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Apr 2018

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Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS
核转运蛋白-β2抑制和ALS / FTD相关蛋白FUS的逆转聚集和液-液相分离   

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Abstract

The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.

Keywords: FUS (FUS), Liquid-liquid phase separation (液-液相分离), Nuclear import receptor (核输入受体), Kapβ2 (Kapβ2), Protein aggregation (蛋白质聚集)

Background

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the mislocalization and aggregation of several RNA-binding proteins (RBP) (Mackenzie et al., 2010; Da Cruz and Cleveland, 2011; King et al., 2012). One of these proteins is FUS (Fused in Sarcoma), a nuclear protein mislocalizes in the cytoplasm of neurons in ALS/FTD patients, where it undergoes liquid-liquid phase transition followed by aberrant phase transition to form insoluble aggregates (Altmeyer et al., 2015; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015) (Figure 1). Karyopherin-β2 (Kapβ2), a nuclear import receptor (NIR), has been established as a chaperone and disaggregase of proteins with PY-nuclear localization sequences (NLS), such as FUS (Guo et al., 2018; Hofweber et al., 2018; Yoshizawa, et al., 2018). The following protocol was developed to test the ability of Kapβ2 to inhibit and reverse FUS aggregation and phase separation ex vivo (Guo et al., 2018).



Figure 1. Aberrant phase transition of FUS (10 μM) leads to the formation of fibrillar hydrogel. A. Domain architecture of FUS. B. DIC images show that FUS undergoes reversible liquid-liquid phase transition to form liquid droplets followed by aberrant phase transition to form hydrogels. Scale bar: 10 μm.

Materials and Reagents

  1. 1.5 ml tube holder
  2. 15 ml tube holder
  3. 50 ml tube holder
  4. Glass beads (MP Biomedicals, catalog number: MP07DP1070 )
  5. Nitrile gloves (FisherBrand, catalog number: 19-130-1597 )
  6. Ethanol spray bottle
  7. MilliQ water spray bottle
  8. Bleach spray bottle
  9. Ice bucket
  10. Foam liquid nitrogen container
  11. 1.5 ml Semi-Micro Plastic Cuvettes (Fisherbrand, catalog number: 14955127 )
  12. 96-well plate (Corning, catalog number: 08-757-230 )
  13. Lens paper (FisherBrand, catalog number: 11-995 )
  14. Glass microscope slides (Fisherbrand, catalog number: 12548B )
  15. 1.5 ml autoclaved Eppendorf tubes (Eppendorf, catalog number: 05-408-129 )
  16. 1.5 ml Protein LoBind Tubes (Eppendorf, catalog number: 22431081 )
  17. 15 ml conical tubes (Thermo Scientific, catalog number: 12-565-268 )
  18. 50 ml conical tubes (Thermo Scientific catalog number: 339652 )
  19. 50 ml plastic round-bottom centrifugal tubes (Thermo Scientific, catalog number: 05-529C )
  20. Pipette tips
    0.1-10 µl (USA Scientific, catalog number: 1121-3810 )
    2-20 µl (Fisherbrand, catalog number: 02-707-432 )
    20-200 µl (Fisherbrand, catalog number: 02-707-430 )
    200-1,000 µl (Fisherbrand, catalog number: 02-707-40 )
  21. Serological pipettor
  22. 5 ml, 10 ml, 25 ml, and 50 ml serological pipettes (Fisherbrand, model: 13-678-11 )
  23. BL21(DE3) Ril cells (Agilent, catalog number: 230245 )
  24. FUS expression construct in pDUET vector (Addgene Plasmid #29629) (Figure 2A)
  25. Kapβ2 expression construct in pGEX-Tev vector (Figure 2B)
  26. LB liquid media (Fisher Chemical, catalog number: BP9723-2 )
  27. Ampicillin (Gold Bio, catalog number: A-301-25 )
  28. LB + ampicillin agar plates
  29. Lysozyme (Gold Bio, catalog number: L-040-25 )
  30. Ice
  31. Liquid nitrogen
  32. Bradford Reagent (Thermo Scientific, catalog number: PI23236 )
  33. Microscope immersion oil (Carl Zeiss, catalog number: 12-070-397 )
  34. Bio-spin Chromatography Columns (Bio-Rad, catalog number: 7326008 )
  35. HiTrap-Q anion exchange chromatography column (GE Healthcare, catalog number: 17115301 )
  36. IPTG (GoldBio, catalog number: 367-93-1 )
  37. Phosphate-Buffered Saline tablets (Fisher Scientific, catalog number: BP2944100 )
  38. D-(+)-trehalose (Fisher Scientific, catalog number: BP2687100 )
  39. Glutathione Sepharose 4 Fast Flow beads (GE Healthcare, catalog number: 17-5132-01 )
  40. NaCl (Fisher Scientific, catalog number: S271-3 )
  41. EDTA (Fisher Scientific, catalog number: BP2482500 )
  42. Glycerol (Fisher Scientific, catalog number: BP2294 )
  43. DTT (Gold Bio, catalog number: DTT10 )
  44. Pepstatin (Gold Bio, catalog number: P-020-25
  45. EGTA (Millipore, catalog number: 324626-25GM )
  46. MgCl2 (Fisher BioReagents, catalog number: BP214-500 )
  47. Protease inhibitor tablets (Thermo Scientific, catalog number: A32955 )
  48. Imidazole (Fisher Scientific, catalog number: O3196-500 )
  49. Reduced glutathione (Fisher Scientific, catalog number: AC120000250 )
  50. FUS Wash Buffer (see Recipes)
  51. FUS Elution Buffer (see Recipes)
  52. Kapβ2 Resuspension Buffer (see Recipes)
  53. Kapβ2 ATP Buffer (see Recipes)
  54. Kapβ2 Buffer A (see Recipes)
  55. Kapβ2 Elution Buffer (see Recipes)
  56. TEV Buffer (see Recipes)


    Figure 2. Plasmid maps for the constructs used in the experiment. A. Plasmid map of FUS expression construct in pDUET vector. B. Plasmid map of Kapβ2 expression construct in pGEX-Tev vector.

