Avanti Polar Lipids,目录号:830841)
  27. 1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)(Avanti Polar Lipids,目录号:850355)
  28. 1,2-二油酰-sn-甘油-3-磷酸胆碱(DOPC)(Avanti Polar Lipids,目录号:850375)
  29. 1,2-二油酰-sn-甘油基-3-磷酸乙醇胺(DOPE)(Avanti Polar Lipids,目录号:850725)
  30. 1,2-二棕榈酰基-sn-甘油基-3-磷酸 - (1'-外消旋甘油)(DPPG)(Avanti Polar Lipids,目录号:840455)
  31. 1,2-二油酰-sn-甘油基-3-磷酸 - (1'-外消旋甘油)(DOPG)(Avanti Polar Lipids,目录号:840475)
  32. 1,2-二棕榈酰-sn-甘油基-3-磷酸-L-丝氨酸(DPPS)(Avanti Polar Lipids,目录号:840037)
  33. 1,2-二油酰-sn-甘油基-3-磷酸-L-丝氨酸(DOPS)(Avanti Polar Lipids,目录号:840035)
  34. 1',3'-双[1,2-二油酰-sn-甘油基-3-磷酸] -sn-甘油(TOCL)(Avanti Polar Lipids,目录号:710335)
  35. 1',3'-双[1,2-二棕榈酰-sn-甘油基-3-磷酸] -sn-甘油TPCL(Echelon Biosciences,目录号:L-C160)
  36. 气氮
  37. SilverQuest TM 银染色试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:LC6070)
  38. Bacto TM 胰蛋白胨(BD,Bacto TM ,目录号:211705)
  39. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  40. Bacto TM酵母提取物(BD,Bacto TM,目录号:212750)
  41. 琼脂(Alfa Aesar,目录号:J10906)
  42. 超纯水(例如,MilliQ)
  43. 氨苄青霉素钠盐(Alfa Aesar,目录号:J11259)
  44. 氯霉素(Mediatech,目录号:61-239-RI)
  45. β-D-吡喃半乳糖苷,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(AMRESCO,目录号:0487)
  46. HEPES(Fisher Scientific,目录号:BP310-1)
  47. 氢氧化钾(KOH)(Avantor Performance Materials,J.T.Baker,目录号:3116-01)
  48. 氯化镁水合物(MgCl 2·6H 2 O)(Avantor,Performance Materials,J.T.Baker,目录号:2444)
  49. 咪唑(Sigma-Aldrich,目录号:I0125或I202)
    注意:产品"I0125"已停止
  50. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  51. 磷酸二氢钠(NaH 2 PO 4·H 2 O)(Fisher Scientific,目录号:S369-500)
  52. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Fisher Scientific,目录号:S373-3)
  53. 氢氧化钠(NaOH)(Avantor Performance Materials,J.T.Baker,目录号:3722)
  54. 考马斯亮蓝R-250(AMRESCO,目录号:M128-50G)
  55. 乙酸(Fisher Scientific,目录号:A38-212)
  56. 甲醇(Fisher Scientific,目录号:A412)
  57. MES(2-吗啉代乙磺酸,一水合物)(Sigma-Aldrich,目录号:M8250)
  58. 蔗糖(Fisher Scientific,目录号:S5-3)
  59. 含有氨苄青霉素和氯霉素的LB板(参见食谱)
  60. LB与氨苄青霉素和氯霉素(参见食谱)
  61. 0.5 M IPTG
  62. 0.5 M HEPES(pH 7.4)(见配方)
  63. 裂解缓冲液(见配方)
  64. 洗涤缓冲液(见配方)
  65. 洗脱缓冲液(见配方)
  66. 10x PBS(参见食谱)
  67. 考马斯亮蓝溶液(见配方)
  68. 100mM MES(pH 7.0)(参见食谱)
  69. 20mM MES(pH 7.0)(参见食谱)
  70. 20mM MES(pH 7.0)/100mM NaCl(参见食谱)
  71. 2.7 M蔗糖/20 mM MES/100 mM NaCl(参见食谱)
  72. 1.25 M蔗糖/20 mM MES/100 mM NaCl(参见食谱)
  73. 0.25 M蔗糖/20 mM MES/100 mM NaCl(参见食谱)

