In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network

Materials and Reagents

1. HEK293F cells (ATCC, catalog number: CRL-1573)

2. Sf9 cells (ATCC, catalog number: CRL-1711)

3. BL21 (DE3) E. coli cells (TransGen Biotech, catalog number: CD601-02)

4. Lab Tek Chambered cover glass (Thermo Fisher Scientific, catalog number: 150682)

5. SMM 293-TII expression medium (Sino Biological, catalog number: M293TII)

6. Sf-900 II SFM medium (Thermo Fisher Scientific, catalog number: 10902096)

7. Polyethyleneimines (PEI) (Polysciences, catalog number: 23966-2)

8. Amylose beads (New England Biolabs, catalog number: E8035S)

9. LB broth powder (Sangon Biotech, catalog number: A507002)

10. Isopropyl-beta-D-thiogalactopyranoside (IPTG) solution (Sangon Biotech, catalog number: B541007)

11. Tris (Sigma-Aldrich, catalog number: 77-86-1)

12. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 329-98-6)

13. Acetic acid (Sangon Biotech, catalog number: A501931)

14. Ammonium acetate (Sangon Biotech, catalog number: A600032)

15. Phosphate buffered saline (PBS) (HyClone, catalog number:SH80255.01)

16. HEPES (Sigma-Aldrich, catalog number: 7365-45-9)

17. Imidazole (Sigma-Aldrich, catalog number: 288-32-4)

18. Glycerol (Sinopharm, catalog number: 56-81-5)

19. DTT (dithiothreitol) (Inaclo, catalog number: 0281)

20. Maltose (Sigma-Aldrich, catalog number: 69-79-4)

21. Rosetta (DE3) pLysS-competent E. coli cells (TIANGEN, catalog number: CB108-02)

22. Strep-Tactin resin (IBA Lifesciences, catalog number: 2-1201-002)

23. D-desthiobiotin (Sigma-Aldrich, catalog number: D1411)

24. HisPur(tm) Ni-NTA Resin (Thermo Fisher Scientific, catalog number: 88222)

25. MBP Trap HP column (GE Healthcare, catalog number: 28-9187-78)

26. TEV protease (Solarbio, catalog number: P2060)

27. Sodium chloride (NaCl) (Sangon Biotech, catalog number: A610476)

28. PIPES (Sangon Biotech, catalog number: A620434)

29. Potassium chloride (KCl) (Sangon Biotech, catalog number: A610440)

30. MgCl₂ (Sangon Biotech, catalog number: B601193)

31. EGTA (MedChemExpress, catalog number: HY-D0861)

32. Ethylenediaminetetraacetic acid (EDTA) (MedChemExpress, catalog number: HY-Y0682)

33. Adenosine 5'-triphosphate (ATP) (MedChemExpress, catalog number: HY-B2176)

34. MOPS (MedChemExpress, catalog number: HY-D0859)

35. Triton X-100 (Solarbio, catalog number: T8200)

36. Protease inhibitor cocktails (MedChemExpress, catalog number: HY-K0012)

37. Glucose oxidase (Solarbio, catalog number: G8030)

38. Catalase (Solarbio, catalog number: C8070)

39. Bovine serum albumin (BSA) (Solarbio, catalog number: A8010)

40. Methyl cellulose (MedChemExpress, catalog number: HY-125861)

41. Sucrose (Solarbio, catalog number: S8271)

42. ATP-Mg (Sigma Aldrich, catalog number: A9187)

43. Buffer A (see Recipes)

44. Buffer B (see Recipes)

45. Buffer C (see Recipes)

46. Lysis buffer (see Recipes)

47. Basic buffer (see Recipes)

48. Loading buffer (see Recipes)

49. High-salt buffer (see Recipes)

50. Wash buffer (see Recipes)

51. TIRF buffer (see Recipes)

52. Elution buffer (see Recipes)

53. Storage buffer (see Recipes)

Equipment

1. Nickel column (Bio-Rad, catalog number: 12009287)

2. Mono Q 5/50 GL column (GE Healthcare, catalog number: 17-5166-01)