Equipment

  1. 50 ml beakers (Pyrex, catalog number: CLS100050 )
  2. 100 ml beakers (Pyrex, catalog number: CLS100050 )
  3. 250 ml Erlenmeyer flask (Pyrex, catalog number: 4980250 )
  4. 2800 ml Erlenmeyer flasks (Pyrex, catalog number: CLS44202XL )
  5. Plastic nalgene 1 L centrifugal jars (Thermo Scientific, catalog number: 11-825B )
  6. 0.1-10 µl, 2-20 µl, 20-200 µl, and 200-1,000 µl pipettes
  7. Centrifuge 5424 R (Eppendorf, catalog number: 2231000655 )
  8. Centrifuge 5810 R (Eppendorf, catalog number: 0 22625004 )
  9. Sorvall Lynx 4000 Superspeed Centrifuge (Thermo Scientific, catalog number: 75-006-580 )
  10. Sorvall BP 8 Centrifuge (Thermo Scientific, catalog number: 75007681 )
  11. ThermomixerTM C with SmartBlock (Eppendorf, catalog number: 2231000667 )
  12. MaxQTM 4450 Benchtop Orbital Shaker (Thermo Scientific, catalog number: SHKE4450 )
  13. Innova® 44 Series Shaking Incubator (New Brunswick, catalog number: M1282 )
  14. HerathermTM General Protocol Microbiological Incubator (Thermo Scientific, catalog number: 51028063 )
  15. Tube Revolver/Rotator (Thermo Scientific, catalog number: 88881001 )
  16. Multi-Purpose Tube Rotator (Fisherbrand, catalog number: 88-861-049 )
  17. Digital Vortex Mixer (Fisherbrand, catalog number: 02-215-418 )
  18. Cimarec+TM Stirring Hotplate S (Thermo Scientific, catalog number: P88857100 )
  19. EntrisTM Precision Balance (SartoriusTM, catalog number: ENTRIS423i-1S )
  20. Accumet® AB150 pH Benchtop Meter (Fisherbrand, catalog number: 13-636-AB150 )
  21. Model 705 Sonic Dismembrator (Fisherbrand, catalog number: FB705110
  22. 160 UV-Vis Spectrophotometer (Biomate, catalog number: 840-301000 )
  23. TecanTM Spark® Multimode Microplate Reader
  24. HP Z230 Tower Workstation
  25. LEICATM DMi8 S Platform Inverted Microscope
  26. Dell Intel Core i7
  27. Cold room
  28. 4 °C Refrigerator
  29. -20 °C freezer
  30. -80 °C freezer
  31. AKTA pure FPLC

Software

  1. Excel (Microsoft Office)
  2. Tecan SparkControl 
  3. Leica LAS X Hardware
  4. AKTA Unicorn

Procedure

  1. FUS Purification
    1. Generate a FUS expression construct in a pDuet vector with a TEV-cleavable site, resulting in a GST-TEV-FUS construct. Express and purify GST-TEV-FUS protein from E. coli BL21-CodonPlus(DE3)-RIL cells (Agilent) as follow.
    2. Grow FUS-expressing cells in 6 L of Lennox Broth (LB) at 37 °C with shaking at 250 rpm until the OD600 is between 0.4-0.6. Induce cells with 1 ml of 1 M IPTG (final conc. 1 mM IPTG), then grow overnight at 15 °C with shaking at 250 rpm.
    3. Spin cells down at 4,000 rpm for 20 min at 4 °C. To lyse cells, resuspend pellet in FUS Wash Buffer (Recipe 1), add 20 µg of lysozyme per 1 ml resuspended cells, and incubate on ice for 30 min. Sonicate the cells for 4 min at amplitude 35 for 30 s intervals on/off. And then spin lysed cells down at 16,000 rpm for 20 min at 4 °C.
    4. To bind and separate GST-tagged proteins, 5 ml Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) is added to lysate supernatant and the mixture was incubated for 90 min at 4 °C. Centrifuge beads at 1,792 x g for 3 min at 4 °C and discard the supernatant. Wash beads three times with Wash Buffer, centrifuging at 1,124 x g for 2 min at 4 °C.
    5. Elute FUS by adding 5 ml FUS Elution Buffer (Recipe 2) and incubating for 20 min at room temperature. Centrifuge the beads in Elution Buffer at 1,124 x g for 2 min at 4 °C in Bio-spin chromatography columns (Bio-Rad) over glass tubes to collect the elution.
    6. Measure purified FUS via Bradford Assay. Flash freeze purified FUS in liquid nitrogen and store at -80 °C. Protein can be used for up to 3 years. Freeze-thaw cycle should be minimized.

  2. Kapβ2 Purification
    1. Generate a Kapβ2 expression construct in a pGEX-TEV vector with a TEV-cleavable site, resulting in a GST-TEV-Kapβ2 construct. Express and purify this Kapβ2 construct from E. coli BL21-CodonPlus(DE3)-RIL cells (Agilent) as follow.
    2. Grow Kapβ2-expressing cells in 6 L of Lennox Broth (LB) at 37°C with shaking at 250 rpm until the OD600 is between 0.6-0.8. Induce cells with 1 ml of 1 M IPTG (final conc. 1 mM IPTG), then grow overnight at 25 °C with shaking at 250 rpm. Spin cells down at 4,000 rpm for 20 min at 4 °C.
    3. To lyse cells, resuspend pellet in Resuspension Buffer (Recipe 3), add 20 µg of lysozyme per 1 ml resuspended cells, and incubate on ice for 30 min. Sonicate cells for 1 min 30 s at amplitude 15, 30 s on/off, and then spin lysed cells down at 16,000 rpm for 60 min at 4 °C.
    4. Incubate lysate supernatant with 5 ml Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) for 90 min at 4 °C. Centrifuge beads at 4,500 x g for 3 min at 4 °C and discard the supernatant.
    5. Wash beads four times with 25 ml Resuspension Buffer each, by centrifuging at 1,124 x g for 3 min at 4 °C. Add 10 ml ATP Buffer (Recipe 4) and let stand at room temperature for 10 min before centrifuging at 1,124 x g for 3 min at 4 °C. Wash beads twice with 20 ml ATP Buffer each, by centrifuging at 1,124 x g for 3 min at 4 °C. Wash beads three time with 10 ml Buffer A each time (Recipe 5), by centrifuging at 1,124 x g for 3 min at 4 °C.
    6. Elute Kapβ2 by adding 20 ml Kapβ2 Elution Buffer (Recipe 6) and incubating for 30 min at 4 °C. Centrifuge the beads in Elution Buffer at 1,124 x g for 2 min at 4 °C in Bio-spin chromatography columns (Bio-Rad) over glass tubes to collect the elution.
    7. Measure the concentration of the purified Kapβ2, then add 1 µg TEV per 10 µg Kapβ2. Aliquot the mixture into Low-Bind Tubes and incubate overnight at 30 °C to cleave the GST tag.
    8. Cleaved Kapβ2 is further purified by HiTrap-Q anion exchange chromatography column (GE healthcare).