设备

  1. 1 ml注射器(Hamilton,目录号:81365)
  2. 500μl注射器(Hamilton,目录号:81265)
  3. 100μl注射器(Hamilton,目录号:81065)
  4. 50μl注射器(Hamilton,目录号:80500)
  5. 25μl注射器(Hamilton,目录号:80400)
  6. 孵化器振荡器(37°C)(Eppendorf,New Brunswick TM,型号:Series 25)
  7. 孵化器振荡器(16°C)(Lab Companion,型号:IS-971R)
  8. 1000毫升瓶(Beckman Coulter,目录号:A98814)
  9. JLA8.1000转子(Beckman Coulter,目录号:969329)
  10. GH3.8转子(Beckman Coulter,目录号:360581)
  11. 费雪科学声波分解器(Fisher Scientific,型号:100)
  12. CS-6R离心机(Beckman Coulter,型号:CS-6R)
  13. Avanti J-E(Beckman Coulter,型号:Avanti J-E,目录号:369001)
  14. SpeedVac SVC100(Savant系统,型号:SpeedVac SVC100)
  15. 加热块(37℃,42℃,95℃)(Fisher Scientific,型号:Fisher Scientific TM Isotemp TM数字和模拟干浴培养箱,目录号: 11-718)
  16. 电动直立荧光显微镜(Olympus,型号:BX61)
  17. POLARstar Omega(BMG LABTECH,型号:POLARstar Omega)
  18. Labquake Shaker Rostisserie(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4152110)
  19. SW55Ti转子(Beckman Coulter,目录号:342196)
  20. Avanti J-26XP(Beckman Coulter,型号:Avanti ® J-26 XP,目录号:393124)
  21. Optima L-100XP超速离心机(Beckman Coulter,型号:Optima TM,L-100XP,目录号:392050)
  22. Scanmaker 8700(Microtek,型号:Scanmaker 8700)
  23. 电源(Bio-Rad Laboratories,型号:PowerPac TM HC)
  24. 多功能旋转器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2309)
  25. 涡旋混合器(Fisher Scientific,型号:Fisher Scientific TM 模拟涡旋混合器,目录号:02215365)

软件

  1. ImageJ(版本1.48, http://imagej.net/

程序

  1. 蛋白质表达和纯化
    1. 将携带Drp1的pET15b载体转化为Rosetta 2(DE3)pLysS感受态细胞。
    2. 将细胞置于含有氨苄青霉素和氯霉素的LB平板上(参见食谱),并在37℃下孵育过夜。
    3. 选择并将菌落放入补充有氨苄青霉素和氯霉素的10ml LB平板(参见食谱)中。在37℃,200rpm下摇动细菌培养过夜(New Brunswick Scientific,25系列)
    4. 将5ml的培养物稀释成1L相同的生长培养基,并使用培养箱振荡器在200rpm下在37℃下继续培养培养3-5小时(New Brunswick Scientific,25系列)。
    5. 将培养的细胞在冰上冷却,加入0.2ml 0.5M IPTG(参见食谱)至1L LB缓冲液。最终IPTG浓度为0.1mM
    6. 使用冷藏培养箱振荡器(The Lab Companion,IS-971R)在200rpm下用冷却系统在16℃下振荡培养物。
    7. 培养后,将培养基移至1000ml瓶(Beckman Coulter),并在4℃下在JLA8.1000转子(Avanti J-26XP)中以5,000×g离心15分钟。 />
    8. 使用CS-6R离心机(Beckman Coulter)在GH3.8转子中以2,400×g离心分离15分钟,并在-80℃下冷冻细胞沉淀,用40ml PBS洗涤细菌两次。
    9. 将冷冻的细胞沉淀重悬于40ml裂解缓冲液(参见食谱)。
    10. 使用Fisher Scientific Sonic分解器模型100,将细菌在冰上超声处理10 x 5秒,然后设置5,然后在设置2下10 x 5秒。
    11. 使用CS-6R离心机,在4℃下,在GH3.8转子中以2,400×g离心细胞匀浆15分钟。
    12. 在JA-20X(Avanti J-E)中,在4℃,20,100xg离心上清液15分钟。
    13. 将上清液通过孔径为0.45μm的Millex-HA注射器过滤器。
    14. 使用Labquake Shaker Rostisserie在4℃下将裂解物与2ml预洗的50%Ni-NTA珠(His-Bind Resin,Novagen)孵育过夜。
    15. 将样品置于15 mL柱(Poly-prep色谱柱,Bio-Rad Laboratories)中,在冷藏室中。
    16. 用3ml裂解缓冲液洗涤珠子,然后用15ml裂解缓冲液洗涤
    17. 用3ml洗涤缓冲液洗涤珠子(参见食谱),然后加入15ml洗涤缓冲液。
    18. 使用0.9ml洗脱缓冲液(参见食谱)将3μl重组Drp1蛋白从柱中洗脱3次,并将洗脱液收集到9个300μl馏分中。
    19. 使用SDS-PAGE和考马斯亮蓝染色鉴定含有Drp1蛋白的级分。合并峰分数(通常为级分#3和4)(图1),并与15ml不含咪唑的裂解缓冲液混合。


