Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS

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. Thermomixer^TM C with SmartBlock (Eppendorf, catalog number: 2231000667 )
12. MaxQ^TM 4450 Benchtop Orbital Shaker (Thermo Scientific, catalog number: SHKE4450 )
13. Innova^® 44 Series Shaking Incubator (New Brunswick, catalog number: M1282 )
14. Heratherm^TM 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. Entris^TM Precision Balance (Sartorius^TM, 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. Tecan^TM Spark^® Multimode Microplate Reader
24. HP Z230 Tower Workstation
25. LEICA^TM 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