  3. Inhibition of FUS aggregation by Kapβ2
    1. Spin down FUS at 16,100 x g for 10 min at 4 °C and transfer supernatant to a fresh tube. Measure the concentration of FUS and Kapβ2 via Bradford Assay with calibration curve generated using BSA.
    2. Set up the aggregation assay in a 96-well plate, with a blank well, a standard FUS well, and experimental wells.
      1. In each experimental well, add 4 µM FUS (Table 1), 4 µM Kapβ2 (Table 1), 5 µl TEV Buffer (Recipe 7), 1.5 µl 100 mM DTT, and 0.8 µg of TEV. Fill to 100 µl with FUS Buffer (Recipe 2). Pipet up and down gently to mix.
      2. In the blank well, add 5 µl TEV Buffer, 1.5 µl 100 mM DTT, and 0.8 µg TEV. Match the volume of Kapβ2 added to the experimental wells with Kapβ2 Buffer. Fill to 100 µl with FUS Buffer. Pipet up and down gently to mix.
      3. In the standard FUS well, add 4 µM FUS, 5 µl TEV Buffer, 1.5 µl 100 mM DTT, and 0.8 µg TEV. Match the volume of Kapβ2 added to the experimental wells with Kapβ2 Buffer. Fill to 100 µl with FUS Buffer. Pipet up and down gently to mix.
    3. Set up the TECAN plate reader. Identify the plate type as Costar 96 Flat Black with no lid and no humidity cassette. Program for a kinetic loop of 90 cycles with fixed intervals of 00:01:00 with no shaking. Program absorbance readings at 395 nm at each cycle. Insert the plate into the plate reader and run the aggregation assay at room temperature.
    4. After 1.5 h, the data will be automatically stored in an excel spreadsheet (Figure 3).


      Figure 3. Turbidity measurement of FUS aggregation shows Kapβ2 inhibits FUS aggregation. A. Raw data as exported from TECAN. B. Data after background subtraction. C. Data after normalization.

  4. Disaggregation of FUS fibrils by Kapβ2
    1. Spin down FUS at 16,100 x g for 10 min at 4 °C and transfer supernatant to a fresh tube. Measure the concentration of FUS and the concentration of Kapβ2.
    2. Set up the aggregation assay in a 96-well plate, with a blank well, a standard FUS well, and experimental wells.
      1. In each experimental well, add 4 µM FUS (Table 1), 5 µl TEV Buffer (Recipe 7), 1.5 µl 100 mM DTT, and 0.8 µg of TEV. Fill with FUS Buffer (Recipe 2) so that adding 4 µM Kapβ2 would give a total volume of 100 µl (Table 1). Pipet up and down gently to mix.
      2. In the blank well, add 5 µl TEV Buffer, 1.5 µl 100 mM DTT, and 0.8 µg TEV. Fill with FUS Buffer so that adding 4 µM Kapβ2 would give a total volume of 100 µl. Pipet up and down gently to mix.
      3. In the standard FUS well, add 4 µM FUS, 5 µl TEV Buffer, 1.5 µl 100 mM DTT, and 0.8 µg TEV. Fill with FUS Buffer so that adding 4 µM Kapβ2 would give a total volume of 100 µl. Pipet up and down gently to mix.
    3. Set up the TECAN plate reader. Identify the plate type as Costar 96 Flat Black with no lid and no humidity cassette. Program for a kinetic loop of 90 cycles with fixed intervals of 00:01:00. Program absorbance readings at 395 nm at each cycle. Insert the plate into the plate reader and run the aggregation assay at room temperature.
    4. At the end of the 90 cycles, add 4 µM of Kapβ2 to each experimental well. Add Kapβ2 Buffer to the blank well and the standard FUS well, match the volume of Kapβ2. Pipet up and down gently to mix. Program for another kinetic loop of 90 cycles with fixed intervals of 00:01:00. Program absorbance readings at 395 nm at each cycle. Insert the plate into the plate reader and run the disaggregation assay (Figure 4).


      Figure 4. Turbidity measurement of FUS disaggregation shows Kapβ2 reverses FUS aggregation. A. Raw data as exported from TECAN. B. Data after background subtraction. C. Data after normalization.

  5. Kapβ2 reverse FUS liquid-liquid phase separation and aberrant phase transition
    1. Spin down FUS at 16,100 x g for 10 min at 4 °C and transfer supernatant to a fresh tube. Measure the concentration.
    2. Dilute FUS to a concentration of 10 µM (Table 1) with FUS Buffer (Recipe 2). Pipette 30 µl of 10 µM FUS into a fresh tube. Leave the tube on the bench at room temperature for approximately 2 h.
    3. Set up the LEICA microscope to observe the liquid droplets using a 100x oil immersion objective.
    4. Pipette 10 µl of the 10 µM FUS sample onto the cover slide above the drop of oil.
    5. Observe liquid-liquid droplet formation. Droplets move fluidly and merge upon contact.
    6. Add 1 µl of 100 μM Kapβ2 (Table 1) into the 10μl FUS sample on the coverslip to disassemble FUS liquid droplets and reverse FUS liquid-liquid phase separation (Figure 5A). Kapβ2 can disassemble preformed FUS liquid droplets within 30 s.
    7. Liquid droplets age into hydrogel after 6 hours of incubation at room temperature. Add 10 µl of 10 µM FUS sample onto the coverslip to observe hydrogel. Add 1 µl of 100 μM Kapβ2 into the 10μl FUS sample on the coverslip to disassemble FUS hydrogel and reverse FUS aberrant phase separation (Figure 5B).