3. HiTrap Q HP (GE Healthcare, catalog number: 17-1154-01)

4. Superdex200 Increase 10/300 GL (GE Healthcare, catalog number: 28990944)

5. Ultrafiltration discs (Sigma-Aldrich, catalog number: PLGC07610)

6. Allegra X-14 centrifuge, type 45 Ti rotor manual, JS-4.750 swinging-bucket rotor and buckets, 4 × 50 mL, 9 × 15 mL adapters (Beckman Coulter, model: Allegra X14, catalog number: A99464)

7. Water bath at 37 °C (Thermo Fisher Scientific, model: TSCIP19)

8. Incubator at 37 °C with 5% CO₂ (Thermo Fisher Scientific, model: HERAcell 150i)

9. Confocal fluorescence microscopy (NIKON, A1 HD25)

10. Nikon SIM/STORM (TIRF)

11. Field emission scanning electron microscopy (FE-SEM) (FEI Quanta 200)

12. Critical Point Dryer (Shianjia Biotechnology, model: SCD-350M)

Software

1. NIS-Elements software (Laboratory Imaging)

2. ImageJ

Procedure

1. Purification of Arp2/3 complex

   1. Construction of expression vector: Sub-clone the full-length open reading frames of seven subunits of human Arp2/3-ArpC1a-ArpC5 complex into pCAG vectors. To facilitate protein purification, add two strep-tags to the N-termini of ArpC1a (Doolittle et al., 2013a and 2013b).

   2. Cell transfection: Grow HEK293F cells in SMM 293-TII expression medium to a density of 1.5–1.8 million cells per milliliter. Transiently transfect the plasmids to HEK293F cells using PEI. Mix 0.25 mg of plasmids of each subunit of complex (1.75 mg in total) and 3 mg of PEI in 50 mL of fresh medium for 25 min, then add to 800 mL of culture. Culture the transfected cells at 37 °C supplemented with 5% CO₂ in a shaker for 48–72 h.

   3. Extraction and purification of target proteins:

      1. Harvest cells by centrifugation and resuspend in ice-cold buffer A.

      2. Solubilize cell membrane using 1% Triton X-100 for 1 h while rotating at 4 °C.

      3. Centrifuge cell lysates at 20,000 × g for 1 h at 4 °C using a type 45 Ti rotor to remove cell debris.

      4. Collect and load the supernatant onto Strep-Tactin resin.

      5. Remove unbound proteins by extensive wash with buffer B.

      6. Elute the bound proteins with buffer B supplemented with 10 mM D-desthiobiotin.

      7. Further purify the target protein by Superdex200 Increase 10/300 GL column using buffer C.

      8. Pool and concentrate peak fractions containing all seven subunits to 10 mg/mL.




2. Purification of PWCA

   1. Construction of vector: Clone the PWCA domain of human WHAMM protein into a pCAG vector containing a N-terminal MBP-tag (Liu et al., 2017). The PWCA domain is expressed and purified from the HEK293F cell line.

   2. When the culture reaches a density of 1.2–1.6 million cells per milliliter, transfect cells with 1 mg of plasmids using 2 mg of PEI.

   3. Extraction and purification of target proteins:

      1. After 48–72 h post transfection, harvest cells and lyse in ice-cold lysis buffer for 1 h while rotating at 4 °C.

      2. Remove the insoluble material by centrifugation at 20,000 × g for 1 h at 4 °C.

      3. Incubate the supernatant with amylose beads for 2 h while rotating at 4 °C.

      4. Remove unbound proteins by extensive wash with the basic buffer and elute the bound proteins using the same buffer with 10 mM maltose.