      图1.通过SDS-PAGE和考马斯亮蓝染色分析纯化的Drp1

    20. 将样品通过Amicon超离心过滤器(50k过滤器,全长Drp1和10k过滤器用于域),使用不含咪唑的裂解缓冲液三次。最终体积通常为1毫升。
    21. 将样品与最终的20%DMSO混合,分装,在液氮中快速冷冻,并储存在-80℃。通常,全长Drp1的浓度为30μM
  2. 浮选分析
    1. 除非另有说明,否则将脂质混合在POPC:罗丹明DPPE:可变脂质比为84:1:15(%mol)中。
    2. 使用氮气将脂质干燥5分钟,并另外在SpeedVac中干燥过夜。
    3. 在脂质膜中加入100μl/20μLMES(pH 7.0)/100 mM NaCl(见食谱)样品,旋转1 h。
    4. 使用干冰和42℃的热块进行五次冻融循环。
    5. 通过挤出通过孔径为400nm的纳米孔膜产生单层脂质体。该过程重复21次。
    6. 确认使用荧光显微镜形成脂质体(图2)

      图2.脂质体的共焦荧光显微镜

    7. 通过POLARstar Omega(波长; Ex 544nm,Em 590nm)检查罗丹明-PE的荧光强度,然后使用20mM MES(pH 7.0)/100mM NaCl,基于罗丹明强度调节脂质的浓度。 >
    8. 将脂质体(5mM脂质)与His6-Drp1(5μM)(终体积:200μl)混合,并在4℃下使用Labquake Shaker Rostisserie在20℃温育下孵育1小时20分钟mM MES(pH 7.0)/100mM NaCl(图3)。


      图3. Drp1和脂质体通过蔗糖梯度离心进行培养和分析。量化与脂质体进入级分1和2的Drp1的量。

    9. 在放置在管底部的最终1.73M蔗糖/20mM MES(pH 7.0)(终体积:1.25ml)中稀释Drp1-脂质体混合物。然后覆盖2.9ml 1.25M蔗糖/20mM MES(pH7.0)和0.85ml 0.25M蔗糖/20mM MES(pH7.0)(图3)。
    10. 在SW55Ti转子(Beckman Coulter)中,在4℃下将蔗糖梯度在287,000×g离心2小时。
    11. 从顶部收集五个级分(0.5,0.75,1.75,1.75和0.25 ml)。
    12. 大多数脂质体通常漂浮在前两个级分。
    13. 使用POLARstar Omega测量罗丹明强度。根据制造商的说明书,使用SDS-PAGE和银染(SilverQuest银染色试剂盒)分析各部分(等体积)。

数据分析

  1. 使用Scanmaker 8700扫描染色的凝胶,并使用NIH ImageJ量化条带强度。基于罗丹明-PE的荧光强度检测脂质体。规范Drp1和脂质体相对于各部分体积的量
  2. 对于统计分析,使用Student's t 测试来计算 P 值。