      Figure 5. Kapβ2 reverses the LLPS and aberrant phase transition of FUS. A. DIC image of FUS liquid droplets before and after addition of Kapβ2. B. DIC image of “aged” FUS droplets (> 6 h) before and after addition of Kapβ2. Scale bars: 10 μm.

Data analysis

Analysis of FUS Aggregation

  1. Upon completion of any turbidity assay, data will automatically be imported from Tecan SparkControl into Microsoft Excel.
  2. Plot the absorbances of each well as a function of time. Subtract absorbances of the blank well from that of the standard well and the experiment wells to get signal.
  3. Normalize the signals to the maximum signal of the standard reaction to determine the relative extent of aggregation/disaggregation (Figures 3 and 4).

Recipes

  1. FUS Wash Buffer
    1. Make 1x PBS prior to purifying FUS
      1. Add 1 L MilliQ H2O to a 1 L bottle
      2. Add 5 Phosphate-Buffered Saline tablets to the MilliQ H2O
      3. Stir until the tablets have fully dissolved. Filter and store at 4 °C
    2. Add fresh reagents to 300 ml 1x PBS the day of purification
      1. Add 6 protease inhibitor tablets to 1x PBS 
      2. Stir until the tablets have fully dissolved
      3. Filter and store at 4 °C
  2. FUS Elution Buffer
    1. Add 1 ml 1 M Tris (pH 8) buffer to a 50 ml beaker
    2. Add 1.513 g of D-(+)-trehalose [final conc. 4.4 mM] to increase solubility of FUS
    3. Add 112.9 mg reduced glutathione [final conc. 364 µM] to remove GST-tagged protein from the Glutathione Sepharose 4 Fast Flow beads
    4. Add Millipore water to a final total volume of 20 ml. Stir until reagents have fully dissolved and pH to pH 8. Filter and store at 4 °C
  3. Kapβ2 Resuspension Buffer
    1. Make a 1 L stock solution prior to purifying Kapβ2
      1. Add 50 ml of 1 M Tris (pH 7.5) [final conc. 50 mM]
      2. Add 5.844 g of NaCl [final conc. 100 mM]
      3. Add 2 ml of 0.5 M EDTA [final conc. 1 mM]
      4. Add 200 ml glycerol [final conc. 20%]
      5. Fill to 1 L with MilliQ water
      6. Stir until reagents have fully dissolved and pH to pH 7.5. Filter and store at 4 °C
    2. Add fresh reagents to 300 ml of stock the day of purification
      1. Add 600 µl 1 M DTT [final conc. 2 mM]
      2. Add 300 µl of 5 mM Pepstatin [final conc. 5 µM]
      3. Add 6 protease inhibitor tablets
      4. Stir solution until protease inhibitor tablets have fully dissolved and pH to pH 7.5. Filter and store at 4 °C
  4. Kapβ2 ATP Buffer
    1. Make a 1 L stock solution prior to purifying Kapβ2
      1. Add 50 ml of 1 M Tris (pH 7.5) [final conc. 50 mM]
      2. Add 5.844 g of NaCl [final conc. 100 mM]
      3. Add 2 ml of 0.5 M EGTA [final conc. 1 mM]
      4. Add 0.10165 of MgCl2 [final conc. 0.5 mM]
      5. Add 200 ml glycerol [final conc. 20%]
      6. Fill to 1 L with MilliQ water
      7. Stir until reagents have fully dissolved and pH to pH 7.5. Filter and store at 4 °C
    2. Add fresh reagents to 100 ml of stock solution the day of purification
      1. Add 200 µl 1 M DTT [final conc. 2 mM]
      2. Add 2 ml 250 mM ATP stock [final conc. 2 mM]
      3. Add 2 protease inhibitor tablets
      4. Stir solution until protease inhibitor tablets have fully dissolved and pH to pH 7.5. Filter and store at room temperature
  5. Kapβ2 Buffer A
    1. Make a 1 L stock solution prior to purifying Kapβ2
      1. Add 50 ml of 1 M Imidazole (pH 6.5) [final conc. 50 mM]
      2. Add 4.383 g of NaCl [final conc. 74 mM]
      3. Add 2 ml of 0.5 M EDTA [final conc. 1 mM]
      4. Add 200 ml glycerol [final conc. 20%]
      5. Fill to 1 L with MilliQ water
      6. Stir until reagents have fully dissolved and pH to pH 6.5. Filter and store at 4 °C
    2. Add fresh reagents to 100 ml of stock solution the day of purification
      1. Add 200 µl 1 M DTT [final conc. 2 mM]
      2. pH to pH 6.5, filter, and store at 4 °C
  6. Kapβ2 Elution Buffer
    1. Add 20 ml of fresh 100 ml Buffer A
    2. Add 122.9 mg reduced glutathione
    3. Stir until reduced glutathione has completely dissolved and pH to pH 6.5. Filter and store at 4 °C
  7. TEV Buffer
    1. Add 1 M Tris-HCl (pH 8.0)
    2. Add 10 mM EDTA
    3. Stir and pH to pH 8.9. Filter and store at room temperature

Table 1. Final Protein Concentrations for Experimental Assays

Acknowledgments

This protocol was described briefly in Guo et al., 2018. This study was supported by grants from the NIH R21NS090205 (to J. Shorter), the G. Harold and Leila Y. Mathers Charitable Foundation (to J. Shorter), Target ALS (to J. Shorter), ALSA (to J. Shorter), and the Packard Center for ALS Research (to J. Shorter). We also acknowledge the Alzheimer's Association (AARF-16-441196) and Target ALS Springboard Fellowship to L.G.