      5. Further purify the eluted proteins by anion exchange using a Mono Q 5/50 GL column.

      6. Load the proteins onto the Mono Q column at 1 mL/min using the loading buffer as running buffer.

      7. Use high-salt buffer for gradient elution.

      8. Pool and load the fractions containing the PWCA domain onto a Superdex 75 10/300 GL column for final purification.




3. Purification of capping protein (CapZ)

   1. Construction of vector: Clone the cDNA of capping protein (α1β2, Mus musculus) into a pET-3d vector containing a N-terminal 6×His-tag.

   2. Transfer the expression vector into Rosetta (DE3) pLysS-competent E. coli cells via transformation for protein expression.

   3. Extraction and purification of target protein:

      1. Grow bacteria in LB medium at 37 °C and induce protein expression at OD₆₀₀ 0.6–1.0 with 0.5 mM IPTG for 3 h.

      2. Collect cell pellets by centrifugation and resuspend in ice-cold lysis buffer.

      3. Lyse cells by ultrasonication; then, remove cell debris by centrifugation at 20,000 × g using the Type 45 Ti rotor at 4 °C for 1 h.

      4. Collect the supernatant and load it onto Ni-NTA resin.

      5. Wash the resin with wash buffer.

      6. Elute the bound proteins with elution buffer.

      7. Further purify the eluted proteins by anion exchange using a Mono Q 5/50 GL column.

      8. Load the proteins onto the Mono Q column at 1 mL/min using the loading buffer as running buffer.

      9. Use high-salt buffer for gradient elution.

      10. Pool and load the fractions containing capping protein onto a Superdex 75 10/300 GL column for final purification.




4. Purification of myosin 1D

   1. Grow Sf9 cells in sf-900 II SFM medium to a density of 2 × 10⁶ cells/mL and incubate with virus harboring full-length Myo1D construct containing a N-terminal 6×His-tag.

   2. After 60 h, collect cells, lyse by freeze-thaw cycles, and centrifuge at 4 °C.

   3. Remove insoluble material by centrifugation at 20,000 × g for 30 min.

   4. Incubate supernatants on a roller at 4 °C for 1 h with 1 mL of Ni-NTA resin and transfer into a column.

   5. Wash the columns with wash buffer.

   6. Elute myosin 1D with elution buffer.

   7. The protein concentration of myosin 1D is determined by OD₂₈₀.

   8. Concentrate the protein by ultrafiltration discs (10 KD).

   9. Store the protein in storage buffer.

   10. Determine the purity of protein by Coomassie blue–stained SDS-poly-acrylamide gels.




5. Expression and purification of Ub8

   1. Grow bacteria transformed with 6×His-Ub8 construct in LB medium at 37 °C and induce at OD₆₀₀ ~0.8 with 0.5 mM IPTG at 18 °C for 16 h.

   2. Re-suspend cells in 50 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM phenylmethylsulphonyl fluoride (PMSF).

   3. Transfer the cell suspension on ice and sonicate to disrupt the cells, followed by centrifugation at 47,850 × g for 1 h at 4 °C.

   4. Isolate the proteins through a Ni-NTA column.

   5. Further purify the proteins using a Hitrap Q HP column.

   6. Acidify the solution of Ub8 using acetic acid.

   7. Apply to a Hitrap SP HP column.

   8. Elute with a linear gradient of NaCl (0–1 M) in 50 mM ammonium acetate pH 4.5.

   9. Further purify Ub8 proteins by gel filtration. Equilibrate the gel filtration columns (Superdex 200 10/300) in 150 mM NaCl, 40 mM Tris-HCl (pH 7.4), 10% glycerol, and 1 mM DTT.

   10. Flash-freeze Ub8 proteins and store at -80 °C.




6. Expression and purification of p62

   1. Transfer the MBP-mCherry-p62 construct (His6-MBP-Tev-mCherry vector backbone) into BL21 (DE3) E. coli cells via transformation and spread to bacterial culture plate for overnight culture at 37 °C.

   2. Pick up a single colony into a liquid LB medium (approximately 15 mL) and incubate at 37 °C in a bacterial shaker at 220 rpm for 14–18 h.