笔记

将样品装入SDS页面时,请用37℃的加热块加热样品。因为样品含有蔗糖,所以样品的流动性根据温度而变化。

食谱

  1. LB板含氨苄青霉素和氯霉素
    10克Bacto TM 胰蛋白胨
    10克NaCl
    5克Bacto TM 酵母提取物
    15克琼脂
    用超纯水加水至1升,121℃高压灭菌30分钟。高压灭菌后,让琼脂溶液冷却至55℃。加入最终100μg/ml氨苄青霉素和25μg/ml氯霉素
  2. LB与氨苄青霉素和氯霉素
    10克Bacto TM 胰蛋白胨
    10克NaCl
    5克Bacto TM 酵母提取物
    用超纯水加水至1升,121℃高压灭菌30分钟。高压灭菌后,将溶液冷却至55℃。加入最终100μg/ml氨苄青霉素和25μg/ml氯霉素
  3. 0.5 M IPTG
    1.19 g IPTG
    用超纯水加满10毫升
  4. 0.5 M HEPES(pH 7.4)
    238.4 g/L HEPES
    用KOH调节pH至7.4,用超纯水将其填充至1升
  5. 裂解缓冲液(10mM咪唑,1mM MgCl 2,500mM NaCl,2mM 2-巯基乙醇,20mM HEPES,pH7.4)
    40 ml/L 0.5 M HEPES(pH 7.4)
    500 ml/L 1 M NaCl
    1ml/L 1M MgCl 2
    5 ml/L 2 M咪唑
    140μl/L 2-巯基乙醇
    用超纯水加满1升
  6. 洗涤缓冲液(40mM咪唑,1mM MgCl 2,500mM NaCl,2mM 2-巯基乙醇,20mM HEPES,pH7.4)
    40 ml/L 0.5 M HEPES(pH 7.4)
    500 ml/L 1 M NaCl
    1ml/L 1M MgCl 2
    20 ml/L 2 M咪唑
    140μl/L 2-巯基乙醇
    用超纯水加满1升
  7. 洗脱缓冲液(250mM咪唑,1mM MgCl 2,500mM NaCl,2mM 2-巯基乙醇,20mM HEPES,pH7.4)
    40 ml/L 0.5 M HEPES(pH 7.4)
    500 ml/L 1 M NaCl
    1ml/L 1M MgCl 2
    50ml/L 2M咪唑
    140μl/L 2-巯基乙醇
    用超纯水加满1升
  8. 10倍PBS(pH 7.4)
    2.56g/L NaH 2 PO 4 H 2 O 2//O 2 22.5g/L Na 2 HPO 4·7H 2 O/
    87.66克/升NaCl
    用超纯水充满1升。用NaOH调节pH至7.0
  9. 考马斯亮蓝解决方案
    2.5 g/L考马斯亮蓝R-250
    100毫升/升酸性酸酸酯
    450毫升/升甲醇 450 ml/L超纯水
  10. 100mM MES(pH 7.0)
    21.538 g/L MES
    3.068 g/L NaOH
    用超纯水加满1升
  11. 20mM MES(pH 7.0)
    200 ml/L 100 mM MES(pH 7.0)
    用超纯水充满1升。用NaOH调节pH至7.0
  12. 20mM MES(pH7.0)/100mM NaCl 200 ml/L 100 mM MES(pH 7.0)
    5.844克/升NaCl
    140μl/L 2-巯基乙醇(最终2mM)
    用超纯水充满1升。根据需要用NaOH将pH调节至7.0
  13. 2.7 M蔗糖/20 mM MES/100 mM NaCl 924.21克/升蔗糖
    用20 mM MES/100 mM NaCl补充1 L
  14. 1.25 M蔗糖/20 mM MES/100 mM NaCl 427.875克/升蔗糖
    用20 mM MES/100 mM NaCl补充1 L
  15. 0.25M蔗糖/20mM MES/100mM NaCl 85.575g/L蔗糖
    用20 mM MES/100 mM NaCl补充1 L

致谢

感谢Iijima和Sesaki实验室的过去和现在的成员进行有益的讨论和技术援助。这项工作得到NIH授予M.I. (GM084015)和美国(GM089853)。

参考

  1. Adachi,Y.,Itoh,K.,Yamada,T.,Cerveny,KL,Suzuki,TL,Macdonald,P.,Frohman,MA,Ramachandran,R.,Iijima,M。和Sesaki,H。(2016)。   重合磷脂酸相互作用抑制线粒体分裂中的Drp1。一个>分子细胞 63(6):1034-1043。
  2. Bustillo-Zabalbeitia,I.,Montessuit,S.,Raemy,E.,Basanez,G.,Terrones,O. and Martinou,JC(2014)。  与心磷脂的特异性相互作用触发Dynamin相关蛋白1的功能激活。 PLoS One 9 (7):e102738。
  3. Macdonald,PJ,Stepanyants,N.,Mehrotra,N.,Mears,JA,Qi,X.,Sesaki,H.and Ramachandran,R。(2014)。二聚平衡中间体将Drp1重组在线粒体膜上进行裂变。分子生物细胞 25(12):1905-1915。
  4. Montessuit,S.,Somasekharan,SP,Terrones,O.,Lucken-Ardjomande,S.,Herzig,S.,Schwarzenbacher,R.,Manstein,DJ,Bossy-Wetzel,E.,Basanez,G.,Meda,P 。和Martinou,JC(2010)。膜重塑诱发通过dynamin相关蛋白Drp1刺激Bax低聚。细胞 142(6):889-901。
  5. Roy,M.,Reddy,PH,Iijima,M。和Sesaki,H。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/线粒体分裂和代谢融合。 33:111-118。
  6. Stephenants,N.,Macdonald,PJ,Francy,CA,Mears,JA,Qi,X.and Ramachandran,R。(2015)。< a class ="ke-insertfile"href ="http:心脑血管相关转移倾向及其由dynamin相关蛋白1重组形成线粒体膜裂变的基础。???????????????????????????????????????????????????????????? 26(17):3104-3116。
  7. Tamura,Y.,Itoh,K。和Sesaki,H。(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21703455" target ="_ blank"> SnapShot:线粒体动力学。 细胞 145(7):1158,1158 e1151。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Adachi, Y., Itoh, K., Iijima, M. and Sesaki, H. (2017). Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes. Bio-protocol 7(9): e2266. DOI: 10.21769/BioProtoc.2266.
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