Competing interests

The Authors declare that there is no conflict of interest.

References

  1. Altmeyer, M., Neelsen, K. J., Teloni, F., Pozdnyakova, I., Pellegrino, S., Grofte, M., Rask, M. B., Streicher, W., Jungmichel, S., Nielsen, M. L. and Lukas, J. (2015). Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nat Commun 6: 8088.
  2. Burke, K. A., Janke, A. M., Rhine, C. L. and Fawzi, N. L. (2015). Residue-by-residue view of in vitro FUS granules that bind the C-terminal domain of RNA polymerase II. Mol Cell 60(2): 231-241.
  3. Da Cruz, S. and Cleveland, D. W. (2011). Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr Opin Neurobiol 21(6): 904-919. 
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简介

[摘要] RNA结合蛋白的研究(RBP)报价洞察病理蛋白聚集的神经变性疾病的机制。我们开发了用于纯化RBP FUS和核输入受体(NIR)Kapβ2 的协议,并测试Kapβ2 减轻FUS聚集和液-液相分离的能力。

[背景] 肌萎缩性脊髓侧索硬化症(ALS)和额颞叶痴呆(FTD)是神经变性疾病,其特征在于所述错误定位几个RNA结合蛋白的和聚集(RBP)(麦肯齐等人,2010;达克鲁斯和克利夫兰,2011; King 等,2012)。其中一种蛋白是FUS(融合在肉瘤中),一种核蛋白在ALS / FTD患者的神经元细胞质中定位不正确,然后经历液-液相转变,然后发生异常的相转变,形成不溶性聚集体(Altmeyer 等, 2015; Burke 等人,2015; Lin 等人,2015; Molliex 等人,2015; Murakami 等人,2015; Patel 等人,2015)(图1)。Karyopherin-β2(甲β2),一个核输入受体(NIR),已经建立了作为伴侣和disaggregase 蛋白与PY-核定位序列(NLS),诸如FUS(过等人,2018; Hofweber 等人。,2018;吉泽,等人。,2018)。以下方案被开发,以测试的能力甲β2抑制和反向FUS聚合和相分离的离体(过等人,2018)。

D:\ Reformatting \ 2020-6-1 \ 2003111--1463林国641755 \ Figs jpg \图1.jpg

图1. FUS(10μM)的异常相变导致原纤维水凝胶的形成。A. FUS的领域架构。B. DIC图像显示FUS经历了可逆的液-液相转变以形成液滴,然后发生异常的相转变以形成水凝胶。比例尺:10μm 。

关键字:FUS, 液-液相分离, 核输入受体, Kapβ2, 蛋白质聚集

材料和试剂
 


1.5 ml管架
15毫升试管架
50 ml管架
玻璃珠(MP Biomedicals,目录号:MP07DP1070)
丁腈手套(FisherBrand ,目录号:19-130-1597)
乙醇喷雾瓶
MilliQ 喷水瓶
漂白剂喷雾瓶
冰桶
泡沫液氮容器
1.5 ml半微量塑料比色杯(Fisherbrand ,目录号14955127)
96孔板(Corning,目录号:08-757-230)
镜头纸(FisherBrand ,目录号:11-995)
玻璃显微镜载玻片(Fisherbrand ,目录号:12548B)
1.5 ml高压灭菌的Eppendorf管(Eppendorf,目录号:05-408-129)
1.5 ml蛋白质LoBind 管(Eppendorf,目录号:22431081)
15 ml锥形管(Thermo Scientific,目录号:12-565-268)
50 ml锥形管(Thermo Scientific 目录号:339652)
50 ml圆底塑料离心管(Thermo Scientific,产品目录号:05-529C)
移液器技巧
0.1-10 µl(USA Scientific,目录号:1121-3810 )


2-20 µl(Fisherbrand ,目录号:02-707-432 )


20-200 µl (Fisherbrand ,目录号:02-707-430 )


200-1 ,000微升(FISHERBRAND ,目录号:02-707-40)


血清移液器
5毫升,10毫升,25毫升和50毫升血清移液器(Fisher b rand ,型号:13-678-11)
BL21(DE3)Ril 电池(安捷伦,目录号:230245)
pDUET vect 或(Addgene Plasmid#29629)中的FUS表达构建体(图2A)
甲在β2表达构建的pGEX-TEV 载体(图2B)
LB液体介质(Fisher Chemical,目录号:BP9723-2)
氨苄青霉素(Gold Bio,目录号:A-301-25)
LB +氨苄西林琼脂板
溶菌酶(Gold Bio,目录号:L-040-25)

液氮
布拉德福德Rea的绅士(热小号系统求解,目录号:PI23236)
显微镜浸油(Carl Zeis s,目录号:12-070-397)
Bio-spin色谱柱(Bio-Rad,目录号:7326008)
HiTrap -Q阴离子交换色谱柱(GE H ealthcare,目录号:17115301)
IPTG(GoldBio ,目录号:367-93-1)
磷酸盐缓冲盐水片(Fisher Scientific,目录号:BP2944100)
D-(+)- 海藻糖(Fisher Scientific,目录号:BP2687100)
谷胱甘肽琼脂糖4快速流动珠(GE Healthcare,目录号:17-5132-01)
NaCl (Fisher Scientific,目录号:S271-3)
EDTA (Fisher Scientific,目录号:BP2482500)
甘油(Fisher Scientific,目录号:BP2294)
DTT (Gold Bio,目录号:DTT10)
抑肽酶(Gold Bio,目录号:P-020-25
EGTA (密理博,目录号:324626-25GM)
MgCl 2 (Fisher BioReagents ,目录号:BP214-500)
蛋白酶抑制剂片剂(Thermo Scientific,目录号:A32955)
咪唑(Fisher Scientific,目录号:O3196-500)
还原型谷胱甘肽(Fisher Scientific,目录号:AC120000250)
FUS洗涤缓冲液(请参阅配方)
FUS洗脱缓冲液(请参阅配方)
Kapβ2 重悬缓冲液(请参阅食谱)
Kapβ2ATP 缓冲液(请参阅食谱)
Kapβ2 缓冲液A(请参阅食谱)
Kapβ2 洗脱缓冲液(请参见配方)
TEV缓冲液(请参阅配方)
 