   3. Inoculate 1% of the bacterial solution into 1 L liquid LB medium and expand the culture at 37 °C.

   4. When OD₆₀₀ reaches ~0.6, add 0.2 mM IPTG to induce protein expression at 16 °C overnight.

   5. Collect bacteria at 1,800 × g for 15 min. Wash again with PBS.

   6. Add 30 mL of lysis buffer, vortex, and shake to fully resuspend the bacteria.

   7. Put the resuspended bacteria into a 50 mL glass beaker on ice.

   8. Ultrasonic crush the bacteria for 10 min, at 60% intensity, sonication for 5 s, interval 3 s.

   9. Centrifuge the sonication solution at 13,000 × g for 30 min at 4 °C.

   10. Collect the supernatant and purify the proteins with MBP Trap HP column.

   11. Use TEV protease to remove MBP tag from MBP-mCherry-p62 fusion protein (Raran-Kurussi et al., 2017). Add the equivalent amount of TEV protease to the standard reaction buffer (50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, and 1 mM DTT) containing MBP-mCherry-p62 fusion protein and incubate overnight (4 °C) or 1–2 h (37 °C).

   12. The resultant mCherry-p62 protein is used for in vitro phase separation assay.




7. In vitro reconstitution of p62 bodies on the Arp2/3-derived actin network (Figure 1)

   1. Prepare actin monomers (1 μM, 50% Oregon-green labeled) as described in another protocol (Jiang and Huang, 2017). The growth of actin filaments can be observed under a Nikon microscope equipped with a 100× oil objective by the TIRF illumination.

   2. To initiate branched actin polymerization, add 1 μM actin (50% Oregon-green labeled), 100 nM Arp2/3 complex, 600 nM CapZ, and 300 nM PWCA to a tube, then add TIRF buffer for a 50 μL mixed solution in total.

   3. Carefully transfer 10 μL of mixed solution to the center of a Lab Tek Chambered cover glass.

   4. In the focal plane of the chamber, robust branched actin polymerization can be observed by TIRF microscopy.

   5. Add 10 μM Myo1D to the solution and continue TIRF imaging.

   6. Add 0.5 μM mCherry-p62 and 3 μM Ub8 to the solution and continue TIRF imaging.

   7. The reconstituted p62 body can also be examined by scanning electron microscopy. For the conventional fixation procedure, grow p62 bodies on coverslips, fix with 2.5% glutaraldehyde in PBS buffer for 2 h at room temperature, wash three times with PBS buffer, and then post-fix with 1% osmium for 20 min at room temperature. Then, dehydrate all samples with a graded series of ethanol (50%, 70%, 80%, 90%, 100%, and 100%) for 2 min each and dry with Critical Point Dryer using CO₂. Coat the dried samples with an approximately 5 nm thick gold film by sputter coating before examination with focused ion beam scanning electron microscopy using a Helios detector at an acceleration voltage of 2.0 kV (Rong et al., 2012).

      
      

      
      Figure 1. Schematic of the p62 body reconstitution assay. Molecules are not drawn to scale.

Data analysis

In this reconstitution assay, all components are derived from purified proteins. Oregon-green-labeled actin monomers can self-assemble into actin filaments (Figure 2, left panel). Addition of Arp2/3 complex, CapZ, and PWCA leads to the formation of branched actin network (Figure 2, right panel). Myo1D, as a motor protein, is not necessary for the branched actin network; however, its inclusion strengthens such branched actin structures. The scaffold proteins of p62 bodies (mCherry-p62 and Ub8) are then introduced on top of branched actin network to reconstitute cytoskeleton-associated p62 bodies, which can be observed by either TIRF microscopy or scanning electron microscopy (Figure 3 and Figure 4). Then, the NIS-Elements software is used for image measurement and co-location analysis. In such in vitro assay, phase separation of p62 bodies is greatly accelerated with lower concentration threshold of scaffold proteins in the presence of branched actin network than that using p62/Ub8 alone (Feng et al., 2022). Use ImageJ to process immunofluorescence images (rotating, cropping, and adjusting brightness and contrast when necessary).