D:\ Reformatting \ 2020-6-1 \ 2003111--1463林国641755 \ Figs jpg \图2.jpg


图2.实验中使用的构建体的质粒图。A.pDUET 载体中FUS表达构建体的质粒图。B.质粒图谱的甲中β2表达构建的pGEX-TEV 载体。              


 


设备


 


50毫升烧杯(派热克斯(Pyrex),货号:CLS100050)
100毫升烧杯(派热克斯(Pyrex),货号:CLS100050)
250 ml锥形瓶(派热克斯(Pyrex),货号:4980250)
2800毫升锥形瓶(派热克斯(Pyrex),目录号:CLS44202XL)
纳尔金1 L 塑料离心罐(Thermo Scientific,目录号:11-825B)
0.1-10微升,2-20微升,20-200微升,和200-1 ,000微升的移液管
5424 R离心机(埃彭多夫(Eppendorf),目录号:2231000655)
离心机5810 R(Eppendorf,目录号:022625004)
Sorvall Lynx 4000超速离心机(Thermo Scientific,目录号:75-006-580)
Sorvall BP 8离心机(Thermo Scientific,目录号:75007681)
带SmartBlock的Thermomixer TM C (Eppendorf,目录号:2231000667)
MaxQ TM 4450台式轨道振动器(Thermo Scientific,目录号:SHKE4450)
伊诺® 44系列摇床(新不伦瑞克省,目录号:M1282)
Heratherm TM 通用协议微生物培养箱(Thermo Scientific,目录号:51028063)
左轮手枪/旋转器(Thermo Scientific,目录号:88881001)
多功能管旋转器(Fisherbrand ,目录号:88-861-049)
数字涡旋混合器(Fisherbrand ,目录号02-215-418)
Cimarec + TM 搅拌加热板S(Thermo Scientific,目录号:P88857100)
Entris TM 精密天平(Sartorius TM ,目录号:ENTRIS423i-1S)
Accumet ® AB150的pH台式计(FISHERBRAND ,目录号:13-636-AB150)
模型705声波粉碎仪(FISHERBRAND ,目录号:FB705110)
160紫外可见分光光度计(Biomate ,目录号:840-301000)
Tecan公司TM 星火® 多模酶标仪
HP Z230塔式工作站
LEICA TM DMi8 S平台倒置显微镜
戴尔英特尔酷睿i7
冷室
4°C冰箱
-20°C冷冻室
-80°C冷冻室
AKTA纯FPLC




软件


 


电子邮件(Microsoft Office)
Tecan SparkControl
徕卡LAS X硬件
AKTA独角兽
 


再修改的è


 


FUS纯化
在带有TEV可切割位点的pDuet 载体中生成FUS表达构建体,从而产生GST-TEV-FUS构建体。从大肠杆菌BL21- CodonPlus(DE3)-RIL细胞(安捷伦)表达和纯化GST-TEV-FUS蛋白的方法如下。
在37 °C的6 L Lennox Broth(LB)中培养表达FUS的细胞,并以250 rpm的速度摇动,直到OD 600 在0.4-0.6之间。用1 ml的1 M IPTG(最终浓缩为1 mM IPTG)诱导细胞,然后在15°C下以250 rpm摇动生长过夜。
在4°C 下以4,000 rpm 旋转细胞20分钟。要裂解细胞,将沉淀物重悬于FUS洗涤缓冲液(配方1)中,每1毫升重悬细胞加入20克溶菌酶,并在冰上孵育30分钟。开/关以30 s的间隔超声处理细胞4分钟(振幅35)。然后在4°C下以16,000 rpm旋转裂解的细胞20分钟。
为了结合和分离带有GST标签的蛋白,将5 ml谷胱甘肽琼脂糖4快速流动珠(GE Healthcare)添加到裂解液上清液中,并将混合物在4°C下孵育90分钟。在4°C下以1,792 xg的速度离心珠3分钟,并丢弃上清液。用洗涤缓冲液洗涤磁珠3次,在4°C下以1,124 xg离心2分钟。
加入5 ml FUS洗脱缓冲液(配方2)洗脱FUS,并在室温下孵育20分钟。在玻璃管中的Bio-spin 色谱柱(Bio-Rad)中于4°C下在1,124 xg的离心缓冲液中将珠粒离心2分钟,以收集洗脱液。
通过Bradford分析测定纯化的FUS。在液氮中快速冷冻纯化的FUS,并储存在-80°C。蛋白质可以使用长达3年。冻融循环应最小化。
 


Kapβ2 纯化
生成甲在β2表达构建的pGEX 与TEV-可切割位点-TEV向量,产生了GST-TEV- 甲β2构建体。表达并纯化此甲从β2构建体大肠杆菌BL21- CodonPlus(DE3)-RIL细胞(安捷伦)如下。
生长甲β2表达细胞在6升伦诺克斯肉汤(LB)的在37℃下以250rpm振荡培养直至OD 600 为0.6-0.8之间。用1 ml的1 M IPTG(最终浓缩为1 mM IPTG)诱导细胞,然后在25°C下以250 rpm摇动生长过夜。在4°C下以4,000 rpm旋转细胞20分钟。
要裂解细胞,请在重悬缓冲液(配方3)中重悬沉淀,每1毫升重悬细胞添加20克溶菌酶,然后在冰上孵育30分钟。在振幅15、30 s开/关下超声处理细胞1分钟30 s,然后在4°C下以16,000 rpm旋转裂解的细胞60分钟。
将裂解液上清液与5 ml谷胱甘肽琼脂糖4快速流动珠(GE Healthcare)在4°C下孵育90分钟。在4离心机珠,500 ×g下在4℃下3分钟,弃去上清液。
洗珠用25ml重悬每个缓冲器四次,通过在1124离心XG 用于在4℃下3分钟。加入10 ml ATP缓冲液(配方4),在室温下静置10分钟,然后在4°C下以1,124 xg离心3分钟。分别用20 ml ATP缓冲液洗涤小珠两次,方法是在4°C下以1,124 xg离心3分钟。通过在4°C下以1,124 xg离心3分钟,每次用10 ml缓冲液A洗涤磁珠3次(配方5)。
洗脱甲β2通过加入20ml 甲β2洗脱缓冲液(配方6),并在4孵育30分钟℃。在玻璃管中的Bio-spin色谱柱(Bio-Rad)中于4°C下以1,124 xg的离心力将珠子在洗脱缓冲液中离心2分钟,以收集洗脱液。
测量纯化的Kapβ2 的浓度,然后每10 µg Kapβ2 加入1 µg TEV 。将混合物分装到低绑定试管中,在30°C下孵育过夜,以裂解GST标签。
裂解的Kapβ2 通过HiTrap -Q阴离子交换色谱柱(GE Healthcare)进一步纯化。
 


通过FUS聚集的抑制甲β2
在4°C 下以16,100 xg 的转速旋转FUS 10分钟,然后将上清液转移到新的试管中。通过Bradford测定法测量FUS和Kapβ2 的浓度,并使用BSA生成校准曲线。
在96孔板,空白孔,标准FUS孔和实验孔中设置聚集测定。
在每个实验孔中,添加4μM的FUS (表1),4μM 甲β2 (表1),5微升缓冲液TEV(配方7),1.5微升100mM DTT,和0.8微克TEV的。用FUS缓冲液(配方2)填充至100 µl。轻轻上下吹打混合。
在空白孔中,加入5 µl TEV缓冲液,1.5 µl 100 mM DTT和0.8 µg TEV。用Kapβ2 缓冲液匹配添加到实验孔中的Kapβ2 的体积。用FUS缓冲液填充至100 µl。轻轻上下吹打混合。
在标准FUS孔中,添加4 µM FUS,5 µl TEV缓冲液,1.5 µl 100 mM DTT和0.8 µg TEV。用Kapβ2 缓冲液匹配添加到实验孔中的Kapβ2 的体积。用FUS缓冲液填充至100 µl。轻轻上下吹打以混合。
设置TECAN p 后期阅读器。将印版类型识别为Costar 96 Flat Black(无盖,无湿度盒)。进行90个循环的动态循环编程,固定间隔为00:01:00,无晃动。在每个循环中在395 nm处编程程序吸光度读数。将板插入板读取器,并在室温下进行聚集测定。
1.5小时后,数据将自动存储在excel电子表格中(图3)。
 


D:\ Reformatting \ 2020-6-1 \ 2003111--1463林果641755 \ Figs jpg \图3.jpg


图3. FUS聚集的浊度测量显示,Kapβ2 抑制FUS聚集。A.从TECAN导出的原始数据。B.背景扣除后的数据。C.标准化后的数据。


 


FUS的纤丝解聚由甲β2
在4°C 下以16,100 xg 的转速旋转FUS 10分钟,然后将上清液转移到新的试管中。测量FUS的浓度和浓度甲β2。
在96孔板,空白孔,标准FUS孔和实验孔中设置聚集测定。
在每个实验孔中,添加4 µM FUS (表1),5 µl TEV缓冲液(配方7),1.5 µl 100 mM DTT和0.8 µg TEV。填充FUS缓冲液(配方2),以便添加4 µM Kapβ2 将产生100 µl的总体积(表1)。轻轻上下吹打混合。  
在空白孔中,加入5 µl TEV缓冲液,1.5 µl 100 mM DTT和0.8 µg TEV。填充FUS缓冲液,以便添加4 µM Kapβ2 将产生100 µl的总体积。轻轻上下吹打混合。
在标准FUS孔中,添加4 µM FUS,5 µl TEV缓冲液,1.5 µl 100 mM DTT和0.8 µg TEV。填充FUS缓冲液,以便添加4 µM Kapβ2 将产生100 µl的总体积。轻轻上下吹打以混合。
设置TECAN读板器。将印版类型标识为Costar 96 Flat Black(无盖,无湿度盒)。编程90个循环的动态循环,固定间隔为00:01:00。在每个循环中在395 nm处编程程序吸光度读数。将板插入板读取器,并在室温下进行聚集测定。
在90个循环的最后,向每个实验孔中加入4 µM Kapβ2 。添加甲β2缓冲空白以及与STA ndard FUS好,匹配的容积金甲β2。轻轻上下吹打以混合。编程另一个90循环的动力学循环,固定间隔为00:01:00。在每个循环中在395 nm处编程程序吸光度读数。将板插入板读取器并进行解离分析(图4)。
 


D:\ Reformatting \ 2020-6-1 \ 2003111--1463林果641755 \ Figs jpg \图4.jpg


图4 的分解FUS显示浊度测量甲β 2点反转FUS聚集。A.从TECAN导出的原始数据。B.背景扣除后的数据。C.标准化后的数据。


Kapβ2 反向FUS液-液相分离和异常相变
在4°C 下以16,100 xg 的转速旋转FUS 10分钟,然后将上清液转移到新的试管中。测量浓度。
用FUS缓冲液(配方2)将FUS 稀释至10 µM (表1)的浓度。移液管加入30μl 的10 μMFUS到一个新的试管中。将试管在室温下放置在工作台上约2小时。
设置LEICA显微镜以使用100倍油浸物镜观察液滴。
将10 µM FUS样品中的10 µl吸到滴油上方的盖玻片上。
观察液-液滴的形成。液滴流动并在接触时融合。
加入1μl100 μM 甲β2 (表1)到各10μlFUS 样品盖玻片上拆卸FUS液滴和反向FUS液-液相分离(图5A)。Kapβ2 可以在30 s内分解预制的FUS液滴。
在室温下孵育6小时后,液滴会老化成水凝胶。在盖玻片上加10 µl 10 µM FUS样品,观察水凝胶。加入1μl100 μM 甲β2到盖玻片上拆卸FUS水凝胶和逆向FUS异常的相分离(图5B)的加入10μlFUS样品。
 