Figure 2. Total internal reflection fluorescence (TIRF) images showing the actin polymerization. Oregon-green-labeled actin filaments (left panel) and reconstituted branched actin network (right panel). Scale bar = 10 μm.

Figure 3. In vitro–reconstituted p62 bodies on branched actin network. Scale bar = 5 µm.

Figure 4. Morphology of reconstituted p62 bodies observed by FE-SEM. Field emission scanning electron microscopy (FE-SEM) image showing the structures of four reconstituted p62 bodies on the Arp2/3-derived actin network. Scale bar = 1 µm.

Notes

1. When mixing the components for branched actin polymerization, it is advised to firstly co-incubate PWCA and Arp2/3 complex for 5 min before mixing with actin and CapZ. Otherwise, the efficiency of branched actin polymerization will be decreased.

2. It is recommended to ultracentrifuge the actin monomers to remove the precipitate before use.

Recipes

1. Buffer A

   20 mM HEPES pH 7.4

   40 mM NaCl

   1 mM DTT

   Store at 4 °C, use within two weeks




2. Buffer B

   20 mM PIPES pH 6.8

   150 mM KCl

   2 mM MgCl₂

   5 mM EGTA

   1mM EDTA

   0.5 mM DTT

   0.2 mM ATP

   5% glycerol

   Store at 4 °C, use within two weeks




3. Buffer C

   20 mM MOPS pH 7.0

   100 mM KCl

   2 mM MgCl₂

   5 mM EGTA

   1 mM EDTA

   0.5 mM DTT

   0.2 mM ATP

   5% glycerol

   Store at 4 °C, use within two weeks




4. Lysis buffer

   20 mM Tris pH 8.0

   150 mM NaCl

   1 mM DTT

   1 mM EDTA

   5% glycerol

   1% Triton X-100

   Protease inhibitor cocktails

   Store at 4 °C, use within two weeks




5. Basic buffer

   20 mM Tris pH 8.0

   150 mM NaCl

   1 mM DTT

   1 mM EDTA

   Store at 4 °C, use within two weeks




6. Loading buffer

   20 mM Tris pH 8.0

   80 mM NaCl

   1 mM DTT

   1 mM EDTA

   Store at -20 °C




7. High-salt buffer

   20 mM Tris pH 8.0

   500 mM NaCl

   1 mM DTT

   1 mM EDTA

   Store at 4 °C, use within two weeks




8. Wash buffer

   20 mM HEPES pH 7.4

   500 mM NaCl

   1 mM DTT

   5% glycerol

   30 mM imidazole

   Store at 4 °C, use within two weeks




9. TIRF buffer

   1 mM MgCl₂

   50 mM KCl

   1 mM EGTA

   10 mM imidazole pH 8.0

   0.2 mM ATP

   100 mM DTT

   100 μg/mL glucose oxidase

   20 μg/mL catalase

   0.2% BSA

   0.5% methyl cellulose

   Store at 4 °C, use within two weeks




10. Elution buffer

    50 mM Tris pH 7.5

    250 mM imidazole

    300 mM NaCl

    0.2 mM EGTA

    Store at 4 °C, use within two weeks




11. Storage buffer

    50 mM HEPES PH7.4

    300 mM NaCl

    50 μM ATP-Mg

    10% sucrose

    Store at 4 °C, use within two weeks

Acknowledgments

We thank Drs. Jiangfeng Shen, Xiaoyu Fu, Wanqing Du, Wenkang Zhao, Xuezhao Feng, Mingrui Ding, Jinpei Zhang and Daxiao Sun for their helpful efforts and suggestions on optimizing this protocol. This work was supported by the Ministry of Science and Technology of China (2017YFA0506300), and the National Natural Science Foundation of China (31771536 and 31860316).

Competing interests

The authors declare no competing interests.

References

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