D:\ Reformatting \ 2020-6-1 \ 2003111--1463林国641755 \ Figs jpg \图5.jpg


图5. 甲β2反转FUS的LLPS和异常相变。前和添加后FUS液滴A. DIC图像的甲β2。B. 在添加Kapβ2 之前和之后的“老化” FUS液滴(> 6小时)的DIC图像。比例尺小号:10 微米。


 


d ATA analysi小号


 


一个FUS聚合的nalysis


完成任何浊度测定后,数据将自动从Tecan SparkControl 导入Microsoft Excel。
绘制每个孔的吸光度与时间的关系。减去空白的吸光度以及从标准井和t 他EXPE riment井获得的信号。
归一化的信号,以标准反应的最大信号,以确定聚集/解聚的相对程度(图小号3和4) 。
 


菜谱


 


FUS洗涤缓冲液
在纯化FUS之前制作1 x PBS
将1升MilliQ H 2 O加到1升瓶中
向MilliQ H 2 O中添加5种磷酸盐缓冲盐水片
搅拌直至片剂完全溶解。过滤并储存在4°C
纯化当天将新鲜试剂加入300 ml 1x PBS中
将6种蛋白酶抑制剂片剂添加到1x PBS中
搅拌直至片剂完全溶解
过滤并储存在4°C
FUS洗脱缓冲液
将1 ml 1 M Tris(pH 8)缓冲液添加到50 ml烧杯中
加入1.513 g D -(+)- 海藻糖[最终浓度。4.4 mM ]以增加FUS的溶解度
加入112.9毫克还原型谷胱甘肽[最终浓度。364 µM]从谷胱甘肽1 Sepharose 4 Fast Flow珠粒中去除带有GST标签的蛋白
加入Millipore水至最终总体积为20 ml。搅拌直至试剂完全溶解并且pH达到pH8。过滤并在4°C储存
Kapβ2 重悬缓冲液
做一个1升原液之前净化金甲β2
加入50 ml 1 M Tris(pH 7.5)[最终浓度。50毫米]
加入5.844 g NaCl [最终浓度。100 mM]
加入2 ml 0.5 M EDTA [最终浓 1毫米]
加入200毫升甘油[最终浓度。20%]
加入MilliQ 水至1 L
搅拌直至试剂完全溶解,并且pH达到pH 7.5。过滤并储存在4°C
在纯化的当天将新鲜的试剂添加到300 ml的储备液中
加入600 µl 1 M DTT [最终浓度。2毫米]
加入300 µl 5 mM 抑肽酶[最终浓度。5 µM]
添加6蛋白酶抑制剂片
搅拌溶液直至蛋白酶抑制剂片剂完全溶解,并且pH达到pH 7.5。过滤并储存在4°C
Kapβ2ATP 缓冲液
做一个1升原液之前净化金甲β2
加入50 ml 1 M Tris(pH 7.5)[最终浓度。50毫米]
加入5.844 g NaCl [最终浓度。100 mM]
加入2 ml 0.5 M EGTA [最终浓 1毫米]
加入0.10165 MgCl 2 [最终浓度。0.5毫米]
加入200毫升甘油[最终浓度。20%]
加入MilliQ 水至1 L
搅拌直至试剂完全溶解,并且pH达到pH 7.5。过滤并储存在4°C
纯化当天将新鲜试剂添加到100 ml储备液中
加入200 µl 1 M DTT [最终浓度。2毫米]
加入2 ml 250 mM ATP储备液[最终浓度。2毫米]
加2蛋白酶抑制剂片
搅拌溶液直至蛋白酶抑制剂片剂完全溶解,并且pH达到pH 7.5。过滤并在室温下储存
Kapβ2 缓冲液A
做一个1升原液之前净化金甲β2
加入50 ml 1 M咪唑(pH 6.5)[最终浓度。50毫米]
加入4.383 g NaCl [最终浓度。74毫米]
加入2 ml 0.5 M EDTA [最终浓 1毫米]
加入200毫升甘油[最终浓度。20%]
加入MilliQ 水至1 L
搅拌直至试剂完全溶解,并且pH达到pH 6.5。过滤并储存在4°C
纯化当天将新鲜试剂添加到100 ml储备液中
加入200 µl 1 M DTT [最终浓度。2毫米]
pH值至pH 6.5,过滤并储存在4°C
Kapβ2 洗脱缓冲液
加入20毫升新鲜的100毫升缓冲液A
加入减少的122.9 mg谷胱甘肽
搅拌直至还原的谷胱甘肽完全溶解并且pH达到pH 6.5。过滤并储存在4°C
TEV缓冲液
加入1 M Tris-HCl(pH 8.0)
加入10 mM EDTA
搅拌并pH至pH 8.9 。过滤并在室温下储存




表1.实验测定的最终蛋白质浓度


 


FUS浓度


Kapβ2 浓度


FUS聚集的抑制由甲β2(实验C)


4微米


4微米


DIS 通过FUS原纤维的聚集甲β2 (实验d)


4微米


4微米


FUS LLPS的逆转金甲β2 (实验E)


10微米


100微米


 


致谢


 


该协议在Guo 等人中进行了简要描述。,2018年。这项研究得到了NIH R21NS090205(授予J. Shorter),G。Harold和Leila Y. Mathers慈善基金会(致J. Shorter),Target ALS(致J. Shorter),ALSA(致J. Shorter)和Packard ALS研究中心(J. Shorter)。我们也感谢阿尔茨海默氏症协会(AARF-16-441196)和LG的目标ALS跳板奖学金


 


利益争夺


 


作者声明没有利益冲突。


 


参考文献


 


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引用:Robinson, E., Shorter, J. and Guo, L. (2020). Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS. Bio-protocol 10(16): e3725. DOI: 10.21769/BioProtoc.3725.
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